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Sample records for macrophage derived factor

  1. Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages.

    PubMed Central

    Lesnik, P; Rouis, M; Skarlatos, S; Kruth, H S; Chapman, M J

    1992-01-01

    Lipid-laden macrophages present as foam cells may contribute to the hyperthrombotic state of human atherosclerotic lesions by the production of tissue factor (TF). We investigated the effect of exogenous nonlipoprotein cholesterol on the expression of TF by human monocyte-derived macrophages in culture. Nonlipoprotein cholesterol at 50 micrograms/ml increased TF activity 4-fold; TF induction was dose- and time-dependent. Expression of TF activity was positively correlated with the free cholesterol content of monocyte-derived macrophages, was increased upon inhibition of cholesterol esterification, and reflected de novo synthesis of TF protein. TF expression in cholesterol-loaded macrophages remained sensitive to stimulation (approximately 12-fold) by bacterial lipopolysaccharide, indicating that intracellular free cholesterol and lipopolysaccharide act by distinct mechanisms in inducing TF procoagulant activity. Our results suggest that loading human monocyte-derived macrophages with free cholesterol induces upregulation of TF expression, thereby contributing to thrombus formation at sites of plaque rupture. Images PMID:1438222

  2. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. PMID:27503803

  3. Recruitment of mesenchymal stem cells and macrophages by dual release of stromal cell-derived factor-1 and a macrophage recruitment agent enhances wound closure.

    PubMed

    Kim, Yang-Hee; Tabata, Yasuhiko

    2016-04-01

    In this study, the wound closure of mouse skin defects was examined in terms of recruitment of mesenchymal stem cells (MSC) and macrophages. For the cells recruitment, stromal derived factor-1 (SDF-1) of a MSC recruitment agent and sphingosine-1 phosphate agonist (SEW2871) of a macrophages recruitment agent were incorporated into gelatin hydrogels, and then released in a controlled fashion. When applied to a skin wound defect of mice, gelatin hydrogels incorporating mixed 500 ng SDF-1 and 0.4, 0.8, or 1.6 mg SEW2871-micelles recruited a higher number of both MSC and macrophages than those incorporating SDF-1 or phosphate buffered saline. However, the number of M1 phenotype macrophages for the hydrogel incorporating mixed SDF-1 and SEW2871-micelles recruited was remarkably low to a significant extent compared with that for those hydrogel incorporating 0.4, 0.8, or 1.6 mg SEW2871-micelles. On the other hand, the number of M2 macrophages 3 days after the implantation of the hydrogels incorporating SDF-1 and 0.4 mg SEW2871-micelles significantly increased compared with that for other hydrogels. In vivo experiments revealed the hydrogels incorporating SDF-1 and 0.4 mg SEW2871-micelles promoted the wound closure of skin defect to a significant stronger extent than those incorporating SEW2871-micelles, SDF-1, and a mixture of SDF-1 and higher doses of SEW2871-micelles. It is concluded that the in vivo recruitment of MSC and macrophages to the defects may contribute to the tissue regeneration of skin wound. PMID:26704185

  4. Alveolar macrophage-derived chemotactic factor: kinetics of in vitro production and partial characterization.

    PubMed Central

    Merrill, W W; Naegel, G P; Matthay, R A; Reynolds, H Y

    1980-01-01

    Alveolar macrophages are the initial phagocytic cells that encounter foreign material and particulates deposited in the terminal airways. We have examined a mechanism by which these cells, after phagocytic challenge, may control or amplify the inflammatory response in lung parenchyma. Normal human alveolar macrophages (AM) were studied from eight subjects. With in vitro culture, AM produced and released two substances into culture media which have potent chemoattractant activity for blood polymorphonuclear granulocytes (PMN) and negligible activity for mononuclear cells. Release of these factors is maximally stimulated by aggregated human immunoglobulin (Ig)G or zymosan particles; however, simple adhesion of the macrophages to plastic surfaces is also sufficient to stimulate release of these chemotactic substances. The larger substance (10,000 daltons) is immunologically distinct from C5a and interacts with a different PMN membrane receptor than that known to exist for formyl-methionyl-leucyl-phenylalanine. Its chemotactic activity is sensitive to the enzymatic effect of trypsin. Although producing a single elution peak on gelfiltration chromatography, electrofocusing in polyacrylamide gels yielded five peaks of radioactivity. Chemotactic activity was localized to a fraction with a pI = 5.0. The smaller molecular weight substance has been less well characterized. Thus, the human AM can produce at least two factors which attract PMN and this capability may augment the local inflammatory response in the lung. PMID:7356678

  5. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

    PubMed

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-07-01

    Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

  6. Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

    PubMed Central

    Tavares-Murta, B. M.; Cunha, F. Q.; Dias-Baruffi, M.; Roque-Barreira, M. C.

    1996-01-01

    In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal−) and bound (D-gal+) fractions, with MNCF being found in the D-gal+ fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal− fraction. Furthermore, the incubation of the D-gal− fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+ activity. Overall, these results suggest that the D-gal− inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal+ fraction. PMID:18475709

  7. Gc-protein-derived macrophage activating factor counteracts the neuronal damage induced by oxaliplatin.

    PubMed

    Morucci, Gabriele; Branca, Jacopo J V; Gulisano, Massimo; Ruggiero, Marco; Paternostro, Ferdinando; Pacini, Alessandra; Di Cesare Mannelli, Lorenzo; Pacini, Stefania

    2015-02-01

    Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce

  8. Modulation of Decidual Macrophage Polarization by Macrophage Colony-Stimulating Factor Derived from First-Trimester Decidual Cells: Implication in Preeclampsia.

    PubMed

    Li, Min; Piao, Longzhu; Chen, Chie-Pein; Wu, Xianqing; Yeh, Chang-Ching; Masch, Rachel; Chang, Chi-Chang; Huang, S Joseph

    2016-05-01

    During human pregnancy, immune tolerance of the fetal semiallograft occurs in the presence of abundant maternal leukocytes. At the implantation site, macrophages comprise approximately 20% of the leukocyte population and act as primary mediators of tissue remodeling. Decidual macrophages display a balance between anti-inflammatory and proinflammatory phenotypes. However, a shift to an M1 subtype is reported in preeclampsia. Granulocyte-macrophage colony-stimulating-factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are major differentiating factors that mediate M1 and M2 polarization, respectively. Previously, we observed the following: i) the preeclamptic decidua contains an excess of both macrophages and GM-CSF, ii) the preeclampsia-associated proinflammatory cytokines, IL-1β and tumor necrosis factor-α, markedly enhance GM-CSF and M-CSF expression in cultured leukocyte-free first-trimester decidual cells (FTDCs), iii) FTDC-secreted GM-CSF polarizes macrophages toward an M1 subtype. The microenvironment is a key determinant of macrophage phenotype. Thus, we examined proinflammatory stimulation of FTDC-secreted M-CSF and its role in macrophage development. Immunofluorescence staining demonstrated elevated M-CSF-positive decidual cell numbers in preeclamptic decidua. In FTDCs, IL-1β and tumor necrosis factor-α signal through the NF-κB pathway to induce M-CSF production, which does the following: i) enhances differentiation of and elevates CD163 expression in macrophages, ii) increases macrophage phagocytic capacity, and iii) inhibits signal-regulatory protein α expression by macrophages. These findings suggest that FTDC-secreted M-CSF modulates the decidual immune balance by inducing M2 macrophage polarization and phagocytic capacity in response to proinflammatory stimuli. PMID:26970370

  9. Macrophage-derived IL-33 is a critical factor for placental growth.

    PubMed

    Fock, Valerie; Mairhofer, Mario; Otti, Gerlinde R; Hiden, Ursula; Spittler, Andreas; Zeisler, Harald; Fiala, Christian; Knöfler, Martin; Pollheimer, Jürgen

    2013-10-01

    IL-33, the most recently discovered member of the IL-1 superfamily and ligand for the transmembrane form of ST2 (ST2L), has been linked to several human pathologies including rheumatoid arthritis, asthma, and cardiovascular disease. Deregulated levels of soluble ST2, the natural IL-33 inhibitor, have been reported in sera of preeclamptic patients. However, the role of IL-33 during healthy pregnancy remains elusive. In the current study, IL-33 was detected in the culture supernatants of human placental and decidual macrophages, identifying them as a major source of secreted IL-33 in the uteroplacental unit. Because flow cytometry and immunofluorescence stainings revealed membranous ST2L expression on specific trophoblast populations, we hypothesized that IL-33 stimulates trophoblasts in a paracrine manner. Indeed, BrdU incorporation assays revealed that recombinant human IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts and cell column trophoblasts in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. Interestingly, Western blot and immunofluorescence analyses demonstrated that IL-33 activates AKT and ERK1/2 in primary trophoblasts and placental explants. Inhibitors against PI3K (LY294002) and MEK1/2 (UO126) efficiently blocked IL-33-induced proliferation in all model systems used. In summary, with IL-33, we define for the first time, to our knowledge, a macrophage-derived regulator of placental growth during early pregnancy. PMID:23997215

  10. Alveolar macrophage-derived vascular endothelial growth factor contributes to allergic airway inflammation in a mouse asthma model.

    PubMed

    Song, C; Ma, H; Yao, C; Tao, X; Gan, H

    2012-06-01

    Vascular endothelial growth factor (VEGF) is a potent proangiogenic factor that correlates with vascular permeability and remodelling in asthma. Recently, alveolar macrophages (AM) were shown to be an important source of VEGF during lung injury. Our previous studies demonstrated that AM are an important subset of macrophages in the initiation of asthmatic symptoms. Here, we further investigated whether AM-derived VEGF was required for allergic airway inflammation in asthma. In this study, we reported that the expression of VEGF in AM was significantly increased after allergen challenge. Depleting AM or neutralizing VEGF in alveolus prevented ovalbumin (OVA)-induced asthma-related inflammation by inhibiting the infiltration of inflammatory cells in the lung, reduced the level of the cytokines, IL-4, IL-5, and IL-13, in the bronchoalveolar lavage fluid (BALF) and decreased airway hyperresponsiveness (AHR). Moreover, the inhibition of miR-20b increased the protein level of VEGF in normal AM; conversely, increasing miR-20b in asthmatic AM resulted in decreased VEGF protein levels. These findings suggest that AM-derived VEGF is necessary for allergic airway inflammation in asthmatic mice and miR-20b negatively regulates this expression. PMID:22324377

  11. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line

    PubMed Central

    TOYOHARA, YUKIYO; HASHITANI, SUSUMU; KISHIMOTO, HIROMITSU; NOGUCHI, KAZUMA; YAMAMOTO, NOBUTO; URADE, MASAHIRO

    2011-01-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model. PMID:22848250

  12. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line.

    PubMed

    Toyohara, Yukiyo; Hashitani, Susumu; Kishimoto, Hiromitsu; Noguchi, Kazuma; Yamamoto, Nobuto; Urade, Masahiro

    2011-07-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model. PMID:22848250

  13. Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats.

    PubMed

    Okubo, Masamichi; Yamanaka, Hiroki; Kobayashi, Kimiko; Dai, Yi; Kanda, Hirosato; Yagi, Hideshi; Noguchi, Koichi

    2016-01-01

    Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats. PMID:27071004

  14. Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats

    PubMed Central

    Okubo, Masamichi; Yamanaka, Hiroki; Kobayashi, Kimiko; Dai, Yi; Kanda, Hirosato; Yagi, Hideshi; Noguchi, Koichi

    2016-01-01

    Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats. PMID:27071004

  15. Transforming growth factor beta reverses the glucocorticoid-induced wound-healing deficit in rats: possible regulation in macrophages by platelet-derived growth factor.

    PubMed

    Pierce, G F; Mustoe, T A; Lingelbach, J; Masakowski, V R; Gramates, P; Deuel, T F

    1989-04-01

    Transforming growth factor beta (TGF-beta) and the platelet-derived growth factor (PDGF) are potent mitogenic polypeptides which enhance rates of wound healing in experimental animals; in contrast, glucocorticoids inhibit wound repair. The potential of TGF-beta and PDGF to reverse this inhibition in healing was tested in methylprednisolone-treated rats with deficits in skin wound strength of 50%. Single applications of TGF-beta (10-40 pmol per wound, 0.25-1 micrograms) applied locally at the time of wounding fully reversed this deficit in a concentration-dependent and highly reproducible manner. Wounds in glucocorticoid-treated animals were characterized by a near total absence of neutrophils and macrophages and by a delayed influx and reduced density of fibroblasts; however, such wounds treated with TGF-beta showed significant increases in wound fibroblasts and in intracellular procollagen type I. PDGF did not reverse the deficit in wound breaking strength in glucocorticoid-treated rats; there were more fibroblasts in the PDGF-treated wounds, but these fibroblasts lacked the enhanced expression of procollagen type I found in TGF-beta-treated wounds. The wound macrophages, required for normal tissue repair, remained absent from both PDGF- and TGF-beta-treated wounds in glucocorticoid-treated animals. This result suggested that macrophages might normally act as an intermediate in the induction of procollagen synthesis in fibroblasts of PDGF-treated wounds and that TGF-beta might bypass the macrophage through its capacity to stimulate directly new synthesis of procollagen type I in fibroblasts. Whereas PDGF does not stimulate procollagen synthesis, in a rodent macrophage cell line, PDGF induced a highly significant, time-dependent enhancement of expression of TGF-beta. PMID:2928327

  16. Patient-derived Granulocyte/Macrophage Colony–Stimulating Factor Autoantibodies Reproduce Pulmonary Alveolar Proteinosis in Nonhuman Primates

    PubMed Central

    Sakagami, Takuro; Beck, David; Uchida, Kanji; Suzuki, Takuji; Carey, Brenna C.; Nakata, Koh; Keller, Gary; Wood, Robert E.; Wert, Susan E.; Ikegami, Machiko; Whitsett, Jeffrey A.; Luisetti, Maurizio; Davies, Stella; Krischer, Jeffrey P.; Brody, Alan; Ryckman, Fred; Trapnell, Bruce C.

    2010-01-01

    Rationale: Granulocyte/macrophage colony–stimulating factor (GM-CSF) autoantibodies (GMAb) are strongly associated with idiopathic pulmonary alveolar proteinosis (PAP) and are believed to be important in its pathogenesis. However, levels of GMAb do not correlate with disease severity and GMAb are also present at low levels in healthy individuals. Objectives: Our primary objective was to determine whether human GMAb would reproduce PAP in healthy primates. A secondary objective was to determine the concentration of GMAb resulting in loss of GM-CSF signaling in vivo (i.e., critical threshold). Methods: Nonhuman primates (Macaca fascicularis) were injected with highly purified, PAP patient-derived GMAb in dose-ranging (2.2–50 mg) single and multiple administration studies, and after blocking antihuman immunoglobulin immune responses, in chronic administration studies maintaining serum levels greater than 40 μg/ml for up to 11 months. Measurements and Main Results: GMAb blocked GM-CSF signaling causing (1) a milky-appearing bronchoalveolar lavage fluid containing increased surfactant lipids and proteins; (2) enlarged, foamy, surfactant-filled alveolar macrophages with reduced PU.1 and PPARγ mRNA, and reduced tumor necrosis factor-α secretion; (3) pulmonary leukocytosis; (4) increased serum surfactant protein-D; and (5) impaired neutrophil functions. GM-CSF signaling varied inversely with GMAb concentration below a critical threshold of 5 μg/ml, which was similar in lungs and blood and to the value observed in patients with PAP. Conclusions: GMAb reproduced the molecular, cellular, and histopathologic features of PAP in healthy primates, demonstrating that GMAb directly cause PAP. These results have implications for therapy of PAP and help define the therapeutic window for potential use of GMAb to treat other disorders. PMID:20224064

  17. Developmental derivation of embryonic and adult macrophages.

    PubMed

    Shepard, J L; Zon, L I

    2000-01-01

    The macrophage cell lineage continually arises from hematopoietic stem cells during embryonic, fetal, and adult life. Previous theories proposed that macrophages are the recent progeny of bone marrow-derived monocytes and that they function primarily in phagocytosis. More recently, however, observations have shown that the ontogeny of macrophages in early mouse and human embryos is different from that occurring during adult development, and that the embryonic macrophages do not follow the monocyte pathway. Fetal macrophages are thought to differentiate from yolk sac-derived primitive macrophages before the development of adult monocytes. Further support for a separate lineage of fetal macrophages has come from studies of several species, including chicken, zebrafish, Xenopus, Drosophila, and C. elegans. The presence of fetal macrophages in PU.1-null mice indicates their independence from monocyte precursors and their existence as an alternative macrophage lineage. PMID:10608497

  18. Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

    PubMed

    Otvos, Balint; Silver, Daniel J; Mulkearns-Hubert, Erin E; Alvarado, Alvaro G; Turaga, Soumya M; Sorensen, Mia D; Rayman, Patricia; Flavahan, William A; Hale, James S; Stoltz, Kevin; Sinyuk, Maksim; Wu, Qiulian; Jarrar, Awad; Kim, Sung-Hak; Fox, Paul L; Nakano, Ichiro; Rich, Jeremy N; Ransohoff, Richard M; Finke, James; Kristensen, Bjarne W; Vogelbaum, Michael A; Lathia, Justin D

    2016-08-01

    Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039. PMID:27145382

  19. Differential expression of HIV-1 interfering factors in monocyte-derived macrophages stimulated with polarizing cytokines or interferons

    NASA Astrophysics Data System (ADS)

    Jiménez, Viviana Cobos; Booiman, Thijs; de Taeye, Steven W.; van Dort, Karel A.; Rits, Maarten A. N.; Hamann, Jörg; Kootstra, Neeltje A.

    2012-10-01

    HIV-1 replication in macrophages can be regulated by cytokines and infection is restricted in macrophages activated by type I interferons and polarizing cytokines. Here, we observed that the expression levels of the cellular factors Trim5α, CypA, APOBEC3G, SAMHD-1, Trim22, tetherin and TREX-1, and the anti-HIV miRNAs miR-28, miR-150, miR-223 and miR-382 was upregulated by IFN-α and IFN-β in macrophages, which may account for the inhibiting effect on viral replication and the antiviral state of these cells. Expression of these factors was also increased by IFN-γ +/- TNF-α, albeit to a lesser extent; yet, HIV-1 replication in these cells was not restricted at the level of proviral synthesis, indicating that these cellular factors only partially contribute to the observed restriction. IL-4, IL-10 or IL-32 polarization did not affect the expression of cellular factors and miRNAs, suggesting only a limited role for these cellular factors in restricting HIV-1 replication in macrophages.

  20. Antitumor effect of vitamin D-binding protein-derived macrophage activating factor on Ehrlich ascites tumor-bearing mice.

    PubMed

    Koga, Y; Naraparaju, V R; Yamamoto, N

    1999-01-01

    Cancerous cells secrete alpha-N-acetylgalactosaminidase (NaGalase) into the blood stream, resulting in deglycosylation of serum vitamin D3-binding protein (known as Gc protein), which is a precursor for macrophage activating factor (MAF). Incubation of Gc protein with immobilized beta-galactosidase and sialidase generates the most potent macrophage activating factor (designated GcMAF). Administration of GcMAF to cancer-bearing hosts can bypass the inactivated MAF precursor and act directly on macrophages for efficient activation. Therapeutic effects of GcMAF on Ehrlich ascites tumor-bearing mice were assessed by survival time and serum NaGalase activity, because serum NaGalase activity was proportional to tumor burden. A single administration of GcMAF (100 pg/mouse) to eight mice on the same day after transplantation of the tumor (5 x 10(5) cells) showed a mean survival time of 21 +/- 3 days for seven mice, with one mouse surviving more than 60 days, whereas tumor-bearing controls had a mean survival time of 13 +/- 2 days. Six of the eight mice that received two GcMAF administrations, at Day 0 and Day 4 after transplantation, survived up to 31 +/- 4 days whereas, the remaining two mice survived for more than 60 days. Further, six of the eight mice that received three GcMAF administrations with 4-day intervals showed an extended survival of at least 60 days, and serum NaGalase levels were as low as those of control mice throughout the survival period. The cure with subthreshold GcMAF-treatments (administered once or twice) of tumor-bearing mice appeared to be a consequence of sustained macrophage activation by inflammation resulting from the macrophage-mediated tumoricidal process. Therefore, a protracted macrophage activation induced by a few administrations of minute amounts of GcMAF eradicated the murine ascites tumor. PMID:9893164

  1. GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients

    PubMed Central

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Branca, Jacopo JV; Morucci, Gabriele; Gulisano, Massimo; Noakes, David; Eslinger, Robert; Pacini, Stefania

    2013-01-01

    α-N-acetylgalactosaminidase (nagalase) accumulates in the serum of cancer patients and its activity correlates with tumor burden, aggressiveness and clinical disease progression. The administration of GC protein-derived macrophage-activating factor (GcMAF) to cancer patients with elevated levels of nagalase has been associated with a decrease of serum nagalase activity and with significant clinical benefits. Here, we report the results of the administration of GcMAF to a heterogeneous cohort of patients with histologically diverse, advanced neoplasms, generally considered as “incurable” diseases. In most cases, GcMAF therapy was initiated at late stages of tumor progression. As this is an open-label, non-controlled, retrospective analysis, caution must be employed when establishing cause-effect relationships between the administration GcMAF and disease outcome. However, the response to GcMAF was generally robust and some trends emerged. All patients (n = 20) presented with elevated serum nagalase activity, well above normal values. All patients but one showed a significant decrease of serum nagalase activity upon weekly GcMAF injections. Decreased nagalase activity was associated with improved clinical conditions and no adverse side effects were reported. The observations reported here confirm and extend previous results and pave the way to further studies aimed at assessing the precise role and indications for GcMAF-based anticancer immunotherapy. PMID:24179708

  2. GC protein-derived macrophage-activating factor decreases α-N-acetylgalactosaminidase levels in advanced cancer patients.

    PubMed

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Branca, Jacopo Jv; Morucci, Gabriele; Gulisano, Massimo; Noakes, David; Eslinger, Robert; Pacini, Stefania

    2013-08-01

    α-N-acetylgalactosaminidase (nagalase) accumulates in the serum of cancer patients and its activity correlates with tumor burden, aggressiveness and clinical disease progression. The administration of GC protein-derived macrophage-activating factor (GcMAF) to cancer patients with elevated levels of nagalase has been associated with a decrease of serum nagalase activity and with significant clinical benefits. Here, we report the results of the administration of GcMAF to a heterogeneous cohort of patients with histologically diverse, advanced neoplasms, generally considered as "incurable" diseases. In most cases, GcMAF therapy was initiated at late stages of tumor progression. As this is an open-label, non-controlled, retrospective analysis, caution must be employed when establishing cause-effect relationships between the administration GcMAF and disease outcome. However, the response to GcMAF was generally robust and some trends emerged. All patients (n = 20) presented with elevated serum nagalase activity, well above normal values. All patients but one showed a significant decrease of serum nagalase activity upon weekly GcMAF injections. Decreased nagalase activity was associated with improved clinical conditions and no adverse side effects were reported. The observations reported here confirm and extend previous results and pave the way to further studies aimed at assessing the precise role and indications for GcMAF-based anticancer immunotherapy. PMID:24179708

  3. Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function.

    PubMed

    Dickerhof, Nina; Schindler, Lisa; Bernhagen, Jürgen; Kettle, Anthony J; Hampton, Mark B

    2015-12-01

    Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF. PMID:26453918

  4. Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

    PubMed

    Branca, Jacopo J V; Morucci, Gabriele; Malentacchi, Francesca; Gelmini, Stefania; Ruggiero, Marco; Pacini, Stefania

    2015-09-01

    The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. PMID:25782915

  5. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid β-induced neurotoxicity

    PubMed Central

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan; Splittgerber, Ryan; Fan, Guo-Huang; Richmond, Ann

    2011-01-01

    Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-β (Aβ). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aβ-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aβ-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aβ led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The Aβ-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F2-isoprostanes, was significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aβ neurotoxicity in CXCR2−/− mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. PMID:21704645

  6. Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).

    PubMed

    Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko

    2009-01-01

    Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years. PMID:19031451

  7. Immunotherapy of metastatic colorectal cancer with vitamin D-binding protein-derived macrophage-activating factor, GcMAF.

    PubMed

    Yamamoto, Nobuto; Suyama, Hirofumi; Nakazato, Hiroaki; Yamamoto, Nobuyuki; Koga, Yoshihiko

    2008-07-01

    Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 microg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/ human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32-50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients. PMID:18058096

  8. Macrophage Migration Inhibitory Factor Inhibits the Migration of Cartilage End Plate-Derived Stem Cells by Reacting with CD74

    PubMed Central

    Xiong, Cheng-jie; Huang, Bo; Zhou, Yue; Cun, Yan-ping; Liu, Lan-tao; Wang, Jian; Li, Chang-qing; Pan, Yong; Wang, Hai

    2012-01-01

    Background Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that regulates inflammatory reactions and the pathophysiology of many inflammatory diseases. Intervertebral disc (IVD) degeneration is characterized by an inflammatory reaction, but the potential role of MIF in IVD degeneration has not been determined. Recent studies have shown that MIF and its receptor, CD74, are involved in regulating the migration of human mesenchymal stem cells (MSCs); Thus, MIF might impair the ability of mesenchymal stem cells (MSCs) to home to injured tissues. Our previous studies indicated that cartilage endplate (CEP)-derived stem cells (CESCs) as a type of MSCs exist in human degenerate IVDs. Here, we investigate the role of MIF in regulating the migration of CESCs. Methods and Findings CESCs were isolated and identified. We have shown that MIF was distributed in human degenerate IVD tissues and was subject to regulation by the pro-inflammatory cytokine TNF-α. Furthermore, in vitro cell migration assays revealed that nucleus pulposus (NP) cells inhibited the migration of CESCs in a number-dependent manner, and ELISA assays revealed that the amount of MIF in conditioned medium (CM) was significantly increased as a function of increasing cell number. Additionally, recombinant human MIF (r-MIF) inhibited the migration of CESCs in a dose-dependent manner. CESCs migration was restored when an antagonist of MIF, (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), was added. Finally, a CD74 activating antibody (CD74Ab) was used to examine the effect of CD74 on CESCs motility and inhibited the migration of CESCs in a dose-dependent manner. Conclusions We have identified and characterized a novel regulatory mechanism governing cell migration during IVD degeneration. The results will benefit understanding of another possible mechanism for IVD degeneration, and might provide a new method to repair degenerate IVD by enhancing CESCs

  9. Effect of salivary gland adenocarcinoma cell-derived alpha-N-acetylgalactosaminidase on the bioactivity of macrophage activating factor.

    PubMed

    Matsuura, Takashi; Uematsu, Takashi; Yamaoka, Minoru; Furusawa, Kiyofumi

    2004-03-01

    The aim of this study was to clarify the effects of alpha-N-acetylgalactosaminidase (alpha-NaGalase) produced by human salivary gland adenocarcinoma (SGA) cells on the bioactivity of macrophage-activating factor (GcMAF). High exo-alpha-NaGalase activity was detected in the SGA cell line HSG. HSG alpha-NaGalase had both exo- and endo-enzyme activities, cleaving the Gal-GalNAc and GalNAc residues linked to Thr/Ser but not releasing the [NeuAc2-6]GalNac residue. Furthermore, GcMAF enzymatically prepared from the Gc protein enhanced the superoxide-generation capacity and phagocytic activity of monocytes/macrophages. However, GcMAF treated with purified alpha-NaGalase did not exhibit these effects. Thus, HSG possesses the capacity to produce larger quantities of alpha-NaGalase, which inactivates GcMAF produced from Gc protein, resulting in reduced phagocytic activity and superoxide-generation capacity of monocytes/macrophages. The present data strongly suggest that HSG alpha-NaGalase acts as an immunodeficiency factor in cancer patients. PMID:14767536

  10. Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis.

    PubMed

    Nijaguna, Mamatha Bangalore; Patil, Vikas; Urbach, Serge; Shwetha, Shivayogi D; Sravani, Kotha; Hegde, Alangar S; Chandramouli, Bangalore A; Arivazhagan, Arimappamagan; Marin, Philippe; Santosh, Vani; Somasundaram, Kumaravel

    2015-09-18

    Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NFκB pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NFκB-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression. PMID:26245897

  11. Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF).

    PubMed

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki; Ushijima, Naofumi

    2008-01-15

    Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years. PMID:17935130

  12. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

    PubMed Central

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-01-01

    Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

  13. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    SciTech Connect

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan; Fan, Guo-Huang; Richmond, Ann

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP-2 or

  14. A Novel Carboline Derivative Inhibits Nitric Oxide Formation in Macrophages Independent of Effects on Tumor Necrosis Factor α and Interleukin-1β Expression

    PubMed Central

    Poola, Bhaskar; Pasupuleti, Nagarekha; Nantz, Michael H.; Lein, Pamela J.; Gorin, Fredric

    2015-01-01

    Neuropathic pain is a maladaptive immune response to peripheral nerve injury that causes a chronic painful condition refractory to most analgesics. Nitric oxide (NO), which is produced by nitric oxide synthases (NOSs), has been implicated as a key factor in the pathogenesis of neuropathic pain. β-Carbolines are a large group of natural and synthetic indole alkaloids, some of which block activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), a predominant transcriptional regulator of NOS expression. Here, we characterize the inhibitory effects of a novel 6-chloro-8-(glycinyl)-amino-β-carboline (8-Gly carb) on NO formation and NF-κB activation in macrophages. 8-Gly carb was significantly more potent than the NOS inhibitor NG-nitro-l-arginine methyl ester in inhibiting constitutive and inducible NO formation in primary rat macrophages. 8-Gly carb interfered with NF-κB–mediated gene expression in differentiated THP1-XBlue cells, a human NF-κB reporter macrophage cell line, but only at concentrations severalfold higher than needed to significantly inhibit NO production. 8-Gly carb also had no effect on tumor necrosis factor α (TNFα)–induced phosphorylation of the p38 mitogen-activated protein kinase in differentiated THP1 cells, and did not inhibit lipopolysaccharide- or TNFα-stimulated expression of TNFα and interleukin-1β. These data demonstrate that relative to other carbolines and pharmacologic inhibitors of NOS, 8-Gly carb exhibits a unique pharmacological profile by inhibiting constitutive and inducible NO formation independent of NF-κB activation and cytokine expression. Thus, this novel carboline derivative holds promise as a parent compound, leading to therapeutic agents that prevent the development of neuropathic pain mediated by macrophage-derived NO without interfering with cytokine expression required for neural recovery following peripheral nerve injury. PMID:25538105

  15. [Adipose-derived stem cells promote the polarization from M1 macrophages to M2 macrophages].

    PubMed

    Yin, Xuehong; Pang, Chunyan; Bai, Li; Zhang, Ying; Geng, Lixia

    2016-03-01

    Objective To investigate the effects of adipose-derived stem cells (ADSCs) on M1/M2 macrophages and whether ADSCs are able to promote the polarization from M1 macrophages to M2 macrophages. Methods M1 macrophages were induced from J774.1 macrophages by 24-hour stimulation of lipopolysaccharide (LPS) and interferon γ (IFN-γ), and M2 macrophages were induced from J774.1 macrophages by interleukin 4 (IL-4) for another 24 hours. Then M1/M2 macrophages were separately cultured in the presence of ADSCs for 24 hours. The M1/M2 macrophages and their corresponding supernatants were collected for further analysis. The expressions of IL-6, tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS), CC chemokine ligand 2 (CCL2), CD86, arginase 1 (Arg1), mannose receptors/CD206 (MR/CD206), IL-10, found in inflammatory zone 1 (FIZZ1), chitinase 3-like 3 (Ym-1) were detected by real-time PCR and ELISA. Results ADSCs significantly decreased the levels of IL-6, TNF-α, iNOS, CCL2 and CD86, and increased the levels of Arg1, CD206 and IL-10 in M1 macrophages. In the supernatant of M1 macrophages, the expressions of IL-6 and TNF-α were reduced, while those of CD206 were enhanced. In M2 macrophages, ADSCs resulted in down-regulation of IL-6, TNF-α, iNOS, CD86 and up-regulation of Arg1, CD206, FIZZ-1, Ym-1 and IL-10. In the supernatant of M2 macrophages, the expression levels of IL-6 and TNF-α were down-regulated and those of CD206 were up-regulated. Conclusion ADSCs can inhibit the gene expression of M1 macrophages and promote the gene expression of M2 macrophages, as well as mediate the polarization from M1 macrophages to M2 macrophages. PMID:26927552

  16. A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

    PubMed

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J V; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

    2013-07-01

    The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects. PMID:23857228

  17. A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

    PubMed Central

    Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J. V.; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

    2013-01-01

    The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects. PMID:23857228

  18. Stromal cell-derived factor-1α and macrophage migration-inhibitory factor induce metastatic behavior in CXCR4-expressing colon cancer cells.

    PubMed

    Shin, Han-Na; Moon, Hyun-Hye; Ku, Ja-Lok

    2012-12-01

    Metastasis of cancer cells is a major cause of death in cancer patients. The process of cancer metastasis includes the proliferation of primary cancer cells, local invasion, intravasation and cancer cell survival in blood flow, extravasation and attachment to secondary organs and metastatic growth in a new environment. In these mechanisms of cancer metastasis, CXC chemokine receptor 4 (CXCR4) and its ligand play an important role. Stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is well known as a ligand of CXCR4, and macrophage migration-inhibitory factor (MIF) has recently become known as a ligand of CXCR4. In many types of cancers including breast, pancreatic and colorectal cancer (CRC), CXCR4/SDF-1α has been investigated in metastasis-related cancer behavior, which include cell proliferation, adhesion, migration and invasion. However, CXCR4/MIF has rarely been investigated in the metastatic behavior of colon cancer cells. In this report, the effect of SDF-1α or MIF was studied on cell cycle, cell proliferation, adhesion and migration of the CXCR4-expressing colon cancer cell line SW480. SDF-1α or MIF caused a decrease in the number of cells in G0/G1 phase and an increase in the numbers of cells in S and G2/M phases. In addition, SDF-1α or MIF caused an increase in cell proliferation, cell adhesion to fibronectin and migration. AMD3100, a CXCR4 antagonist, attenuated these effects, which included increased cell proliferation, adhesion and migration due to treatment of CXCR4-expressing colon cancer cells with SDF-1α or MIF. In conclusion, SDF-1α or MIF affects the metastasis-related behaviors of CXCR4-expressing colon cancer cells. PMID:23023114

  19. Is chondroitin sulfate responsible for the biological effects attributed to the GC protein-derived Macrophage Activating Factor (GcMAF)?

    PubMed

    Ruggiero, Marco; Reinwald, Heinz; Pacini, Stefania

    2016-09-01

    We hypothesize that a plasma glycosaminoglycan, chondroitin sulfate, may be responsible for the biological and clinical effects attributed to the Gc protein-derived Macrophage Activating Factor (GcMAF), a protein that is extracted from human blood. Thus, Gc protein binds chondroitin sulfate on the cell surface and such an interaction may occur also in blood, colostrum and milk. This interpretation would solve the inconsistencies encountered in explaining the effects of GcMAF in vitro and in vivo. According to our model, the Gc protein or the GcMAF bind to chondroitin sulfate both on the cell surface and in bodily fluids, and the resulting multimolecular complexes, under the form of oligomers trigger a transmembrane signal or, alternatively, are internalized and convey the signal directly to the nucleus thus eliciting the diverse biological effects observed for both GcMAF and chondroitin sulfate. PMID:27515218

  20. Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

    PubMed

    Yamamoto, N; Naraparaju, V R

    1998-06-01

    Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice. PMID:9682967

  1. Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

    PubMed

    Yamamoto, N

    1996-10-01

    Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. PMID:9070663

  2. Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6.

    PubMed Central

    Mühlradt, P F; Frisch, M

    1994-01-01

    Mycoplasmal products may exert a number of diverse in vitro effects on cells of the immune system. A macrophage-activating substance from Mycoplasma fermentans was described in this laboratory and named mycoplasma-derived high-molecular-weight material (MDHM). Using synthesis of nitric oxide by peritoneal cells from endotoxin low-responder mice as an assay system, MDHM was purified as follows. After freeze-thawing of M. fermentans, MDHM activity was sedimented with the membrane fraction. Membranes were delipidated with chloroform-methanol, and MDHM activity was extracted with octyl glucoside. Coextracted proteins were degraded by proteinase K. MDHM was further purified by reversed-phase high-pressure liquid chromatography and eluted in one major and one minor peak of activity. Neither carbohydrates nor amino acids were found as constituents. MDHM had the following properties: it partitioned into the phenol phase upon phenol-water extraction and into the Triton phase after extraction with Triton X-114. MDHM was not inactivated by either phospholipase A2 or triglyceride lipases. However, mild periodate treatment led to a > 95% loss of activity. Also, alkaline hydrolysis at 25 degrees C completely abolished MDHM activity with a half-life of 2 min. MDHM activity was spread out over a wide molecular weight range upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes, whereas after proteinase treatment MDHM activity migrated close to the front. These features of MDHM, taken together, speak in favor of an amphiphilic molecule with a lipid moiety carrying fatty acids in ester linkage and a polyol moiety of unknown character. MDHM was active in the nanogram-per-milliliter range, activating macrophages to release nitric oxide, interleukin-6, and tumor necrosis factor. Images PMID:8063396

  3. Involvement of insulin-like growth factor-1 and its binding proteins in proliferation and differentiation of murine bone marrow-derived macrophage precursors.

    PubMed

    Long, E; Huynh, H T; Zhao, X

    1998-10-01

    Insulin-like growth factor 1 (IGF-1) and its binding proteins (IGFBPs) are involved in proliferation and differentiation of many cell types. In the present study, the involvement of IGF-1 and IGFBPs in proliferation and differentiation of murine bone marrow-derived macrophages (BMDM) was investigated. L929-conditioned media (LCM) containing abundant macrophage colony-stimulating factor CSF-1 were used to stimulate BMDM development from their bone marrow precursors. The alteration of IGF-1 and IGFBPs during LCM-induced BMDM proliferation and differentiation was first studied. The cells were cultured in RPMI complete media containing 20% LCM for different time periods and then incubated in serum-free media for 24 h. The supernatants were collected for Western ligand blotting and immunoblotting analyses, and the cell pellets for Northern blotting analyses. The mRNA level of IGF-1 increased in a time-dependent manner. An increase of IGFBP-4 accumulation in the conditioned media was also observed during this process. However the mRNA expression of IGFBP-4 remained constant, indicating a posttranscriptional regulation of IGFBP-4 secretion and/or stability. The effects of exogenous recombinant human IGF-1 (rhIGF-1) on BMDM proliferation and differentiation were further studied. Two IGF-1 analogs (long R3 IGF-1 and des [1-3] IGF-1) were also used in parallel with regular IGF-1 to indicate the involvement of IGFBPs in BMDM development. Cells were cultured in complete media containing 20% LCM for different time periods, and then incubated in serum-free media in the presence of rhIGF-1 or its analogs for 24 h. These three forms of IGF-1 all potentiated the proliferation of freshly isolated BMDM precursors (d 0). rhIGF-1 and long R3 IGF-1, but not des (1-3) IGF-1, continued to stimulate the cell proliferation on d 1. The effects of these three forms of IGF-1 on BMDM differentiation were investigated using mannose receptor expression as a marker. Long R3 IGF-1 and des (1-3) IGF

  4. Modulation of adhesion molecule expression on endothelial cells during the late asthmatic reaction: role of macrophage-derived tumour necrosis factor-alpha.

    PubMed Central

    Lassalle, P; Gosset, P; Delneste, Y; Tsicopoulos, A; Capron, A; Joseph, M; Tonnel, A B

    1993-01-01

    In a previous work we have demonstrated that in patients exhibiting a late allergic reaction (LAR), alveolar macrophages (AM) collected 18 h after bronchial allergen challenge produced high levels of IL-6 and tumour necrosis factor-alpha (TNF) which is known to up-regulate the endothelial cell expression of adhesion molecules participating in the development of the inflammatory reaction in bronchial asthma. For these reasons, we evaluated the effect of AM supernatants from asthmatic patients developing an LAR on intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1) expression by human endothelial cells. The expression of adhesion molecules was assessed by an ELISA method and compared with the effect of an optimal dose of human recombinant (hr) TNF. Results showed that AM supernatants, from challenged asthmatics developing an LAR, increased significantly the ICAM-1 and ELAM-1 expression on endothelial cells to a level similar to that obtained in the presence of hrTNF (500 U/ml) (P < 0.001 in both cases, respectively 90.4% and 75.2% of the level obtained with hrTNF). In contrast, AM supernatants from asthmatics at baseline or exhibiting, after challenge, a single early reaction had no significant effect on these parameters (P = NS in both cases, respectively 23.5% and 24.7% of the ICAM-1 expression, 22.7% and 15.3% of the ELAM-1 expression obtained with hrTNF). AM-derived TNF present in these supernatants was thought to play a key role in endothelial cell stimulation, since: (i) TNF concentration in AM supernatants correlated with its effect on ICAM-1 (r = 0.80, P < 10(-4)) and ELAM-1 expression (r = 0.88, P < 10(-5)); and (ii) a neutralizing anti-TNF antibody decreased their effect (68% and 80% respectively on ICAM-1 and ELAM-1 expression). Moreover, the role of IL-6 was excluded on the basis both of the hrIL-6 inefficiency to induce ICAM-1 and ELAM-1 synthesis, even in costimulation with hrTNF, and of anti-IL-6 antibody

  5. Prostate Field Cancerization: Deregulated Expression of Macrophage Inhibitory Cytokine 1 (MIC-1) and Platelet Derived Growth Factor A (PDGF-A) in Tumor Adjacent Tissue

    PubMed Central

    Jones, Anna C.; Shoshan, Dor S.; Fischer, Edgar G.; Trujillo, Kristina A.; Bisoffi, Marco

    2015-01-01

    Prostate field cancerization denotes molecular alterations in histologically normal tissues adjacent to tumors. Such alterations include deregulated protein expression, as we have previously shown for the key transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS). Here we add the two secreted factors macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) to the growing list of protein markers of prostate field cancerization. Expression of MIC-1 and PDGF-A was measured quantitatively by immunofluorescence and comprehensively analyzed using two methods of signal capture and several groupings of data generated in human cancerous (n = 25), histologically normal adjacent (n = 22), and disease-free (n = 6) prostate tissues. A total of 208 digitized images were analyzed. MIC-1 and PDGF-A expression in tumor tissues were elevated 7.1x to 23.4x and 1.7x to 3.7x compared to disease-free tissues, respectively (p<0.0001 to p = 0.08 and p<0.01 to p = 0.23, respectively). In support of field cancerization, MIC-1 and PDGF-A expression in adjacent tissues were elevated 7.4x to 38.4x and 1.4x to 2.7x, respectively (p<0.0001 to p<0.05 and p<0.05 to p = 0.51, respectively). Also, MIC-1 and PDGF-A expression were similar in tumor and adjacent tissues (0.3x to 1.0x; p<0.001 to p = 0.98 for MIC-1; 0.9x to 2.6x; p<0.01 to p = 1.00 for PDGF-A). All analyses indicated a high level of inter- and intra-tissue heterogeneity across all types of tissues (mean coefficient of variation of 86.0%). Our data shows that MIC-1 and PDGF-A expression is elevated in both prostate tumors and structurally intact adjacent tissues when compared to disease-free specimens, defining field cancerization. These secreted factors could promote tumorigenesis in histologically normal tissues and lead to tumor multifocality. Among several clinical applications, they could also be exploited as indicators of disease in false negative

  6. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.

    PubMed

    Gomez Perdiguero, Elisa; Klapproth, Kay; Schulz, Christian; Busch, Katrin; Azzoni, Emanuele; Crozet, Lucile; Garner, Hannah; Trouillet, Celine; de Bruijn, Marella F; Geissmann, Frederic; Rodewald, Hans-Reimer

    2015-02-26

    Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs), however, macrophages in adult tissues can self-maintain independently of HSCs. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs, and fetal macrophages can develop independently of Myb, a transcription factor required for HSC, and can persist in adult tissues. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2(+) (also known as Tek) cellular pathway generating Csf1r(+) erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages. PMID:25470051

  7. Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype

    PubMed Central

    Campbell, Gillian M.; Nicol, Marlynne Q.; Dransfield, Ian; Shaw, Darren J.; Nash, Anthony A.

    2015-01-01

    The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection. PMID:26297234

  8. Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

    PubMed

    Campbell, Gillian M; Nicol, Marlynne Q; Dransfield, Ian; Shaw, Darren J; Nash, Anthony A; Dutia, Bernadette M

    2015-10-01

    The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection. PMID:26297234

  9. Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro.

    PubMed

    Revoltella, R P; Laricchia Robbio, L; Liberati, A M; Reato, G; Foa, R; Funaro, A; Vinante, F; Pizzolo, G

    2000-09-15

    We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing. PMID:11069719

  10. Monocyte/Macrophage-derived IGF-1 Orchestrates Murine Skeletal Muscle Regeneration and Modulates Autocrine Polarization.

    PubMed

    Tonkin, Joanne; Temmerman, Lieve; Sampson, Robert D; Gallego-Colon, Enrique; Barberi, Laura; Bilbao, Daniel; Schneider, Michael D; Musarò, Antonio; Rosenthal, Nadia

    2015-07-01

    Insulin-like growth factor 1 (IGF-1) is a potent enhancer of tissue regeneration, and its overexpression in muscle injury leads to hastened resolution of the inflammatory phase. Here, we show that monocytes/macrophages constitute an important initial source of IGF-1 in muscle injury, as conditional deletion of the IGF-1 gene specifically in mouse myeloid cells (ϕIGF-1 CKO) blocked the normal surge of local IGF-1 in damaged muscle and significantly compromised regeneration. In injured muscle, Ly6C+ monocytes/macrophages and CD206+ macrophages expressed equivalent IGF-1 levels, which were transiently upregulated during transition from the inflammation to repair. In injured ϕIGF-1 CKO mouse muscle, accumulation of CD206+ macrophages was impaired, while an increase in Ly6C+ monocytes/macrophages was favored. Transcriptional profiling uncovered inflammatory skewing in ϕIGF-1 CKO macrophages, which failed to fully induce a reparative gene program in vitro or in vivo, revealing a novel autocrine role for IGF-1 in modulating murine macrophage phenotypes. These data establish local macrophage-derived IGF-1 as a key factor in inflammation resolution and macrophage polarization during muscle regeneration. PMID:25896247

  11. Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

    PubMed

    Lathrop, Stephanie K; Binder, Kelsey A; Starr, Tregei; Cooper, Kendal G; Chong, Audrey; Carmody, Aaron B; Steele-Mortimer, Olivia

    2015-07-01

    Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. PMID:25895967

  12. Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

    PubMed Central

    Lathrop, Stephanie K.; Binder, Kelsey A.; Starr, Tregei; Cooper, Kendal G.; Chong, Audrey; Carmody, Aaron B.

    2015-01-01

    Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. PMID:25895967

  13. Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

    PubMed Central

    Yeung, A. T. Y.; Hale, C.; Xia, J.; Tate, P. H.; Goulding, D.; Keane, J. A.; Mukhopadhyay, S.; Forrester, L.; Billker, O.; Skarnes, W. C.; Hancock, R. E. W.; Dougan, G.

    2015-01-01

    The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection. PMID:25752829

  14. Candida albicans-Derived β-1,2-Linked Mannooligosaccharides Induce Desensitization of Macrophages

    PubMed Central

    Jouault, Thierry; Fradin, Chantal; Trinel, Pierre-André; Poulain, Daniel

    2000-01-01

    Candida albicans β-1,2-oligomannosides stimulate macrophage tumor necrosis factor alpha (TNF-α) but not NO release. This stimulation desensitized macrophages by altering β-1,2-oligomannoside-dependent TNF-α production and lipopolysaccharide-dependent TNF-α and NO secretion. Desensitization was not related to tyrosine phosphorylation signal transduction but was transferred by culture supernatants in which arachidonic acid derivatives were evidenced. PMID:10639473

  15. Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor.

    PubMed

    Yamamoto, N; Naraparaju, V R

    1997-06-01

    Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90

  16. Effect of cytokines on Siglec-1 and HIV-1 entry in monocyte-derived macrophages: the importance of HIV-1 envelope V1V2 region.

    PubMed

    Jobe, Ousman; Trinh, Hung V; Kim, Jiae; Alsalmi, Wadad; Tovanabutra, Sodsai; Ehrenberg, Philip K; Peachman, Kristina K; Gao, Guofen; Thomas, Rasmi; Kim, Jerome H; Michael, Nelson L; Alving, Carl R; Rao, Venigalla B; Rao, Mangala

    2016-06-01

    Monocytes and monocyte-derived macrophages express relatively low levels of CD4. Despite this, macrophages can be effectively infected with human immunodeficiency virus type 1. Macrophages have a critical role in human immunodeficiency virus type 1 transmission; however, the mechanism or mechanisms of virus infection are poorly understood. We report that growth factors, such as granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor affect the phenotypic profile and permissiveness of macrophages to human immunodeficiency virus type 1. Human immunodeficiency virus type 1 infection of monocyte-derived macrophages derived from granulocyte macrophage and macrophage colony-stimulating factors was predominantly facilitated by the sialic acid-binding immunoglobulin-like lectin-1. The number of sialic acid-binding immunoglobulin-like lectin receptors on macrophage colony-stimulating factor-derived monocyte-derived macrophages was significantly greater than on granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages, and correspondingly, human immunodeficiency virus type 1 infection was greater in the macrophage colony-stimulating factor-derived monocyte-derived macrophages. Single-genome analysis and quantitative reverse transcriptase-polymerase chain reaction revealed that the differences in infectivity was not due to differences in viral fitness or in viral variants with differential infectivity but was due to reduced viral entry into the granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages. Anti-sialic acid-binding immunoglobulin-like lectin, trimeric glycoprotein 145, and scaffolded V1V2 proteins were bound to sialic acid-binding immunoglobulin-like lectin and significantly reduced human immunodeficiency virus type 1 entry and infection. Furthermore, sialic acid residues present in the V1V2 region of the envelope protein mediated human immunodeficiency virus type 1

  17. Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide.

    PubMed

    Pereira, Sandra Rodrigues; Faça, Vitor Marcel; Gomes, Glauce Gaspar; Chammas, Roger; Fontes, Aparecida Maria; Covas, Dimas Tadeu; Greene, Lewis Joel

    2005-04-01

    Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation. PMID:15800872

  18. Polydatin Inhibits Formation of Macrophage-Derived Foam Cells

    PubMed Central

    Wu, Min; Liu, Meixia; Guo, Gang; Zhang, Wengao; Liu, Longtao

    2015-01-01

    Rhizoma Polygoni Cuspidati, a Chinese herbal medicine, has been widely used in traditional Chinese medicine for a long time. Polydatin, one of the major active ingredients in Rhizoma Polygoni Cuspidati, has been recently shown to possess extensive cardiovascular pharmacological activities. In present study, we examined the effects of Polydatin on the formation of peritoneal macrophage-derived foam cells in Apolipoprotein E gene knockout mice (ApoE−/−) and explored the potential underlying mechanisms. Peritoneal macrophages were collected from ApoE−/− mice and cultured in vitro. These cells sequentially were divided into four groups: Control group, Model group, Lovastatin group, and Polydatin group. Our results demonstrated that Polydatin significantly inhibits the formation of foam cells derived from peritoneal macrophages. Further studies indicated that Polydatin regulates the metabolism of intracellular lipid and possesses anti-inflammatory effects, which may be regulated through the PPAR-γ signaling pathways. PMID:26557864

  19. Alternatively activated macrophages derived from monocytes and tissue macrophages are phenotypically and functionally distinct

    PubMed Central

    Gundra, Uma Mahesh; Girgis, Natasha M.; Ruckerl, Dominik; Jenkins, Stephen; Ward, Lauren N.; Kurtz, Zachary D.; Wiens, Kirsten E.; Tang, Mei San; Basu-Roy, Upal; Mansukhani, Alka; Allen, Judith E.

    2014-01-01

    Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the interleukin-4receptor(R)α. AAMs can be derived either from proliferation of tissue resident macrophages or recruited inflammatory monocytes, but it is not known whether these different sources generate AAMs that are phenotypically and functionally distinct. By transcriptional profiling analysis, we show here that, although both monocyte and tissue-derived AAMs expressed high levels of Arg1, Chi3l3, and Retnla, only monocyte-derived AAMs up-regulated Raldh2 and PD-L2. Monocyte-derived AAMs were also CX3CR1-green fluorescent protein (GFP)high and expressed CD206, whereas tissue-derived AAMs were CX3CR1-GFP and CD206 negative. Monocyte-derived AAMs had high levels of aldehyde dehydrogenase activity and promoted the differentiation of FoxP3+ cells from naïve CD4+ cells via production of retinoic acid. In contrast, tissue-derived AAMs expressed high levels of uncoupling protein 1. Hence monocyte-derived AAM have properties associated with immune regulation, and the different physiological properties associated with AAM function may depend on the distinct lineage of these cells. PMID:24695852

  20. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  1. Macrophage Migration Inhibitory Factor in Acute Adipose Tissue Inflammation.

    PubMed

    Kim, Bong-Sung; Rongisch, Robert; Hager, Stephan; Grieb, Gerrit; Nourbakhsh, Mahtab; Rennekampff, Hans-Oliver; Bucala, Richard; Bernhagen, Juergen; Pallua, Norbert

    2015-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and has been implicated in inflammatory diseases. However, little is known about the regulation of MIF in adipose tissue and its impact on wound healing. The aim of this study was to investigate MIF expression in inflamed adipose and determine its role in inflammatory cell recruitment and wound healing. Adipose tissue was harvested from subcutaneous adipose tissue layers of 24 healthy subjects and from adipose tissue adjacent to acutely inflamed wounds of 21 patients undergoing wound debridement. MIF protein and mRNA expression were measured by ELISA and RT-PCR. Cell-specific MIF expression was visualized by immunohistochemistry. The functional role of MIF in cell recruitment was investigated by a chemotaxis assay and by flow cytometry of labeled macrophages that were injected into Mif-/-and wildtype mice. Wound healing was evaluated by an in vitro scratch assay on human fibroblast monolayers. MIF protein levels of native adipose tissue and supernatants from acutely inflamed wounds were significantly elevated when compared to healthy controls. MIF mRNA expression was increased in acutely inflamed adipose tissue indicating the activation of MIF gene transcription in response to adipose tissue inflammation. MIF is expressed in mature adipocytes and in infiltrated macrophages. Peripheral blood mononuclear cell migration was significantly increased towards supernatants derived from inflamed adipose tissue. This effect was partially abrogated by MIF-neutralizing antibodies. Moreover, when compared to wildtype mice, Mif-/-mice showed reduced infiltration of labeled macrophages into LPS-stimulated epididymal fat pads in vivo. Finally, MIF antibodies partially neutralized the detrimental effect of MIF on fibroblast wound healing. Our results indicate that increased MIF expression and rapid activation of the MIF gene in fat tissue adjacent to acute wound healing disorders may play a role in cell

  2. Macrophage Migration Inhibitory Factor in Acute Adipose Tissue Inflammation

    PubMed Central

    Kim, Bong-Sung; Rongisch, Robert; Hager, Stephan; Grieb, Gerrit; Nourbakhsh, Mahtab; Rennekampff, Hans-Oliver; Bucala, Richard; Bernhagen, Juergen; Pallua, Norbert

    2015-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and has been implicated in inflammatory diseases. However, little is known about the regulation of MIF in adipose tissue and its impact on wound healing. The aim of this study was to investigate MIF expression in inflamed adipose and determine its role in inflammatory cell recruitment and wound healing. Adipose tissue was harvested from subcutaneous adipose tissue layers of 24 healthy subjects and from adipose tissue adjacent to acutely inflamed wounds of 21 patients undergoing wound debridement. MIF protein and mRNA expression were measured by ELISA and RT-PCR. Cell-specific MIF expression was visualized by immunohistochemistry. The functional role of MIF in cell recruitment was investigated by a chemotaxis assay and by flow cytometry of labeled macrophages that were injected into Mif–/–and wildtype mice. Wound healing was evaluated by an in vitro scratch assay on human fibroblast monolayers. MIF protein levels of native adipose tissue and supernatants from acutely inflamed wounds were significantly elevated when compared to healthy controls. MIF mRNA expression was increased in acutely inflamed adipose tissue indicating the activation of MIF gene transcription in response to adipose tissue inflammation. MIF is expressed in mature adipocytes and in infiltrated macrophages. Peripheral blood mononuclear cell migration was significantly increased towards supernatants derived from inflamed adipose tissue. This effect was partially abrogated by MIF-neutralizing antibodies. Moreover, when compared to wildtype mice, Mif–/–mice showed reduced infiltration of labeled macrophages into LPS-stimulated epididymal fat pads in vivo. Finally, MIF antibodies partially neutralized the detrimental effect of MIF on fibroblast wound healing. Our results indicate that increased MIF expression and rapid activation of the MIF gene in fat tissue adjacent to acute wound healing disorders may play a role in cell

  3. Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow.

    PubMed

    Dong, Yifei; Arif, Arif A; Poon, Grace F T; Hardman, Blair; Dosanjh, Manisha; Johnson, Pauline

    2016-01-01

    Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function. PMID:27404290

  4. Adipocyte-derived lipids increase angiotensin-converting enzyme (ACE) expression and modulate macrophage phenotype.

    PubMed

    Kohlstedt, Karin; Trouvain, Caroline; Namgaladze, Dmitry; Fleming, Ingrid

    2011-03-01

    Human monocytes/macrophages express the angiotensin-converting enzyme (ACE) but nothing is known about its role under physiological conditions. As adipose tissue contains resident macrophages that have been implicated in the generation of insulin resistance in expanding fat mass, we determined whether adipocytes release factors that affect ACE expression and function in monocytes. Incubation of human monocyte-derived macrophages with conditioned medium from freshly isolated human adipocytes (BMI = 25.4 ± 0.96) resulted in a 4-fold increase in ACE expression. The effect was insensitive to denaturation and different proteases but abolished after lipid extraction. mRNA levels of the major histocompatibility complex class II protein increased in parallel with ACE, whereas the expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, and cyclooxygenase-2 decreased. As a consequence of the reduction in MCP-1, monocyte recruitment was also attenuated. Moreover, adipocyte-conditioned medium prevented the interferon (IFN)-γ induced formation of TNF-α, IL-6, and MCP-1, all markers of classically-activated (M1 type) macrophages. The decrease in cytokine expression in adipocyte-conditioned medium-treated macrophages was sensitive to ACE silencing by small interfering RNA (siRNA). Accordingly, ACE overexpression in THP-1 cells mimicked the effect of adipocyte-conditioned medium. In both cell types, ACE inhibition failed to affect the changes induced by adipocyte conditioned-medium treatment and ACE overexpression. Thus, the modulation of macrophage polarization by ACE appears to be mediated independently of enzyme activity, probably via intracellular signaling. Interestingly, human macrophage ACE expression was also upregulated by IL-4 and IL-13, which promote the "alternative" activation of macrophages and decreased by LPS and IFN-γ. Mechanistically, adipocyte-conditioned medium stimulated the phosphorylation of

  5. Functional characterization of the turkey macrophage migration inhibitory factor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characte...

  6. The transcription factor NR4A1 is essential for the development of a novel macrophage subset in the thymus

    PubMed Central

    Tacke, Robert; Hilgendorf, Ingo; Garner, Hannah; Waterborg, Claire; Park, Kiwon; Nowyhed, Heba; Hanna, Richard N.; Wu, Runpei; Swirski, Filip K.; Geissmann, Frederic; Hedrick, Catherine C.

    2015-01-01

    Tissue macrophages function to maintain homeostasis and regulate immune responses. While tissue macrophages derive from one of a small number of progenitor programs, the transcriptional requirements for site-specific macrophage subset development are more complex. We have identified a new tissue macrophage subset in the thymus and have discovered that its development is dependent on transcription factor NR4A1. Functionally, we find that NR4A1-dependent macrophages are critically important for clearance of apoptotic thymocytes. These macrophages are largely reduced or absent in mice lacking NR4A1, and Nr4a1-deficient mice have impaired thymocyte engulfment and clearance. Thus, NR4A1 functions as a master transcription factor for the development of this novel thymus-specific macrophage subset. PMID:26091486

  7. Differential utilization of Ras signaling pathways by macrophage colony-stimulating factor (CSF) and granulocyte-macrophage CSF receptors during macrophage differentiation.

    PubMed

    Guidez, F; Li, A C; Horvai, A; Welch, J S; Glass, C K

    1998-07-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) independently stimulate the proliferation and differentiation of macrophages from bone marrow progenitor cells. Although the GM-CSF and M-CSF receptors are unrelated, both couple to Ras-dependent signal transduction pathways, suggesting that these pathways might account for common actions of GM-CSF and M-CSF on the expression of macrophage-specific genes. To test this hypothesis, we have investigated the mechanisms by which GM-CSF and M-CSF regulate the expression of the macrophage scavenger receptor A (SR-A) gene. We demonstrate that induction of the SR-A gene by M-CSF is dependent on AP-1 and cooperating Ets domain transcription factors that bind to sites in an M-CSF-dependent enhancer located 4.1 to 4.5 kb upstream of the transcriptional start site. In contrast, regulation by GM-CSF requires a separate enhancer located 4.5 to 4.8 kb upstream of the transcriptional start site that confers both immediate-early and sustained transcriptional responses. Results of a combination of DNA binding experiments and functional assays suggest that immediate transcriptional responses are mediated by DNA binding proteins that are constitutively bound to the GM-CSF enhancer and are activated by Ras. At 12 to 24 h after GM-CSF treatment, the GM-CSF enhancer becomes further occupied by additional DNA binding proteins that may contribute to sustained transcriptional responses. In concert, these studies indicate that GM-CSF and M-CSF differentially utilize Ras-dependent signal transduction pathways to regulate scavenger receptor gene expression, consistent with the distinct functional properties of M-CSF- and GM-CSF-derived macrophages. PMID:9632769

  8. Amyloid fibril formation by macrophage migration inhibitory factor

    SciTech Connect

    Lashuel, Hilal A. . E-mail: hilal.lashuel@epfl.ch; Aljabari, Bayan; Sigurdsson, Einar M.; Metz, Christine N.; Leng Lin; Callaway, David J.E.; Bucala, Richard

    2005-12-16

    We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins {beta}-amyloid and {alpha}-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.

  9. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    PubMed

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF. PMID:21170647

  10. Krüppel-like factor KLF10 deficiency predisposes to colitis through colonic macrophage dysregulation.

    PubMed

    Papadakis, Konstantinos A; Krempski, James; Svingen, Phyllis; Xiong, Yuning; Sarmento, Olga F; Lomberk, Gwen A; Urrutia, Raul A; Faubion, William A

    2015-12-01

    Krüppel-like factor (KLF)-10 is an important transcriptional regulator of TGF-β1 signaling in both CD8(+) and CD4(+) T lymphocytes. In the present study, we demonstrate a novel role for KLF10 in the regulation of TGFβRII expression with functional relevance in macrophage differentiation and activation. We first show that transfer of KLF10(-/-) bone marrow-derived macrophages into wild-type (WT) mice leads to exacerbation of experimental colitis. At the cell biological level, using two phenotypic strategies, we show that KLF10-deficient mice have an altered colonic macrophage phenotype with higher frequency of proinflammatory LyC6(+)MHCII(+) cells and a reciprocal decrease of the anti-inflammatory LyC6(-)MHCII(+) subset. Additionally, the anti-inflammatory CD11b(+)CX3CR1(hi) subset of colonic macrophages is significantly decreased in KLF10(-/-) compared with WT mice under inflammatory conditions. Molecularly, CD11b(+) colonic macrophages from KLF10(-/-) mice exhibit a proinflammatory cytokine profile with increased production of TNF-α and lower production of IL-10 in response to LPS stimulation. Because KLF10 is a transcription factor, we explored how this protein may regulate macrophage function. Consequently, we analyzed the expression of TGFβRII expression in colonic macrophages and found that, in the absence of KLF10, macrophages express lower levels of TGFβRII and display an attenuated Smad-2 phosphorylation following TGF-β1 stimulation. We further show that KLF10 directly binds to the TGFβRII promoter in macrophages, leading to enhanced gene expression through histone H3 acetylation. Collectively, our data reveal a critical role for KLF10 in the epigenetic regulation of TGFβRII expression in macrophages and the acquisition of a "regulatory" phenotype that contributes to intestinal mucosal homeostasis. PMID:26472224

  11. Host and Bacterial Factors Involved in the Innate Ability of Mouse Macrophages To Eliminate Internalized Unopsonized Escherichia coli

    PubMed Central

    Hamrick, Terri S.; Havell, Edward A.; Horton, John R.; Orndorff, Paul E.

    2000-01-01

    In an effort to better understand genetic and cellular factors that influence innate immunity, we examined host and bacterial factors involved in the nonopsonic phagocytosis and killing of Escherichia coli K-12 by mouse macrophages. Unelicited (resident) peritoneal macrophages from five different mouse strains, BALB/c, C57BL/6, CD-1, C3H/HeJ, and C3H/HeN, were employed. Additional macrophage populations were obtained from CD-1 mice (bone marrow-derived macrophages). Also, for BALB/c and C57BL/6 mice, peritoneal macrophages elicited with either thioglycolate or proteose peptone, bone marrow-derived macrophages, and macrophage-like cell lines derived from the two strains were employed. Two E. coli K-12 strains that differed specifically in their abilities to produce type 1 pili containing the adhesive protein FimH were examined. The parameters used to assess macrophage bacteriocidal activity were (i) the killing of internalized (gentamicin-protected) E. coli during the approximately 4-h assay and (ii) the initial rate at which internalized E. coli were eliminated. Data on these parameters allowed the following conclusions: (i) unelicited or proteose peptone-elicited peritoneal macrophages were significantly better at eliminating internalized bacteria than thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages, or macrophage cell lines; (ii) the host genetic background had no significant effect upon the ability of unelicited peritoneal macrophages to kill E. coli (even though the mouse strains differ widely in their in vivo susceptibilities to bacterial infection); and (iii) the FimH phenotype had no significant effect upon E. coli survival once the bacterium was inside a macrophage. Additionally, there was no correlation between the bacteriocidal effectiveness of a macrophage population and the number of bacteria bound per macrophage. However, macrophage populations that were the least bacteriocidal tended to bind higher ratios of FimH+ to Fim

  12. Superinduction of interleukin 8 mRNA in activated monocyte derived macrophages from rheumatoid arthritis patients

    PubMed Central

    Rodenburg, R.; van den Hoogen, F. H J; Barrera, P.; van Venrooij, W. J; van de Putte, L. B A

    1999-01-01

    OBJECTIVE—Synovial inflammation in patients with rheumatoid arthritis (RA) is characterised by the presence of large numbers of highly activated monocytes and macrophages. The importance of these cells in the aethiopathogenesis and prognosis of RA is increasingly recognised. The object of this report is to determine whether monocytes and monocyte derived macrophages of RA patients produce increased cytokine mRNA levels.
METHODS—Monocyte derived macrophages from RA patients and healthy controls were cultured either in the absence or presence of lipopolysaccharide. The expression levels of the mRNAs encoding GAPDH, interleukin 1β (IL1β), IL8, and α2 macroglobulin in these cells were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR).
RESULTS—Activated monocyte derived macrophages from RA patients produce significantly higher IL8 mRNA levels than activated macrophages from healthy controls. By contrast, resting RA and control macrophages produce similar levels of IL8 mRNA. Culturing of activated macrophages in the presence of RA or control sera has no effect on the expression levels of IL8 mRNA. No significant differences between RA and control macrophages were observed in the expression levels of IL1β and α2 macroglobulin mRNAs.
CONCLUSION—These data indicate that the increased IL8 mRNA production capacity of RA macrophages upon activation is an intrinsic property of these cells, and is not attributable to factors present in the circulation. Based on these observations, it is postulated that this innate hyper-responsiveness of RA macrophages contributes to the high IL8 levels present in the synovial fluid of rheumatoid joints, and is implicated in the chemotactic gradient leading to the homing of leucocytes to the joints.

 PMID:10491366

  13. Multipotent hematopoietic cell lines derived from C/EBPalpha(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte- colony-stimulating factor, and retinoic acid-induced granulocytic differentiation.

    PubMed

    Collins, S J; Ulmer, J; Purton, L E; Darlington, G

    2001-10-15

    The transcription factor C/EBPalpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPalpha knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor-dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPalpha knockout animals (C/EBPalpha(-/-)) and their heterozygous littermates (C/EBPalpha(+/-)). Surprisingly, the C/EBPalpha(-/-) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)-CSF. This GM-CSF-mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPalpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPalpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPalpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPalpha-responsive genes can occur in the absence of C/EBPalpha, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes. PMID:11588034

  14. Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway.

    PubMed

    Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

    2016-03-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, β-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/β-arrestin 2/Rho A signaling pathway. PMID:26852662

  15. Loss of autophagy enhances MIF/macrophage migration inhibitory factor release by macrophages.

    PubMed

    Lee, Jacinta P W; Foote, Andrew; Fan, Huapeng; Peral de Castro, Celia; Lang, Tali; Jones, Sarah A; Gavrilescu, Nichita; Mills, Kingston H G; Leech, Michelle; Morand, Eric F; Harris, James

    2016-06-01

    MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers. PMID:27163877

  16. Effects of Two Fullerene Derivatives on Monocytes and Macrophages

    PubMed Central

    Pacor, Sabrina; Grillo, Alberto; Đorđević, Luka; Zorzet, Sonia; Da Ros, Tatiana; Prato, Maurizio

    2015-01-01

    Two fullerene derivatives (fullerenes 1 and 2), bearing a hydrophilic chain on the pyrrolidinic nitrogen, were developed with the aim to deliver anticancer agents to solid tumors. These two compounds showed a significantly different behaviour on human neoplastic cell lines in vitro in respect to healthy leukocytes. In particular, the pyrrolidinium ring on the fullerene carbon cage brings to a more active compound. In the present work, we describe the effects of these fullerenes on primary cultures of human monocytes and macrophages, two kinds of immune cells representing the first line of defence in the immune response to foreign materials. These compounds are not recognized by circulating monocytes while they get into macrophages. The evaluation of the pronecrotic or proapoptotic effects, analysed by means of analysis of the purinergic receptor P2X7 activation and of ROS scavenging activity, has allowed us to show that fullerene 2, but not its analogue fullerene 1, displays toxicity, even though at concentrations higher than those shown to be active on neoplastic cells. PMID:26090460

  17. Macrophage-secreted factors induce adipocyte inflammation and insulin resistance

    SciTech Connect

    Permana, Paska A. . E-mail: Paska.Permana@med.va.gov; Menge, Christopher; Reaven, Peter D.

    2006-03-10

    Macrophage infiltration into adipose tissue increases with obesity, a condition associated with low-grade inflammation and insulin resistance. We investigated the direct effects of macrophage-secreted factors on adipocyte inflammation and insulin resistance. 3T3-L1 adipocytes incubated with media conditioned by RAW264.7 macrophages (RAW-CM) showed dramatically increased transcription of several inflammation-related genes, greater nuclear factor kappa B (NF-{kappa}B) activity, and enhanced binding of U937 monocytes. All of these effects were prevented by co-incubation with pyrrolidinedithiocarbamate, an NF-{kappa}B inhibitor. Adipocytes incubated with RAW-CM also released more non-esterified fatty acids and this increased lipolysis was not suppressed by insulin. In addition, RAW-CM treatment decreased insulin-stimulated glucose uptake in adipocytes. Taken together, these results indicate that macrophage-secreted factors induce inflammatory responses and reduce insulin responsiveness in adipocytes. These effects of macrophage-secreted factors on adipocytes may contribute significantly to the systemic inflammation and insulin resistance associated with obesity.

  18. Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of Mycobacterium tuberculosis in Human Macrophages

    PubMed Central

    Chmura, Kathryn; Ovrutsky, Alida R.; Su, Wen-Lin; Griffin, Laura; Pyeon, Dohun; McGibney, Mischa T.; Strand, Matthew J.; Numata, Mari; Murakami, Seiji; Gaido, Loretta; Honda, Jennifer R.; Kinney, William H.; Oberley-Deegan, Rebecca E.; Voelker, Dennis R.; Ordway, Diane J.; Chan, Edward D.

    2013-01-01

    Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy. PMID:23634218

  19. Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

    PubMed

    Bai, Xiyuan; Feldman, Nicole E; Chmura, Kathryn; Ovrutsky, Alida R; Su, Wen-Lin; Griffin, Laura; Pyeon, Dohun; McGibney, Mischa T; Strand, Matthew J; Numata, Mari; Murakami, Seiji; Gaido, Loretta; Honda, Jennifer R; Kinney, William H; Oberley-Deegan, Rebecca E; Voelker, Dennis R; Ordway, Diane J; Chan, Edward D

    2013-01-01

    Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy. PMID:23634218

  20. Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment.

    PubMed

    Pagán, Antonio J; Yang, Chao-Tsung; Cameron, James; Swaim, Laura E; Ellett, Felix; Lieschke, Graham J; Ramakrishnan, Lalita

    2015-07-01

    The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis. PMID:26159717

  1. Tumour necrosis factor (TNF) as a mediator of macrophage helminthotoxic activity.

    PubMed

    James, S L; Glaven, J; Goldenberg, S; Meltzer, M S; Pearce, E

    1990-01-01

    Lymphokine-activated macrophages are cytotoxic for larvae of the helminth parasite Schistosoma mansoni. That soluble secreted factors may mediate this cytotoxicity was suggested by the observation that culture supernatant fluids from stimulated macrophages also exhibited larvacidal activity. These fluids contain the monokine tumour necrosis factor (TNF). Several observations indicated that TNF is directly toxic to schistosome larvae. Cytotoxic sera taken from BCG- or S. mansoni-immunized mice after endotoxin challenge killed schistosomula in vitro, and upon gel filtration the larvacidal factor(s) in the sera co-eluted with the tumoricidal activity defined as TNF. Recombinant-derived TNF exhibited direct toxicity to schistosomula at high concentrations, or at lower concentrations in the presence of IFN gamma. The larvacidal activity of macrophage supernatant fluids was abrogated by addition of either anti-TNF antisera or Zn+2, which has been shown to inhibit TNF-induced damage of tumour cells. Anti-TNF and Zn+2 likewise suppressed schistosomulum killing by lymphokine-activated peritoneal macrophages or the IC-21 macrophage line, indicating that TNF also plays a role in the effector mechanism of larval killing by whole cells. PMID:2314921

  2. LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

    SciTech Connect

    Cleary, S.F.; Marciano-Cabral, F.

    1986-03-01

    Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNA or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.

  3. Granulocyte macrophage colony stimulating factor therapy for pulmonary alveolar proteinosis.

    PubMed

    Shende, Ruchira P; Sampat, Bhavin K; Prabhudesai, Pralhad; Kulkarni, Satish

    2013-03-01

    We report a case of 58 year old female diagnosed with Pulmonary Alveolar Proteinosis (PAP) with recurrence of PAP after 5 repeated whole lung lavage, responding to subcutaneous injections of Granulocyte Macrophage Colony Stimulating Factor therapy (GM-CSF). Thus indicating that GM-CSF therapy is a promising alternative in those requiring repeated whole lung lavage PMID:24475687

  4. Characterization of Neospora caninum macrophage migration inhibitory factor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in three forms, NcMIF (mature protein), NcMI...

  5. Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing

    PubMed Central

    Okuno, Yuji; Nakamura-Ishizu, Ayako; Kishi, Kazuo; Suda, Toshio

    2011-01-01

    Bone marrow-derived cells (BMDCs) contribute to postnatal vascular growth by differentiating into endothelial cells or secreting angiogenic factors. However, the extent of their endothelial differentiation highly varies according to the angiogenic models used. Wound healing is an intricate process in which the skin repairs itself after injury. As a process also observed in cancer progression, neoangiogenesis into wound tissues is profoundly involved in this healing process, suggesting the contribution of BMDCs. However, the extent of the differentiation of BMDCs to endothelial cells in wound healing is unclear. In this study, using the green fluorescent protein-bone marrow chim-eric experiment and high resolution confocal microscopy at a single cell level, we observed no endothelial differentiation of BMDCs in 2 acute wound healing models (dorsal excisional wound and ear punch) and a chronic wound healing model (decubitus ulcer). Instead, a major proportion of BMDCs were macrophages. Indeed, colony-stimulating factor 1 (CSF-1) inhibition depleted approximately 80% of the BMDCs at the wound healing site. CSF-1–mutant (CSF-1op/op) mice showed significantly reduced neoangiogenesis into the wound site, supporting the substantial role of BMDCs as macrophages. Our data show that the proangiogenic effects of macrophages, but not the endothelial differentiation, are the major contribution of BMDCs in wound healing. PMID:21411758

  6. Macrophage-mediated myelin-related mitogenic factor for cultured Schwann cells.

    PubMed Central

    Baichwal, R R; Bigbee, J W; DeVries, G H

    1988-01-01

    Conditioned medium from cultured peritoneal macrophages that have phagocytosed a myelin membrane fraction is mitogenic for cultured Schwann cells. Production of the mitogenic supernatant was time- and dose-dependent with a maximal Schwann cell-proliferative response from supernatants after 48-hr incubation of cultured macrophages with myelin-enriched fraction (200 micrograms of protein per ml). The response was specific for myelin membrane: supernatants derived from macrophages incubated with axolemma, liver microsomes, polystyrene beads, or lipopolysaccharide were not mitogenic. Lysosomal processing of the myelin membrane was necessary for the production of the mitogenic factor, which was shown to be heat labile and trypsin sensitive. There was no species specificity because myelin membranes isolated from the central and peripheral nervous systems of rat, bovine, and human were equally potent in eliciting mitogenic supernatant. However, supernatants derived from central nervous system myelin membranes were two to three times more mitogenic than those obtained from peripheral nervous system fractions of the same species. Previous observations that myelin is mitogenic for cultured Schwann cells may, in part, involve the intermediate processing of myelin by macrophages that are present in Schwann cell cultures. These results suggest that macrophages play a crucial role in Schwann cell proliferation during Wallerian degeneration. Images PMID:3422757

  7. Simultaneous Addition of Shikonin and Its Derivatives with Lipopolysaccharide Induces Rapid Macrophage Death.

    PubMed

    Koike, Atsushi; Shibano, Makio; Mori, Hideya; Kohama, Kiyoko; Fujimori, Ko; Amano, Fumio

    2016-01-01

    Macrophages play pivotal roles in inflammatory responses. Previous studies showed that various natural products exert antiinflammatory effects by regulating macrophage activation. Recent studies have shown that shikonin (SHK) and its derivatives (β-hydroxyisovalerylshikonin, acetylshikonin, and isobutylshikonin), which are 1,4-naphthoquinone pigments extracted from the roots of Lithospermum erythrorhizon, have various pharmacological, including antiinflammatory and antitumor, effects. Even though there have been many studies on the antiinflammatory activities of SHK derivatives, only a few have described their direct effects on macrophages. We investigated the effects of SHK derivatives on lipopolysaccharide (LPS)-treated macrophages. Low doses of SHK derivatives induced significant macrophage cytotoxicity (mouse macrophage-like J774.1/JA-4 cells and mouse peritoneal macrophages) in the presence of LPS. SHK activated caspases-3 and -7, which led to DNA fragmentation, but this cytotoxicity was prevented through a pan-caspase inhibitor in LPS-treated JA-4 cells. Maximal cytotoxic effects were achieved when SHK was added immediately before LPS addition. These results indicate that SHK derivatives induce caspase-dependent apoptotic cell death of LPS-treated macrophages and suggest that SHK acts during an early stage of LPS signaling. PMID:27251498

  8. Differential Responses Between Monocytes and Monocyte-Derived Macrophages for Lipopolysaccharide Stimulation of Calves

    PubMed Central

    Guo, Yijie; Zhao, Guoqi; Tanaka, Sachi; Yamaguchi, Takahiro

    2009-01-01

    In this experiment Toll-like receptor expression pattern in monocytes and monocyte-derived macrophages by lipopolysaccharide (LPS) stimulation was examined. Jugular venous blood was collected from four Japanese calves, and the peripheral blood mononuclear cells (PBMCs) were isolated. The cells were directly used for collecting monocytes by magnetic cell sorting or cultured for 7 days to collect monocyte-derived macrophages in Repcell. Then we analyzed the mRNA expression pattern of TLRs and cytokines in monocytes and monocyte-derived macrophages after LPS stimulation for 24 h. LPS stimulation of both monocytes and monocyte-derived macrophages resulted in an increase in the levels of mRNA transcripts for TNF-α, IL-6 and IL-8. Moreover, TNF-α and IL-6 mRNA expressions were significantly augmented by LPS stimulation in monocyte-derived macrophages. TLRs mRNA expressions were unchanged after LPS stimulation of monocytes, while TLRs mRNA expressions in monocyte-derived macrophages were complicated. TLR1, 3, 5, 8 and 10 were significantly decreased after LPS stimulation and there were no differences in the mRNA expressions of TLR2, 4, 6 and 7 between the groups of control and LPS stimulation. Besides, no expression of TLR9 was found. As antigen presenting cells, monocytes and monocyte-derived macrophages respond differently to LPS, so they may have different functions in the innate immune system. PMID:19567206

  9. The role of macrophage-derived IL-1 in induction and maintenance of angiogenesis.

    PubMed

    Carmi, Yaron; Voronov, Elena; Dotan, Shahar; Lahat, Nitza; Rahat, Michal A; Fogel, Mina; Huszar, Monika; White, Malka R; Dinarello, Charles A; Apte, Ron N

    2009-10-01

    Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1beta, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1beta knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1beta has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value. PMID:19752225

  10. Lipocalin 2 from macrophages stimulated by tumor cell-derived sphingosine 1-phosphate promotes lymphangiogenesis and tumor metastasis.

    PubMed

    Jung, Michaela; Ören, Bilge; Mora, Javier; Mertens, Christina; Dziumbla, Sarah; Popp, Rüdiger; Weigert, Andreas; Grossmann, Nina; Fleming, Ingrid; Brüne, Bernhard

    2016-01-01

    Tumor cell-derived factors skew macrophages toward a tumor-supporting phenotype associated with the secretion of protumorigenic mediators. Apoptosing tumor cells release sphingosine 1-phosphate (S1P), which stimulates the production of lipocalin 2 (LCN2) in tumor-associated macrophages and is associated with tumor metastasis. We explored the mechanism by which S1P induces LCN2 in macrophages and investigated how this contributed to tumor growth and metastasis. Knockdown of S1P receptor 1 (S1PR1) in primary human macrophages and experiments with bone marrow-derived macrophages from S1PR1-deficient mice showed that S1P signaled through S1PR1 to induce LCN2 expression. The LCN2 promoter contains a consensus sequence for signal transducer and activator of transcription 3 (STAT3), and deletion of the STAT3 recognition sequence reduced expression of an LCN2-controlled reporter gene. Conditioned medium from coculture experiments indicated that the release of LCN2 from macrophages induced tube formation and proliferation in cultures of primary human lymphatic endothelial cells in a manner dependent on the kinase PI3K and subsequent induction of the growth factor VEGFC, which functioned as an autocrine signal stimulating the receptor VEGFR3. Knockout of Lcn2 attenuated tumor-associated lymphangiogenesis and breast tumor metastasis both in the breast cancer model MMTV-PyMT mice and in mice bearing orthotopic wild-type tumors. Our findings indicate that macrophages respond to dying tumor cells by producing signals that promote lymphangiogenesis, which enables metastasis. PMID:27353364

  11. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide.

    PubMed

    Fei, Fan; Lee, Keith M; McCarry, Brian E; Bowdish, Dawn M E

    2016-01-01

    Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6-8 wk) and old (18-22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age. PMID:26940652

  12. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide

    NASA Astrophysics Data System (ADS)

    Fei, Fan; Lee, Keith M.; McCarry, Brian E.; Bowdish, Dawn M. E.

    2016-03-01

    Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6–8 wk) and old (18–22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age.

  13. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide

    PubMed Central

    Fei, Fan; Lee, Keith M.; McCarry, Brian E.; Bowdish, Dawn M. E.

    2016-01-01

    Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6–8 wk) and old (18–22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age. PMID:26940652

  14. Generation and characterization of bovine bone marrow-derived macrophage cell line.

    PubMed

    Xiao, Jiajia; Xie, Rongxia; Li, Qiaoqiao; Chen, Wuju; Zhang, Yong

    2016-05-01

    Macrophages, as the forefront of innate immune defense, have an important role in the host responses to mycobacterial infection. Therefore, a stable macrophage cell line is needed for future bovine immune system research on the bacterial infection. In this study, we established a bovine macrophage cell line by introducing the human telomerase reverse transcriptase (hTERT) gene into bovine bone marrow-derived macrophages (bBMMs). The TERT-bBMMs cells expressed macrophage surface antigen (CD11b, CD282) and upregulated expression of the cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α in response to bacterial invasion. These results demonstrate that this cell line provide reliable cell model system for future studies on interactions between the bovine macrophages and Mycobacterium tuberculosis. PMID:26936441

  15. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.

    PubMed

    Daigneault, Marc; Preston, Julie A; Marriott, Helen M; Whyte, Moira K B; Dockrell, David H

    2010-01-01

    Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD(3)) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD(3) and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells. PMID:20084270

  16. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.

    PubMed

    Epelman, Slava; Lavine, Kory J; Beaudin, Anna E; Sojka, Dorothy K; Carrero, Javier A; Calderon, Boris; Brija, Thaddeus; Gautier, Emmanuel L; Ivanov, Stoyan; Satpathy, Ansuman T; Schilling, Joel D; Schwendener, Reto; Sergin, Ismail; Razani, Babak; Forsberg, E Camilla; Yokoyama, Wayne M; Unanue, Emil R; Colonna, Marco; Randolph, Gwendalyn J; Mann, Douglas L

    2014-01-16

    Cardiac macrophages are crucial for tissue repair after cardiac injury but are not well characterized. Here we identify four populations of cardiac macrophages. At steady state, resident macrophages were primarily maintained through local proliferation. However, after macrophage depletion or during cardiac inflammation, Ly6c(hi) monocytes contributed to all four macrophage populations, whereas resident macrophages also expanded numerically through proliferation. Genetic fate mapping revealed that yolk-sac and fetal monocyte progenitors gave rise to the majority of cardiac macrophages, and the heart was among a minority of organs in which substantial numbers of yolk-sac macrophages persisted in adulthood. CCR2 expression and dependence distinguished cardiac macrophages of adult monocyte versus embryonic origin. Transcriptional and functional data revealed that monocyte-derived macrophages coordinate cardiac inflammation, while playing redundant but lesser roles in antigen sampling and efferocytosis. These data highlight the presence of multiple cardiac macrophage subsets, with different functions, origins, and strategies to regulate compartment size. PMID:24439267

  17. Protective effects of macrophage-derived interferon against encephalomyocarditis virus-induced diabetes mellitus in mice.

    PubMed

    Hirasawa, K; Ogiso, Y; Takeda, M; Lee, M J; Itagaki, S; Doi, K

    1995-12-01

    The involvement of macrophages in protection against diabetes mellitus in mice of BALB/c (susceptible) and C57BL (resistant) strains infected with the B (non-diabetogenic) or D (highly diabetogenic) variant of encephalomyocarditis (EMC) virus was examined. Pretreatment with the B variant of EMC virus (EMC-B), avirulent interferon (IFN) inducer, or Corynebacterium parvum inhibited diabetes in BALB/c mice infected with the D variant of EMC virus (EMC-D). Treatment of C57BL mice with carrageenan to compromise macrophage function rendered C57BL mice susceptible to EMC-D-induced diabetes. In macrophage culture for BALB/c mice, EMC-B induced IFN at an earlier stage than did EMC-D. The C57BL mouse-derived macrophages produced more IFN than did BALB/c mouse-derived macrophages after stimulation with EMC-D. Moreover, C. parvum increased IFN production in macrophage cultures from BALB/c mice, whereas carrageenan inhibited that in macrophage cultures from C57BL mice. These results suggest that IFN derived from macrophages may have an important role in protecting mice against EMC virus infection. PMID:8746525

  18. Matrix metalloproteinase-12 gene regulation by a PPAR alpha agonist in human monocyte-derived macrophages

    SciTech Connect

    Souissi, Imen Jguirim; Billiet, Ludivine; Cuaz-Perolin, Clarisse; Rouis, Mustapha

    2008-11-01

    MMP-12, a macrophage-specific matrix metalloproteinase with large substrate specificity, has been reported to be highly expressed in mice, rabbits and human atherosclerotic lesions. Increased MMP-12 from inflammatory macrophages is associated with several degenerative diseases such as atherosclerosis. In this manuscript, we show that IL-1{beta}, a proinflammatory cytokine found in atherosclerotic plaques, increases both mRNA and protein levels of MMP-12 in human monocyte-derived macrophages (HMDM). Since peroxisome proliferator-activated receptors (PPARs), such as PPAR{alpha} and PPAR{gamma}, are expressed in macrophages and because PPAR activation exerts an anti-inflammatory effect on vascular cells, we have investigated the effect of PPAR{alpha} and {gamma} isoforms on MMP-12 regulation in HMDM. Our results show that MMP-12 expression (mRNA and protein) is down regulated in IL-1{beta}-treated macrophages only in the presence of a specific PPAR{alpha} agonist, GW647, in a dose-dependent manner. In contrast, this inhibitory effect was abolished in IL-1{beta}-stimulated peritoneal macrophages isolated from PPAR{alpha}{sup -/-} mice and treated with the PPAR{alpha} agonist, GW647. Moreover, reporter gene transfection experiments using different MMP-12 promoter constructs showed a reduction of the promoter activities by {approx} 50% in IL-1{beta}-stimulated PPAR{alpha}-pre-treated cells. However, MMP-12 promoter analysis did not reveal the presence of a PPRE response element. The IL-1{beta} effect is known to be mediated through the AP-1 binding site. Mutation of the AP-1 site, located at - 81 in the MMP-12 promoter region relative to the transcription start site, followed by transfection analysis, gel shift and ChIP experiments revealed that the inhibitory effect was the consequence of the protein-protein interaction between GW 647-activated PPAR{alpha} and c-Fos or c-Jun transcription factors, leading to inhibition of their binding to the AP-1 motif. These studies

  19. Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis

    SciTech Connect

    Walker, E.B.; Akporiaye, E.T.; Warner, N.L.; Stewart, C.C.

    1985-01-01

    Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogenous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2K/sup k/) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and ..gamma..2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages. 37 references, 5 figures, 5 tables.

  20. FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism.

    PubMed

    Jiang, Yideng; Ma, Shengchao; Zhang, Huiping; Yang, Xiaoling; Lu, Guan Jun; Zhang, Hui; He, Yangyang; Kong, Fanqi; Yang, Anning; Xu, Hua; Zhang, Minghao; Jiao, Yun; Li, Guizhong; Cao, Jun; Jia, Yuexia; Jin, Shaoju; Wei, Jun; Shi, Yingkang

    2016-07-01

    Hyperhomocysteinemia (HHcy) is an independent risk factor for the development of atherosclerosis (AS), according to overwhelming number of clinical and epidemiological studies. However, the underlying pathogenic molecular mechanisms by which HHcy promotes AS remain to be fully elucidated. Fatty acid binding protein 4 (FABP4) has been shown to be important in macrophage cholesterol trafficking. The objective of the present study was to determine whether homocysteine (Hcy) accelerates AS through regulating FABP4, and then mediates cholesterol accumulation in macrophages. Hcy concentrations of 0, 50, 100, 200 and 500 µM, and 100 µM Hcy+30 µM vitamin B12 (VB12)+30 µM folic acid (FA) were respectively added to cultured THP‑1 monocyte‑derived macrophages for 24 h. The levels of FABP4, which acts as a key factor connecting cellular lipid accumulation to inflammation, were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses in the macrophages. The present study used a nested touchdown methylation‑specific PCR assay to detect the DNA methylation status of the FABP4 promoter region. In addition, the FABP4 gene fragment was inserted into the cloning vector, pcDNA3.1‑EGFP, to construct the recombinant plasmid, pcDNA3.1‑EGFP/FABP4, which was identified using restriction endonuclease digestion analysis and DNA sequencing. The pcDNA3.1‑EGFP/FABP4 expression plasmid was transfected into THP‑1 monocyte‑derived macrophages, mediated by liposome reagent, following which the expression levels of FABP4 were detected using RT‑qPCR and western blot analyses. The present study also determined the intracellular accumulation of total cholesterol in the macrophages. The results indicated that Hcy decreased the levels of FABP4 promoter methylation, but increased the mRNA and protein expression levels of FABP4 in the macrophages, compared with the control group (0 µM Hcy). However, no dose

  1. A Leishmania Ortholog of Macrophage Migration Inhibitory Factor Modulates Host Macrophage Responses1

    PubMed Central

    Kamir, Daniela; Zierow, Swen; Leng, Lin; Cho, Yoonsang; Diaz, Yira; Griffith, Jason; McDonald, Courtney; Merk, Melanie; Mitchell, Robert A.; Trent, John; Chen, Yibang; Kwong, Yuen-Kwan Amy; Xiong, Huabao; Vermeire, Jon; Cappello, Michael; McMahon-Pratt, Diane; Walker, John; Bernhagen, Jurgen; Lolis, Elias; Bucala, Richard

    2009-01-01

    Parasitic organisms have evolved specialized strategies to evade immune defense mechanisms. We describe herein an ortholog of the cytokine, macrophage migration inhibitory factor (MIF), which is produced by the obligate intracellular parasite, Leishmania major. The Leishmania MIF protein, Lm1740MIF, shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 Å). Differences between the two proteins in the N-terminal tautomerization site are evident, and we provide evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site. Lm1740MIF shows significant binding interaction with the MIF receptor, CD74 (Kd = 2.9 × 10−8 M). Like its mammalian counterpart, Lm1740MIF induces ERK1/2 MAP kinase activation in a CD74-dependent manner and inhibits the activation-induced apoptosis of macrophages. The ability of Lm1740MIF to inhibit apoptosis may facilitate the persistence of Leishmania within the macrophage and contribute to its evasion from immune destruction. PMID:18523291

  2. A Leishmania Ortholog of Macrophage Migration Inhibitory Factor Modulates Host Macrophage Responses

    SciTech Connect

    Kamir,D.; Zierow, S.; Leng, L.; Cho, Y.; Diaz, Y.; Griffith, J.; McDonald, C.; Merk, M.; Mitchell, R.; et al

    2008-01-01

    Parasitic organisms have evolved specialized strategies to evade immune defense mechanisms. We describe herein an ortholog of the cytokine, macrophage migration inhibitory factor (MIF), which is produced by the obligate intracellular parasite, Leishmania major. The Leishmania MIF protein, Lm1740MIF, shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 A). Differences between the two proteins in the N-terminal tautomerization site are evident, and we provide evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site. Lm1740MIF shows significant binding interaction with the MIF receptor, CD74 (K(d) = 2.9 x 10(-8) M). Like its mammalian counterpart, Lm1740MIF induces ERK1/2 MAP kinase activation in a CD74-dependent manner and inhibits the activation-induced apoptosis of macrophages. The ability of Lm1740MIF to inhibit apoptosis may facilitate the persistence of Leishmania within the macrophage and contribute to its evasion from immune destruction.

  3. Fine-tuning of macrophage activation using synthetic rocaglate derivatives

    PubMed Central

    Bhattacharya, Bidisha; Chatterjee, Sujoy; Devine, William G.; Kobzik, Lester; Beeler, Aaron B.; Porco, John A.; Kramnik, Igor

    2016-01-01

    Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource. PMID:27086720

  4. Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74.

    PubMed

    Djudjaj, Sonja; Lue, Hongqi; Rong, Song; Papasotiriou, Marios; Klinkhammer, Barbara M; Zok, Stephanie; Klaener, Ole; Braun, Gerald S; Lindenmeyer, Maja T; Cohen, Clemens D; Bucala, Richard; Tittel, Andre P; Kurts, Christian; Moeller, Marcus J; Floege, Juergen; Ostendorf, Tammo; Bernhagen, Jürgen; Boor, Peter

    2016-06-01

    Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow-derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN. PMID:26453615

  5. Effect of a standardized liver and spleen fraction of peptides on the differentiation of human monocyte-derived macrophages.

    PubMed

    Spessotto, P; Bulla, R; Mittenzwei, H; Dri, P

    1994-06-01

    The effect of Factor AF2 (AF2), a standardized fraction of peptides with a molecular weight of < 10,000 Dalton obtained from livers and spleens of newborn lambs, on the differentiation of human monocyte-derived macrophages was studied, in view of the central role played by these cells in inflammation and tumor cytotoxicity. The results show that the drug 1. increases the cell density of cultures, 2. favours the morphologic differentiation of monocytes into macrophages, and 3. increases the macrophages phagocytic capacity. The first two effects are observed when monocytes are cultured in 1% serum but not in 10% serum while the enhancement of phagocytic activity is detected at both serum concentrations. PMID:8053979

  6. An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.

    PubMed Central

    Bacher, M; Metz, C N; Calandra, T; Mayer, K; Chesney, J; Lohoff, M; Gemsa, D; Donnelly, T; Bucala, R

    1996-01-01

    The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755565

  7. Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages

    PubMed Central

    Abu Khweek, Arwa; Fernández Dávila, Natalia S.; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A.; Tazi, Mia; Hassan, Hoda; Novotny, Laura A.; Bakaletz, Lauren O.; Amer, Amal O.

    2013-01-01

    Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human. PMID:23750338

  8. Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages.

    PubMed

    Abu Khweek, Arwa; Fernández Dávila, Natalia S; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A; Tazi, Mia; Hassan, Hoda; Novotny, Laura A; Bakaletz, Lauren O; Amer, Amal O

    2013-01-01

    Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human. PMID:23750338

  9. Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors

    PubMed Central

    2011-01-01

    Background Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages. Results Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494. Conclusions This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages. PMID:21281514

  10. The immune-microenvironment confers resistance to MAP kinase pathway inhibitors through macrophage-derived TNFα

    PubMed Central

    O’Brien, Kate; Brunton, Holly; Ferguson, Jennifer; Young, Helen; Dhomen, Nathalie; Flaherty, Keith T.; Frederick, Dennie T.; Cooper, Zachary A.; Wargo, Jennifer A.; Marais, Richard; Wellbrock, Claudia

    2014-01-01

    Recently the rationale for combining targeted therapy with immunotherapy has come to light, but our understanding of the immune response during MAPK pathway inhibitor treatment is limited. We discovered that the immune-microenvironment can act as source of resistance to MAPK pathway-targeted therapy, and moreover during treatment this source becomes reinforced. In particular, we identified macrophage-derived TNFα as a crucial melanoma-growth factor that provides resistance to MAPK pathway inhibitors through the lineage-transcription factor MITF. Most strikingly, in BRAF mutant melanomas of patients and BRafV600E-melanoma allografts MAPK pathway inhibitors increased the number of tumor-associated macrophages, and TNFα and MITF expression. Inhibiting TNFα-signaling with IκB-kinase inhibitors profoundly enhanced the efficacy of MAPK pathway inhibitors by targeting not only the melanoma cells, but also the microenvironment. In summary, we identify the immune-microenvironment as a novel source of resistance and reveal a new strategy to improve the efficacy of targeted therapy in melanoma. PMID:25256614

  11. Bone marrow-derived macrophages exclusively expressed caveolin-2: The role of inflammatory activators and hypoxia.

    PubMed

    Maceckova, Michaela; Martiskova, Hana; Koudelka, Adolf; Kubala, Lukas; Lojek, Antonin; Pekarova, Michaela

    2015-11-01

    Caveolins are specific proteins involved in regulation of signal transduction to intracellular space. Still, their contribution to immune functions has not been completely clarified. Thus, we decided to characterize the expression of caveolins in bone marrow-derived macrophages (BMDMs) under resting and inflammatory conditions. The effect of classical activators (lipopolysaccharide, LPS; interferon-gamma, IFN-γ) was further potentiated with hypoxic (5% O2) conditions. The activation of p44/42-extracellular signal-regulated kinases 1 and 2 (ERK1/2) and expression of caveolin-1, -2, and -3, hypoxia inducible factor-1 alpha (HIF-1α), as well as inducible nitric oxide synthase (iNOS) was monitored using the Western blot technique. The production of nitric oxide (NO) and tumor necrosis factor-alpha (TNFα) was analyzed by Griess method or ELISA, respectively. BMDMs were also transfected with siRNA against caveolin-2. Importantly, our study showed for the first time that BMDMs expressed only caveolin-2, and its level decreased after activation of macrophages with LPS, IFN-γ, and/or hypoxia. The expression of caveolin-2 negatively correlates with the iNOS and HIF-1α protein levels, as well as with the LPS/IFN-γ- and hypoxia-induced activation of ERK1/2. We concluded that caveolin-2 is most probably involved in regulation of pro-inflammatory responses of BMDMs, triggered via activation of ERK1/2. PMID:26215374

  12. Reduced Simian Immunodeficiency Virus Replication in Macrophages of Sooty Mangabeys Is Associated with Increased Expression of Host Restriction Factors

    PubMed Central

    Mir, Kiran D.; Mavigner, Maud; Wang, Charlene; Paiardini, Mirko; Sodora, Donald L.; Chahroudi, Ann M.; Bosinger, Steven E.

    2015-01-01

    ABSTRACT Macrophages are target cells of HIV/SIV infection that may play a role in AIDS pathogenesis and contribute to the long-lived reservoir of latently infected cells during antiretroviral therapy (ART). In previous work, we and others have shown that during pathogenic SIV infection of rhesus macaques (RMs), rapid disease progression is associated with high levels of in vivo macrophage infection. In contrast, during nonpathogenic SIV infection of sooty mangabeys (SMs), neither spontaneous nor experimental CD4+ T cell depletion results in substantial levels of in vivo macrophage infection. To test the hypothesis that SM macrophages are intrinsically more resistant to SIV infection than RM macrophages, we undertook an in vitro comparative assessment of monocyte-derived macrophages (MDMs) from both nonhuman primate species. Using the primary isolate SIVM949, which replicates well in lymphocytes from both RMs and SMs, we found that infection of RM macrophages resulted in persistent SIV-RNA production while SIV-RNA levels in SM macrophage cultures decreased 10- to 100-fold over a similar temporal course of in vitro infection. To explore potential mechanisms responsible for the lower levels of SIV replication and/or production in macrophages from SMs we comparatively assessed, in the two studied species, the expression of the SIV coreceptor as well as the expression of a number of host restriction factors. While previous studies showed that SM monocytes express lower levels of CCR5 (but not CD4) than RM monocytes, the level of CCR5 expression in MDMs was similar in the two species. Interestingly, we found that SM macrophages exhibited a significantly greater increase in the expression of tetherin (P = 0.003) and TRIM22 (P = 0.0006) in response to alpha interferon stimulation and increased expression of multiple host restriction factors in response to lipopolysaccharide stimulation and exposure to SIV. Overall, these findings confirm, in an in vitro infection system

  13. Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages.

    PubMed

    Ryzhova, Elena V; Vos, Robin M; Albright, Andrew V; Harrist, Alexia V; Harvey, Thomas; González-Scarano, Francisco

    2006-03-01

    Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM. PMID:16501079

  14. Macrophage-derived Lipocalin-2 contributes to ischemic resistance mechanisms by protecting from renal injury

    PubMed Central

    Jung, Michaela; Brüne, Bernhard; Hotter, Georgina; Sola, Anna

    2016-01-01

    Renal ischemia-reperfusion injury triggers an inflammatory response associated to infiltrating macrophages which determines the further outcome of disease. Brown Norway rats are known to show endogenous resistance to ischemia-induced renal damage. By contrast, Sprague Dawley rats exhibit a higher susceptibility to ischemic injury. In order to ascertain cytoprotective mechanisms, we focused on the implication of lipocalin-2 protein in main resistance mechanisms in renal ischemia/reperfusion injury by using adoptive macrophage administration, genetically modified ex vivo either to overexpress or to knockdown lipocalin-2. In vitro experiments with bone marrow-derived macrophages both from Brown Norway rats and from Sprague Dawley rats under hypoxic conditions showed endogenous differences regarding cytokine and lipocalin-2 expression profile in the two strains. Most interestingly, we observed that macrophages of the resistant strain express significantly more lipocalin-2. In vivo studies showed that tubular epithelial cell apoptosis and renal injury significantly increased and reparative markers decreased in Brown Norway rats after injection of lipocalin-2-knockdown macrophages, while the administration of lipocalin-2-overexpressing cells significantly decreased Sprague Dawley susceptibility. These data point to a crucial role of macrophage-derived lipocalin-2 in endogenous cytoprotective mechanisms. We conclude that expression of lipocalin-2 in tissue-infiltrating macrophages is pivotal for kidney-intrinsic cytoprotective pathways during ischemia reperfusion injury. PMID:26911537

  15. SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions.

    PubMed

    Pucci, Ferdinando; Garris, Christopher; Lai, Charles P; Newton, Andita; Pfirschke, Christina; Engblom, Camilla; Alvarez, David; Sprachman, Melissa; Evavold, Charles; Magnuson, Angela; von Andrian, Ulrich H; Glatz, Katharina; Breakefield, Xandra O; Mempel, Thorsten R; Weissleder, Ralph; Pittet, Mikael J

    2016-04-01

    Tumor-derived extracellular vesicles (tEVs) are important signals in tumor-host cell communication, yet it remains unclear how endogenously produced tEVs affect the host in different areas of the body. We combined imaging and genetic analysis to track melanoma-derived vesicles at organismal, cellular, and molecular scales to show that endogenous tEVs efficiently disseminate via lymphatics and preferentially bind subcapsular sinus (SCS) CD169(+) macrophages in tumor-draining lymph nodes (tdLNs) in mice and humans. The CD169(+) macrophage layer physically blocks tEV dissemination but is undermined during tumor progression and by therapeutic agents. A disrupted SCS macrophage barrier enables tEVs to enter the lymph node cortex, interact with B cells, and foster tumor-promoting humoral immunity. Thus, CD169(+) macrophages may act as tumor suppressors by containing tEV spread and ensuing cancer-enhancing immunity. PMID:26989197

  16. Human lung-resident macrophages express CB1 and CB2 receptors whose activation inhibits the release of angiogenic and lymphangiogenic factors.

    PubMed

    Staiano, Rosaria I; Loffredo, Stefania; Borriello, Francesco; Iannotti, Fabio Arturo; Piscitelli, Fabiana; Orlando, Pierangelo; Secondo, Agnese; Granata, Francescopaolo; Lepore, Maria Teresa; Fiorelli, Alfonso; Varricchi, Gilda; Santini, Mario; Triggiani, Massimo; Di Marzo, Vincenzo; Marone, Gianni

    2016-04-01

    Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung-resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce 2-arachidonoylglycerol,N-arachidonoyl-ethanolamine,N-palmitoyl-ethanolamine, andN-oleoyl-ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize 2-arachidonoylglycerol in a calcium-dependent manner. Human lung macrophages express cannabinoid receptor types 1 and 2, and their activation induces ERK1/2 phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH-133 (but not the endogenous agonist 2-arachidonoylglycerol) markedly inhibits LPS-induced production of vascular endothelial growth factor-A, vascular endothelial growth factor-C, and angiopoietins and modestly affects IL-6 secretion. No significant modulation of TNF-α or IL-8/CXCL8 release was observed. The production of vascular endothelial growth factor-A by human monocyte-derived macrophages is not modulated by activation of cannabinoid receptor types 1 and 2. Given the prominent role of macrophage-assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer-associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte-derived macrophages. Activation of cannabinoid receptors on tissue-resident macrophages might be a novel strategy to modulate macrophage-assisted vascular remodeling

  17. Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

    PubMed Central

    Sánchez-Reyes, Karina; Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Jave-Suárez, Luis Felipe; Gómez-Lomelí, Paulina; de Celis, Ruth; Aguilar-Lemarroy, Adriana; Domínguez-Rodríguez, Jorge Ramiro; Ortiz-Lazareno, Pablo Cesar

    2014-01-01

    Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages. PMID:25309919

  18. Protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis

    PubMed Central

    Heinrichs, Daniel; Berres, Marie-Luise; Coeuru, Melanie; Knauel, Meike; Nellen, Andreas; Fischer, Petra; Philippeit, Claudia; Bucala, Richard; Trautwein, Christian; Wasmuth, Hermann E.; Bernhagen, Jürgen

    2014-01-01

    , P., Philippeit, C., Bucala, R., Trautwein, C., Wasmuth, H. E., Bernhagen, J. Protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis. PMID:25122558

  19. Activated Human Mast Cells Induce LOX-1-Specific Scavenger Receptor Expression in Human Monocyte-Derived Macrophages

    PubMed Central

    Alanne-Kinnunen, Mervi; Lappalainen, Jani; Öörni, Katariina; Kovanen, Petri T.

    2014-01-01

    Objective Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs). Results Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell –induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages. Conclusions Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis. PMID:25250731

  20. Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF).

    PubMed

    Zhao, Bin; Li, Yifu; Buono, Chiara; Waldo, Stephen W; Jones, Nancy L; Mori, Masahiro; Kruth, Howard S

    2006-06-01

    Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors. PMID:16606620

  1. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

    SciTech Connect

    Gardner, Carol R.; Hankey, Pamela; Mishin, Vladimir; Francis, Mary; Yu, Shan; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  2. Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells.

    PubMed

    Sheng, Jianpeng; Ruedl, Christiane; Karjalainen, Klaus

    2015-08-18

    Macrophages are one of the most diverse cell populations in terms of their anatomical location and functional specialization during both homeostasis and disease. Although it has been shown in different fate mapping models that some macrophages present in adult tissues are already established during fetal development, their exact origins are still under debate. In the current study, we developed a fate mapping strain, based on the Kit locus, which allowed us to readdress "the origins" question. Different types of macrophages from various adult tissues were traced to their fetal or adult sources by inducing labeling in precursors at several time points either during fetal development or in adult mice. We show that all adult macrophages, resident or infiltrating, are progenies of classical hematopoietic stem cells (HSC) with the exception of microglia and, partially epidermal Langerhans cells, which are yolk sac (YS)-derived. PMID:26287683

  3. Diverse HLA-I Peptide Repertoires of the APC Lines MUTZ3-Derived Immature and Mature Dendritic Cells and THP1-Derived Macrophages.

    PubMed

    Nyambura, Lydon Wainaina; Jarmalavicius, Saulius; Baleeiro, Renato Brito; Walden, Peter

    2016-09-15

    Dendritic cells (DCs) and macrophages are specialized APCs that process and present self-Ags for induction of tolerance and foreign Ags to initiate T cell-mediated immunity. Related to differentiation states they have specific phenotypes and functions. However, the impact of these differentiations on Ag processing and presentation remains poorly defined. To gain insight into this, we analyzed and compared the HLA-I peptidomes of MUTZ3-derived human immature and mature DC lines and THP1-derived macrophages by liquid chromatography tandem mass spectrometry. We found that the HLA-I peptidomes were heterogeneous and individualized and were dominated by nonapeptides with similar HLA-I binding affinities and anchor residues. MUTZ3-derived DCs and THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and were involved in various cellular functions in similar proportion, with preference to proteins involved in cell communication, signal transduction, protein metabolism, and transcription factor/regulator activity. PMID:27543614

  4. Particulate matter phagocytosis induces tissue factor in differentiating macrophages.

    PubMed

    Milano, M; Dongiovanni, P; Artoni, A; Gatti, S; Rosso, L; Colombo, F; Bollati, V; Maggioni, M; Mannucci, P M; Bertazzi, P A; Fargion, S; Valenti, L

    2016-01-01

    Airborne exposure to particulate matter with diameter < 10 mcM (PM10) has been linked to an increased risk of thromboembolic events, but the mechanisms are not completely understood. The aim of this study was to evaluate the effect of PM10 phagocytosis on the release of procoagulant molecules in human differentiating macrophages, and that of PM10 inhalation in an experimental model in rats. Human monocytes were separated from the peripheral blood by the lymphoprep method, differentiated in vitro and treated with standard PM10 or vehicle. Sprague-Dawley rats were instilled intratracheally with PM10 or vehicle alone. The outcome was expression of proinflammatory genes and of tissue factor (TF). In human differentiating macrophages, PM10 exposure upregulated inflammatory genes, but most consistently induced TF mRNA and protein levels, but not TF protein inhibitor, resulting in increased TF membrane expression and a procoagulant phenotype. Differentiation towards the anti-inflammatory M2 phenotype inhibited PM10 -mediated TF expression. TF induction required phagocytosis of PM10 , whereas phagocytosis of inert particles was less effective. PM10 phagocytosis was associated with a gene expression profile consistent with intracellular retention of iron, inducing oxidative stress. Both PM10 and iron activated the stress kinases ERK1/2 pathway, involved in the induction of TF expression. In rats, alveolar exposure to PM10 was associated with pulmonary recruitment of inflammatory cells and resulted in local, but not systemic, induction of TF expression, which was sufficient to increase circulating TF levels. In conclusion, TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure. PMID:25858758

  5. Bastadins, brominated-tyrosine derivatives, suppress accumulation of cholesterol ester in macrophages.

    PubMed

    Eguchi, Keisuke; Kato, Hikaru; Fujiwara, Yukio; Losung, Fitje; Mangindaan, Remy E P; de Voogd, Nicole J; Takeya, Motohiro; Tsukamoto, Sachiko

    2015-11-15

    The formation of foam cells in macrophages has been suggested to play an essential role in the progression of early atherosclerotic lesions in vivo and, thus, its suppression is considered to be one of the major approaches for the treatment of atherosclerosis. We isolated eight brominated-tyrosine derivatives, bastadins, from the EtOH extract of the marine sponge Ianthella vasta as inhibitors of the formation of foam cells induced by acetylated low-density lipoproteins in human monocyte-derived macrophages. Bastadin 6 was the strongest inhibitor of foam cell formation due to its suppression of acyl-coenzyme A:cholesterol acyltransferase. PMID:26403929

  6. Macrophage-derived LIF and IL1B regulate alpha(1,2)fucosyltransferase 2 (Fut2) expression in mouse uterine epithelial cells during early pregnancy.

    PubMed

    Jasper, Melinda J; Care, Alison S; Sullivan, Brad; Ingman, Wendy V; Aplin, John D; Robertson, Sarah A

    2011-01-01

    Macrophages accumulate within stromal tissue subjacent to the luminal epithelium in the mouse uterus during early pregnancy after seminal fluid exposure at coitus. To investigate their role in regulating epithelial cell expression of fucosylated structures required for embryo attachment and implantation, fucosyltransferase enzymes Fut1, Fut2 (Enzyme Commission number [EC] 2.4.1.69), and Fut4 (EC 2.4.1.214) and Muc1 and Muc4 mRNAs were quantified by quantitative real-time PCR in uterine epithelial cells after laser capture microdissection in situ or after epithelial cell coculture with macrophages or macrophage-secreted factors. When uterine macrophage recruitment was impaired by mating with seminal plasma-deficient males, epithelial cell Fut2 expression on Day 3.5 postcoitus (pc) was reduced compared to intact-mated controls. Epithelial cell Fut2 was upregulated in vitro by coculture with macrophages or macrophage-conditioned medium (MCM). Macrophage-derived cytokines LIF, IL1B, and IL12 replicated the effect of MCM on Fut2 mRNA expression, and MCM-stimulated expression was inhibited by anti-LIF and anti-IL1B neutralizing antibodies. The effects of acute macrophage depletion on fucosylated structures detected with lectins Ulex europaeus 1 (UEA-1) and Lotus tetragonolobus purpureas (LTP), or LewisX immunoreactivity, were quantified in vivo in Cd11b-dtr transgenic mice. Depletion of macrophages caused a 30% reduction in luminal epithelial UEA-1 staining and a 67% reduction in LewisX staining in uterine tissues of mice hormonally treated to mimic early pregnancy. Together, these data demonstrate that uterine epithelial Fut2 mRNA expression and terminal fucosylation of embryo attachment ligands is regulated in preparation for implantation by factors including LIF and IL1B secreted from macrophages recruited during the inflammatory response to insemination. PMID:20864644

  7. FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice.

    PubMed

    Bosma, Madeleen; Gerling, Marco; Pasto, Jenny; Georgiadi, Anastasia; Graham, Evan; Shilkova, Olga; Iwata, Yasunori; Almer, Sven; Söderman, Jan; Toftgård, Rune; Wermeling, Fredrik; Boström, Elisabeth Almer; Boström, Pontus Almer

    2016-01-01

    FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases. PMID:27066907

  8. FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice

    PubMed Central

    Bosma, Madeleen; Gerling, Marco; Pasto, Jenny; Georgiadi, Anastasia; Graham, Evan; Shilkova, Olga; Iwata, Yasunori; Almer, Sven; Söderman, Jan; Toftgård, Rune; Wermeling, Fredrik; Boström, Elisabeth Almer; Boström, Pontus Almer

    2016-01-01

    FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases. PMID:27066907

  9. Granulocyte macrophage colony-stimulating factor and the intestinal innate immune cell homeostasis in Crohn's disease.

    PubMed

    Däbritz, Jan

    2014-03-01

    Current literature consolidates the view of Crohn's disease (CD) as a form of immunodeficiency highlighting dysregulation of intestinal innate immunity in the pathogenesis of CD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine, suggesting that the monocyte/macrophage compartment might be an attractive therapeutic target for the management of CD. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that also promotes myeloid cell activation, proliferation, and differentiation. GM-CSF has a protective effect in human CD and mouse models of colitis. However, the role of GM-CSF in immune and inflammatory reactions in the intestine is not well defined. Beneficial effects exerted by GM-CSF during intestinal inflammation could relate to modulation of the mucosal barrier function in the intestine, including epithelial cell proliferation, survival, restitution, and immunomodulatory actions. The aim of this review is to summarize potential mechanistic roles of GM-CSF in intestinal innate immune cell homeostasis and to highlight its central role in maintenance of the intestinal immune barrier in the context of immunodeficiency in CD. PMID:24503766

  10. Human umbilical cord blood-derived f-macrophages retain pluripotentiality after thrombopoietin expansion

    SciTech Connect

    Zhao Yong . E-mail: yongzhao@uic.edu; Mazzone, Theodore

    2005-11-01

    We have previously characterized a new type of stem cell from human peripheral blood, termed fibroblast-like macrophage (f-M{phi}). Here, using umbilical cord blood as a source, we identified cells with similar characteristics including expression of surface markers (CD14, CD34, CD45, CD117, and CD163), phagocytosis, and proliferative capacity. Further, thrombopoietin (TPO) significantly stimulated the proliferation of cord blood-derived f-M{phi} (CB f-M{phi}) at low dosage without inducing a megakaryocytic phenotype. Additional experiments demonstrated that TPO-expanded cord blood-derived f-M{phi} (TCB f-M{phi}) retained their surface markers and differentiation ability. Treatment with vascular endothelial cell growth factor (VEGF) gave rise to endothelial-like cells, expressing Flt-1, Flk-1, von Willebrand Factor (vWF), CD31, acetylated low density lipoprotein internalization, and the ability to form endothelial-like cell chains. In the presence of lipopolyssacharide (LPS) and 25 mM glucose, the TCB f-M{phi} differentiated to express insulin mRNA, C-peptide, and insulin. In vitro functional analysis demonstrated that these insulin-positive cells could release insulin in response to glucose and other secretagogues. These findings demonstrate a potential use of CB f-M{phi} and may lead to develop new therapeutic strategy for treating dominant disease.

  11. Potentiation of photodynamic therapy by granulocyte-macrophage colony stimulating factor immunotherapy

    NASA Astrophysics Data System (ADS)

    Krosl, Gorazd; Korbelik, Mladen; Krosl, Jana; Dougherty, Graeme J.

    1995-03-01

    The murine squamous carcinoma cell line (SCCVII) was genetically engineered to produce high levels of granulocyte-macrophage colony stimulating factor (GM-CSF). Lethally irradiated GM-CSF producing cells were injected under the subcutaneously growing parental SCCVII tumor at various times before and/or after PDT. Even a single treatment with GM- CSF producing cells injected two days before PDT markedly enhanced the tumor cure rate when compared to the PDT treatment alone. Effective potentiation was observed with PDT mediated either by Photofrin or by benzoporphyrin derivative.

  12. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ

    PubMed Central

    2014-01-01

    Background In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. Methods Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/− IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. Results M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ–activated M2-like macrophages had low anti-tumor activity, IL-10–polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. Conclusion This study demonstrates that human macrophages can be induced to exert direct anti

  13. The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages.

    PubMed

    Libby, S J; Lesnick, M; Hasegawa, P; Weidenhammer, E; Guiney, D G

    2000-02-01

    The pathogenesis of serious systemic Salmonella infections is characterized by survival and proliferation of bacteria inside macrophages. Infection of human monocyte-derived macrophages in vitro with S. typhimurium or S. dublin produces cytopathology characterized by detachment of cells that contain large numbers of proliferating bacteria. This cytopathology is dependent on the expression of the bacterial spv genes, a virulence locus previously shown to markedly enhance the ability of Salmonella to produce systemic disease. After 24 h of infection, macrophage cultures contain two populations of bacteria: (i) proliferating organisms present in a detached cell fraction; and (ii) a static bacterial population in macrophages remaining attached to the culture well. Mutations in either the essential transcriptional activator SpvR or the key SpvB protein markedly reduce the cytopathic effect of Salmonella infection. The spv-dependent cytopathology in macrophages exhibits characteristics of apoptosis, with release of nucleosomes into the cytoplasm, nuclear condensation and DNA fragmentation. The current findings suggest that the mechanism of the spv effect is through induction of increased cytopathology in host macrophages. PMID:11207562

  14. CSF-1–dependant donor-derived macrophages mediate chronic graft-versus-host disease

    PubMed Central

    Alexander, Kylie A.; Flynn, Ryan; Lineburg, Katie E.; Kuns, Rachel D.; Teal, Bianca E.; Olver, Stuart D.; Lor, Mary; Raffelt, Neil C.; Koyama, Motoko; Leveque, Lucie; Le Texier, Laetitia; Melino, Michelle; Markey, Kate A.; Varelias, Antiopi; Engwerda, Christian; Serody, Jonathan S.; Janela, Baptiste; Ginhoux, Florent; Clouston, Andrew D.; Blazar, Bruce R.; Hill, Geoffrey R.; MacDonald, Kelli P.A.

    2014-01-01

    Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17–dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS–). Cutaneous cGVHD developed in a CSF-1/CSF-1R–dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r–/– mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti–CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD. PMID:25157821

  15. Orthologs of macrophage migration inhibitory factor from parasitic nematodes

    PubMed Central

    Vermeire, Jon J.; Cho, Yoonsang; Lolis, Elias; Bucala, Richard; Cappello, Michael

    2013-01-01

    Chronic helminth infections are associated with modulation of host cellular immune responses, presumably to prolong parasite survival within the mammalian host. This phenomenon is attributed, at least in part, to the elaboration of parasite molecules, including orthologs of host cytokines and receptors, at the host–parasite interface. This review describes recent progress in the characterization of macrophage migration inhibitory factor (MIF) orthologs from parasitic nematodes. The roles of these molecules in parasite developmental biology and pathogenesis are discussed. Further knowledge of the species-specific activities and three-dimensional structures of human and parasitic nematode MIF molecules should make them ideal targets for drug- and/or vaccine-based strategies aimed at nematode disease control. PMID:18603473

  16. Molecular Cloning and Functional Characterization of the Avian Macrophage Migration Inhibitory Factor (MIF)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor produced by sensitized T lymphocytes and inhibits the random migration of macrophages. Recent studies have revealed a more prominent role for MIF as a multi-functional cytokine mediating both innate and adaptive immune r...

  17. Killing of Leishmania parasites in activated murine macrophages is based on an L-arginine-dependent process that produces nitrogen derivatives

    SciTech Connect

    Maul, J.R.; Ransijn, A.; Buchmueller-Rouiller, Y. )

    1991-01-01

    The experiments described in this report were aimed at determining whether L-arginine (L-arg)-derived nitrogen oxidation products (nitric oxide, nitrous acid, nitrites) are involved in the intracellular killing of Leishmania parasites by activated murine macrophages in vitro. Peritoneal or bone marrow-derived macrophages were infected with L. enriettii or L. major, then activated by exposure to recombinant murine interferon-gamma or to macrophage activating factor (MAF)-rich media in the presence of lipopolysaccharide. Activation of macrophages in regular (i.e., arginine-containing) culture medium led to complete destruction of the microorganisms within 24 h (L. enriettii) or 48 h (L. major), concomitant with accumulation of nitrites (NO2-) in the culture fluids. When macrophage activation was carried out in L-arg-free medium, however, neither parasite killing nor NO2- production was obtained. A similar inhibition of macrophage leishmanicidal activity and of NO2- release was observed using media treated with arginase (which converts L-arg to urea and ornithine), or supplemented with NG-monomethyl-L-arg or guanidine (which inhibit the conversion of L-arg to nitrogen oxidation products). In all these situations, an excellent correlation between the levels of NO2- production by macrophages and intracellular killing of Leishmania was observed, whereas no strict correlation was detectable between leishmanicidal activity and superoxide production. Intracellular parasite killing by activated macrophages could be prevented by addition of iron salts to the incubation fluids. Incubation of free parasites with NaNO2 at acid pH led to immobilisation, multiplication arrest, and morphological degeneration of the microorganisms. Similarly, exposure of infected cells to NaNO2 led to killing of the intracellular parasite without affecting macrophage viability.

  18. Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumour necrosis factor-alpha and nitrite release.

    PubMed

    Formica, S; Roach, T I; Blackwell, J M

    1994-05-01

    The murine resistance gene Lsh/Ity/Bcg regulates activation of macrophages for tumour necrosis factor-alpha (TNF-alpha)-dependent production of nitric oxide mediating antimicrobial activity against Leishmania, Salmonella and Mycobacterium. As Lsh is differentially expressed in macrophages from different tissue sites, experiments were performed to determine whether interaction with extracellular matrix (ECM) proteins would influence the macrophage TNF-alpha response. Plating of bone marrow-derived macrophages onto purified fibrinogen or fibronectin-rich L929 cell-derived matrices, but not onto mannan, was itself sufficient to stimulate TNF-alpha release, with significantly higher levels released from congenic B10.L-Lshr compared to C57BL/10ScSn (Lshs) macrophages. Only macrophages plated onto fibrinogen also released measurable levels of nitrites, again higher in Lshr compared to Lshs macrophages. Addition of interferon-gamma (IFN-gamma), but not bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as a second signal enhanced the TNF-alpha and nitrite responses of macrophages plated onto fibrinogen, particularly in the Lshr macrophages. Interaction with fibrinogen and fibronectin also primed macrophages for an enhanced TNF-alpha response to leishmanial parasites, but this was only translated into enhanced nitrite responses in the presence of IFN-gamma. In these experiments, Lshr macrophages remained superior in their TNF-alpha responses throughout, but to a degree which reflected the magnitude of the difference observed on ECM alone. Hence, the specificity for the enhanced TNF-alpha responses of Lshr macrophages lay in their interaction with fibrinogen and fibronectin ECM, while a differential nitrite response was only observed with fibrinogen and/or IFN-gamma. The results are discussed in relation to the possible function of the recently cloned candidate gene Nramp, which has structural identity to eukaryote transporters and an N-terminal cytoplasmic

  19. Xylitol, an Anticaries Agent, Exhibits Potent Inhibition of Inflammatory Responses in Human THP-1-Derived Macrophages Infected With Porphyromonas gingivalis

    PubMed Central

    Park, Eunjoo; Na, Hee Sam; Kim, Sheon Min; Wallet, Shannon; Cha, Seunghee; Chung, Jin

    2016-01-01

    Background Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. Methods Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis–induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. Results Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1β, in a multiplicity of infection– and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ–induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis– induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted anti-phagocytic activity against both Escherichia coli and P. gingivalis. Conclusion These findings suggest that xylitol acts as an antiinflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis. PMID:24592909

  20. Functional characterization of the turkey macrophage migration inhibitory factor.

    PubMed

    Park, Myeongseon; Kim, Sungwon; Fetterer, Raymond H; Dalloul, Rami A

    2016-08-01

    Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characterize its basic function. The full-length TkMIF gene was amplified from total RNA extracted from turkey spleen, followed by cloning into a prokaryotic (pET11a) expression vector. Sequence analysis revealed that TkMIF consists of 115 amino acids with 12.5 kDa molecular weight. Multiple sequence alignment revealed 100%, 65%, 95% and 92% identity with chicken, duck, eagle and zebra finch MIFs, respectively. Recombinant TkMIF (rTkMIF) was expressed in Escherichia coli and purified through HPLC and endotoxin removal. SDS-PAGE analysis revealed an approximately 13.5 kDa of rTkMIF monomer containing T7 tag in soluble form. Western blot analysis showed that anti-chicken MIF (ChMIF) polyclonal antisera detected a monomer form of TkMIF at approximately 13.5 kDa size. Further functional analysis revealed that rTkMIF inhibits migration of both mononuclear cells and splenocytes in a dose-dependent manner, but was abolished by the addition of anti-ChMIF polyclonal antisera. qRT-PCR analysis revealed elevated transcripts of pro-inflammatory cytokines by rTkMIF in LPS-stimulated monocytes. rTkMIF also led to increased levels of IFN-γ and IL-17F transcripts in Con A-activated splenocytes, while IL-10 and IL-13 transcripts were decreased. Overall, the sequences of both the turkey and chicken MIF have high similarity and comparable biological functions with respect to migration inhibitory activities of macrophages and enhancement of pro-inflammatory cytokine expression, suggesting that turkey and chicken MIFs would be biologically cross-reactive. PMID:27062968

  1. Lipopolysaccharide-Related Stimuli Induce Expression of the Secretory Leukocyte Protease Inhibitor, a Macrophage-Derived Lipopolysaccharide Inhibitor

    PubMed Central

    Jin, Fenyu; Nathan, Carl F.; Radzioch, Danuta; Ding, Aihao

    1998-01-01

    Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-κB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417–426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins—interleukin-10 (IL-10) and IL-6—also induced SLPI, while TNF and IL-1β did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules—taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls—also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI’s slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria. PMID:9596701

  2. Oxidative metabolism in cord blood monocytes and monocyte-derived macrophages.

    PubMed Central

    Speer, C P; Ambruso, D R; Grimsley, J; Johnston, R B

    1985-01-01

    Little is known about phagocytosis-associated oxidative metabolism in mononuclear phagocytes from the human neonate. We investigated this phenomenon in monocytes from the cord blood of term newborn infants by measuring generation of superoxide anion (O2-) and hydroxyl radical (X OH) after stimulation with opsonized zymosan or phorbol myristate acetate. Production of these microbicidal oxygen metabolites by monocytes from neonates and healthy adult volunteers was equivalent. When cultured in the presence of the macrophage activator lipopolysaccharide or muramyl dipeptide, monocytes from neonates and adults differentiated into cells with the appearance of macrophages and with an enhanced capacity to release O2- compared with cells cultured in the absence of an activator. Monocyte-derived macrophages from neonates produced only slightly less O2- than did adult cells. Thus, unlike the cord blood neutrophil, which exhibits abnormalities in oxidative metabolism, the cord blood mononuclear phagocyte has a respiratory burst that is quantitatively comparable to that of the adult cell. PMID:2999001

  3. Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

    PubMed

    Karpurapu, Manjula; Ranjan, Ravi; Deng, Jing; Chung, Sangwoon; Lee, Yong Gyu; Xiao, Lei; Nirujogi, Teja Srinivas; Jacobson, Jeffrey R; Park, Gye Young; Christman, John W

    2014-01-01

    The role of different lineage specific transcription factors in directing hematopoietic cell fate towards myeloid lineage is well established but the status of epigenetic modifications has not been defined during this important developmental process. We used non proliferating, PU.1 inducible myeloid progenitor cells and differentiating bone marrow derived macrophages to study the PU.1 dependent KLF4 transcriptional regulation and its promoter demethylation during monocyte/macrophage differentiation. Expression of KLF4 was regulated by active demethylation of its promoter and PU.1 specifically bound to KLF4 promoter oligo harboring the PU.1 consensus sequence. Methylation specific quantitative PCR and Bisulfite sequencing indicated demethylation of CpG residues most proximal to the transcription start site of KLF4 promoter. Cloned KLF4 promoter in pGL3 Luciferase and CpG free pcpgf-bas vectors showed accentuated reporter activity when co-transfected with the PU.1 expression vector. In vitro methylation of both KLF4 promoter oligo and cloned KLF4 promoter vectors showed attenuated in vitro DNA binding activity and Luciferase/mouse Alkaline phosphotase reporter activity indicating the negative influence of KLF4 promoter methylation on PU.1 binding. The Cytosine deaminase, Activation Induced Cytidine Deaminase (AICDA) was found to be critical for KLF4 promoter demethylation. More importantly, knock down of AICDA resulted in blockade of KLF4 promoter demethylation, decreased F4/80 expression and other phenotypic characters of macrophage differentiation. Our data proves that AICDA mediated active demethylation of the KLF4 promoter is necessary for transcriptional regulation of KLF4 by PU.1 during monocyte/macrophage differentiation. PMID:24695324

  4. Anti-bis(monoacylglycero)phosphate antibody accumulates acetylated LDL-derived cholesterol in cultured macrophages.

    PubMed

    Delton-Vandenbroucke, Isabelle; Bouvier, Jerome; Makino, Asami; Besson, Nelly; Pageaux, Jean-François; Lagarde, Michel; Kobayashi, Toshihide

    2007-03-01

    Bis(monoacylglycero)phosphate (BMP), also called lysobisphosphatidic acid, is a phospholipid highly enriched in the internal membranes of multivesicular late endosomes, in which it forms specialized lipid domains. It has been suggested that BMP-rich membranes regulate cholesterol transport. Here, we examine the effects of an anti-BMP antibody on cholesterol metabolism and transport in two macrophage cell lines, RAW 264.7 and THP-1, during loading with acetylated low density lipoprotein (AcLDL). Anti-BMP antibody was internalized and accumulated in both macrophage cell types. Cholesterol staining with filipin and mass measurements indicate that AcLDL-stimulated accumulation of free cholesterol (FC) was enhanced in macrophages that had accumulated the antibody. Unlike the hydrophobic amine U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), esterification of AcLDL-derived cholesterol by ACAT was not modified after anti-BMP treatment. AcLDL loading led to an increase of FC in the plasma membrane. This increase was further enhanced in anti-BMP-treated macrophages. However, cholesterol efflux to HDL was reduced in antibody-treated cells. These results suggest that the accumulation of anti-BMP antibody alters cholesterol homeostasis in AcLDL-loaded macrophages. PMID:17146116

  5. Expression and Function of Semaphorin 3A and Its Receptors in Human Monocyte-derived Macrophages

    PubMed Central

    Ji, Jong-Dae; Park-Min, Kyung-Hyun; Ivashkiv, Lionel B.

    2016-01-01

    Semaphorins are a large family of secreted and membrane-bound proteins. Recently, several roles of semaphorins in the immune system have emerged. Several semaphorins and their receptors are expressed in a variety of lymphoid and myeloid cells and affect immune cell functions, including cell proliferation, differentiation, chemotaxis, and cytokine production. However, the roles of class 3 semaphorins in human myeloid cells are not well known. Here we examined the regulation of expression of class 3 semaphorins and their receptors by inflammatory stimuli and their function in human macrophages. We show that the expression of Sema3A receptors (neuropilin-1 (NRP-1), NRP-2, plexin A1, plexin A2 and plexin A3) significantly increased during M-CSF-mediated differentiation of monocytes into macrophages under conditions that promote an M2 alternatively activated macrophage phenotype. Consistent with increased NRP-1 expression, cell surface binding of Sema3A increased during M2 differentiation. IFN-γ and LPS that promote classical M1 macrophage activation affected expression of NRP-1, NRP-2 and plexin A1. IFN-γ decreased NRP-1 expression and LPS suppressed NRP-2 and plexin A1 expression. Furthermore we show that Sema3A induced apoptosis in monocyte-derived macrophages, and cooperated with anti-Fas CH11 antibody to augment apoptosis. Our results suggest Sema3A plays a role in induction of apoptosis in monocyte-derived macrophages that are resistant to Fas-induced apoptosis and that its function can be modulated in inflammatory conditions. PMID:19480842

  6. Production of tumor necrosis factor and interleukin-1 by macrophages from human atheromatous plaques.

    PubMed Central

    Tipping, P. G.; Hancock, W. W.

    1993-01-01

    The production of cytokines by atheromatous plaque macrophages from human endarterectomy tissue was assessed in vitro by short-term cell culture and in situ by immunohistology. Macrophages were isolated from plaques of 14 patients undergoing carotid endarterectomy and 7 patients undergoing reconstructive procedures on atheromatous distal aortic and femoral arteries. Tumor necrosis factor (TNF) and interleukin 1 (IL-1) production by plaque macrophages and blood monocytes isolated concurrently from these patients was assessed. TNF release by macrophages from carotid plaques (0.39 +/- 0.12 ng/10(6) cells/24 hours) was significantly augmented compared to the release by corresponding blood monocytes (0.014 +/- 0.011 ng/10(6) cells/24 hours, P = 0.03), and by macrophages from noncarotid lesions (0.038 +/- 0.036 ng/10(6) cells/24 hours, P < 0.04). Cellular TNF expression by macrophages within carotid plaques was also more prominent than in noncarotid lesions. By contrast, IL-1 production by plaque macrophages from both carotid and noncarotid plaques was not augmented compared to blood monocytes, and only infrequent and low-intensity labeling for IL-1 was present on macrophages within plaques from either group. These results provide functional and immunohistological evidence for increased production of TNF but not IL-1 by activated macrophages, indicating local and selective augmentation of cytokine production within carotid plaques. This suggests that macrophages play an active role in the inflammatory response within atheromatous carotid plaques. Images Figure 3 PMID:8506944

  7. Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4–CXCR7

    PubMed Central

    Chatterjee, M; von Ungern-Sternberg, S N I; Seizer, P; Schlegel, F; Büttcher, M; Sindhu, N A; Müller, S; Mack, A; Gawaz, M

    2015-01-01

    Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4–CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4–CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1–M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4–CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163+ macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies

  8. Stimulation of monocytes, macrophages, and microglia by amphotericin B and macrophage colony-stimulating factor promotes remyelination.

    PubMed

    Döring, Axinia; Sloka, Scott; Lau, Lorraine; Mishra, Manoj; van Minnen, Jan; Zhang, Xu; Kinniburgh, David; Rivest, Serge; Yong, V Wee

    2015-01-21

    Approaches to stimulate remyelination may lead to recovery from demyelinating injuries and protect axons. One such strategy is the activation of immune cells with clinically used medications, since a properly directed inflammatory response can have healing properties through mechanisms such as the provision of growth factors and the removal of cellular debris. We previously reported that the antifungal medication amphotericin B is an activator of circulating monocytes, and their tissue-infiltrated counterparts and macrophages, and of microglia within the CNS. Here, we describe that amphotericin B activates these cells through engaging MyD88/TRIF signaling. When mice were subjected to lysolecithin-induced demyelination of the spinal cord, systemic injections of nontoxic doses of amphotericin B and another activator, macrophage colony-stimulating factor (MCSF), further elevated the representation of microglia/macrophages at the site of injury. Treatment with amphotericin B, particularly in combination with MCSF, increased the number of oligodendrocyte precursor cells and promoted remyelination within lesions; these pro-regenerative effects were mitigated in mice treated with clodronate liposomes to reduce circulating monocytes and tissue-infiltrated macrophages. Our results have identified candidates among currently used medications as potential therapies for the repair of myelin. PMID:25609628

  9. Induction of prostanoid, nitric oxide, and cytokine formation in rat bone marrow derived macrophages by activin A.

    PubMed

    Nüsing, R M; Barsig, J

    1999-06-01

    1. In this study we describe that activin A, a transforming growth factor (TGF) beta-like polypeptide affects the expression of inflammatory response genes and their products. 2. In rat bone marrow derived macrophages 15 nM activin A caused the stimulation of prostaglandin (PG) E2 and thromboxane (TX) A2 formation, production of nitrite as a marker for nitric oxide (NO) and the release of the cytokines tumour necrosis factor (TNF) alpha and interleukin (IL) -1beta. As shown by mRNA analysis induction of cyclo-oxygenase-2 and inducible nitric oxide synthase by activin A gave rise to the enhanced release of prostanoids and NO. 3. Costimulation of bone marrow derived macrophages with 15 nM activin A and 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA) potentiated the synthesis of prostanoids in a synergistic manner. With respect to NO formation the effect of activin A and TPA was additive. 4. In contrast to the nitrite production activin A induced PGE2 synthesis was susceptible to tyrosine kinase inhibition by genistein and tyrphostin 46 (IC50 was 10 and 20 microM, respectively). This observed inhibition was caused by the selective suppression of activin A induced cyclo-oxygenase-2 mRNA expression. Further, the release of TNFalpha in the presence of activin A was potentiated by tyrosine kinase inhibition. 5. In summary, we report that activin A exerts proinflammatory activity which results in the formation of prostanoids, NO and cytokines in rat bone marrow derived macrophages. Tyrosine kinase dependent and independent signalling pathways are involved leading to the increased synthesis of these metabolites. Based upon these results, we speculate that activin A may be considered as a possible component of inflammatory processes affecting at least the haematopoietic system. PMID:10433499

  10. Adipocyte-derived PAMM suppresses macrophage inflammation by inhibiting MAPK signalling.

    PubMed

    Guo, Fang; He, Hui; Fu, Zhi-Chao; Huang, Shengping; Chen, Tingtao; Papasian, Christopher J; Morse, Leslie R; Xu, Yan; Battaglino, Ricardo A; Yang, Xiao-Feng; Jiang, Zhisheng; Xin, Hong-Bo; Fu, Mingui

    2015-12-15

    Macrophages within adipose tissue play a key role in mediating inflammatory responses in adipose tissue that are associated with obesity-related metabolic complications. In an effort to identify novel proteins secreted from adipocytes that may negatively regulate macrophage inflammation, we found that peroxiredoxin (PRX)-like 2 activated in M-CSF stimulated monocytes (PAMM), a CXXC-type PRX-like 2 domain-containing redox regulatory protein, is a novel secreted protein with potent anti-inflammatory properties. PAMM is secreted from mature human adipocytes but not preadipocytes. Overexpression of PAMM significantly attenuated lipopolysaccharide (LPS)-induced macrophage inflammation. Incubation of macrophages with adipocyte-conditional medium treated with anti-PAMM antibody significantly enhanced LPS-induced interleukin-12 (IL-12) expression in Raw264.7 cells. In addition, incubation of Raw264.7 cells with purified PAMM protein had a similar anti-inflammatory effect. Moreover, forced expression of PAMM in Raw264.7 cells resulted in decreased LPS-induced ERK1/2, p38 and c-Jun N-terminal kinase (JNK) phosphorylation, suggesting that PAMM exerted the anti-inflammatory function probably by suppressing the mitogen-activated protein kinase (MAPK) signalling pathway. Mutations in the CXXC motif of PAMM that suppressed its anti-redox activity were still able to suppress production of inflammatory cytokines in LPS-stimulated macrophages, suggesting that PAMM's anti-inflammatory properties may be independent of its antioxidant properties. Finally, PAMM was highly expressed in both white (WAT) and brown adipose tissues (BAT) and further increased in obesity status. Our results suggest that adipocyte-derived PAMM may suppress macrophage activation by inhibiting MAPK signalling pathway. PMID:26438880

  11. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    SciTech Connect

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  12. Macrophage Migration Inhibitory Factor in Clinical Kidney Disease

    PubMed Central

    Bruchfeld, Annette; Wendt, Mårten; Miller, Edmund J.

    2016-01-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine implicated in acute and chronic inflammatory conditions, including sepsis, autoimmune disease, atherogenesis, plaque instability, and pulmonary arterial hypertension. MIF in plasma and urine is significantly elevated in patients with acute kidney injury (AKI) and elevated MIF in serum is associated with markers of oxidative stress, endothelial dysfunction, arterial stiffness, and markers of myocardial damage in chronic kidney disease (CKD). Furthermore, MIF seems to be involved in vascular processes and cardiovascular disease associated with CKD, glomerulonephritis, autosomal dominant polycystic kidney disease, and possibly also in progression to renal failure. Moreover, in active anti-neutrophil cytoplasmatic antibody-associated vasculitis, plasma MIF levels have been shown to be significantly elevated as compared with samples from patients in remission. A significant difference in the genotype frequency of high production MIF -173 G/C genotype has been found in end-stage renal disease, compared to controls. Inhibition of MIF in a diabetic nephropathy model ameliorated blood glucose and albuminuria and in a model of adult polycystic kidney disease cyst growth was delayed. Preclinical studies support a potential therapeutic role for MIF in AKI and in a number of CKDs, whereas these data in human disease are still observational. Future interventional studies are needed to delineate the role of MIF as a treatment target in clinical kidney disease. PMID:26858715

  13. An integrated signal transduction network of macrophage migration inhibitory factor.

    PubMed

    Subbannayya, Tejaswini; Variar, Prathyaksha; Advani, Jayshree; Nair, Bipin; Shankar, Subramanian; Gowda, Harsha; Saussez, Sven; Chatterjee, Aditi; Prasad, T S Keshava

    2016-06-01

    Macrophage migration inhibitory factor (MIF) is a glycosylated multi-functional protein that acts as an enzyme as well as a cytokine. MIF mediates its actions through a cell surface class II major histocompatibility chaperone, CD74 and co-receptors such as CD44, CXCR2, CXCR4 or CXCR7. MIF has been implicated in the pathogenesis of several acute and chronic inflammatory diseases. Although MIF is a molecule of biomedical importance, a public resource of MIF signaling pathway is currently lacking. In view of this, we carried out detailed data mining and documentation of the signaling events pertaining to MIF from published literature and developed an integrated reaction map of MIF signaling. This resulted in the cataloguing of 68 molecules belonging to MIF signaling pathway, which includes 24 protein-protein interactions, 44 post-translational modifications, 11 protein translocation events and 8 activation/inhibition events. In addition, 65 gene regulation events at the mRNA levels induced by MIF signaling have also been catalogued. This signaling pathway has been integrated into NetPath ( http://www.netpath.org ), a freely available human signaling pathway resource developed previously by our group. The MIF pathway data is freely available online in various community standard data exchange formats. We expect that data on signaling events and a detailed signaling map of MIF will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on MIF. PMID:27139435

  14. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages.

    PubMed Central

    Drapier, J C; Hirling, H; Wietzerbin, J; Kaldy, P; Kühn, L C

    1993-01-01

    Biosynthesis of nitric oxide (NO) from L-arginine modulates activity of iron-dependent enzymes, including mitochondrial acontiase, an [Fe-S] protein. We examined the effect of NO on the activity of iron regulatory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murine macrophages were activated with interferon-gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG-substituted analogues of L-arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria-free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN-gamma and/or LPS stimulation. Authentic NO gas as well as the NO-generating compound 3-morpholinosydnonimine (SIN-1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post-transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe-S] cluster of IRF mediates iron-dependent regulation. Images PMID:7504626

  15. Non-identical twins - microglia and monocyte-derived macrophages in acute injury and autoimmune inflammation.

    PubMed

    Jung, Steffen; Schwartz, Michal

    2012-01-01

    The brain has been commonly regarded as a "tissue behind walls." Appearance of immune cells in the brain has been taken as a sign of pathology. Moreover, since infiltrating monocyte-derived macrophages and activated resident microglia were indistinguishable by conventional means, both populations were considered together as inflammatory cells that should be mitigated. Yet, because the microglia permanently reside in the brain, attributing to them negative properties evoked an ongoing debate; why cells that are supposed to be the brain guardians acquire only destructive potential? Studies over the last two decades in the immune arena in general, and in the context of central nervous system pathology in particular, have resulted in a paradigm shift toward a more balanced appreciation of the contributions of immune cells in the context of brain maintenance and repair, and toward the recognition of distinct roles of resident microglia and infiltrating monocyte-derived macrophages. PMID:22566968

  16. BDMC33, A Curcumin Derivative Suppresses Inflammatory Responses in Macrophage-Like Cellular System: Role of Inhibition in NF-κB and MAPK Signaling Pathways

    PubMed Central

    Lee, Ka-Heng; Chow, Yuh-Lit; Sharmili, Vidyadaran; Abas, Faridah; Alitheen, Noorjahan Banu Mohamed; Shaari, Khozirah; Israf, Daud Ahmad; Lajis, Nordin Haji; Syahida, Ahmad

    2012-01-01

    Our preliminary screening has shown that curcumin derivative BDMC33 [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] exerted promising nitric oxide inhibitory activity in activated macrophages. However, the molecular basis and mechanism for its pharmacological action is yet to be elucidated. The aim of this study was to investigate the anti-inflammatory properties of BDMC33 and elucidate its underlying mechanism action in macrophage cells. Our current study demonstrated that BDMC33 inhibits the secretion of major pro-inflammatory mediators in stimulated macrophages, and includes NO, TNF-α and IL-1β through interference in both nuclear factor kappaB (NF-κB) and mitogen activator protein kinase (MAPK) signaling cascade in IFN-γ/LPS-stimulated macrophages. Moreover, BDMC33 also interrupted LPS signaling through inhibiting the surface expression of CD-14 accessory molecules. In addition, the inhibitory action of BDMC33 not only restricted the macrophages cell (RAW264.7), but also inhibited the secretion of NO and TNF-α in IFN-γ/LPS-challenged microglial cells (BV-2). The experimental data suggests the inflammatory action of BDMC33 on activated macrophage-like cellular systems, which could be used as a future therapeutic agent in the management of chronic inflammatory diseases. PMID:22489138

  17. Choroid plexus macrophages proliferate and release toxic factors in response to feline immunodeficiency virus.

    PubMed

    Bragg, D C; Hudson, L C; Liang, Y H; Tompkins, M B; Fernandes, A; Meeker, R B

    2002-06-01

    Recent observations have suggested that lentiviruses stimulate the proliferation and activation of microglia. A similar effect within the dense macrophage population of the choroid plexus could have significant implications for trafficking of virus and inflammatory cells into the brain. To explore this possibility, we cultured fetal feline macrophages and examined their response to feline immunodeficiency virus (FIV) or the T-cell-derived protein, recombinant human CD40-ligand trimer (rhuCD40-L). The rhCD40-L was the most potent stimulus for macrophage proliferation, often inducing a dramatic increase in macrophage density. Exposure to FIV resulted in a small increase in the number of macrophages and macrophage nuclei labeled with bromodeoxyuridine. The increase in macrophage density after FIV infection also correlated with an increase in neurotoxic activity of the macrophage-conditioned medium. Starting at 16-18 weeks postinfection, well after the peak of viremia, a similar toxic activity was detected in cerebrospinal fluid (CSF) from FIV-infected cats. Toxicity in the CSF increased over time and was paralleled by strong CD18 staining of macrophages/microglia in the choroid plexus and adjacent parenchyma. These results suggest that lentiviral infection of the choroid plexus can induce a toxic inflammatory response that is fueled by local macrophage proliferation. Together with the observation of increasing toxic activity in the CSF and increased CD18 staining in vivo, these observations suggest that choroid plexus macrophages may contribute to an inflammatory cascade in the brain that progresses independently of systemic and CSF viral load. PMID:12053277

  18. Resistance of LPS-activated bone marrow derived macrophages to apoptosis mediated by dexamethasone

    PubMed Central

    Haim, Yasmin Ohana; Unger, Naamit Deshet; Souroujon, Miriam C.; Mittelman, Moshe; Neumann, Drorit

    2014-01-01

    Glucocorticoids (GC) display pleiotropic effects on the immune system. Macrophages are a major target for GC action. Here we show that dexamethasone (DEX), a synthetic GC, decreased viability of naïve bone marrow-derived macrophages (BMDM), involving an apoptotic mechanism. Administration of DEX together with lipopolysaccharide (LPS) protected BMDM against DEX-mediated cell death, suggesting that activated BMDM respond to DEX differently than naïve BMDM. An insight to the molecular basis of LPS actions was provided by a 7 fold increase in mRNA levels of glucocorticoid receptor beta (GRβ), a GR dominant-negative splice variant which inhibits GRα's transcriptional activity. LPS did not inhibit all DEX-mediated effects on BMDM; DEX significantly reduced the percentage of BMDM expressing high levels of the cell surface markers F4/80 and CD11b and led to a decrease in macrophage inflammatory protein 1 alpha (MIP1-α) mRNA and protein levels. These two DEX-mediated effects were not prevented by LPS. Our finding that LPS did not reduce the DEX-induced elevation of glucocorticoid-induced leucine zipper (GILZ), a mediator of GCs anti-inflammatory actions, may provide an underlying mechanism. These findings enable a better understanding of clinical states, such as sepsis, in which macrophages are activated by endotoxins and treatment by GCs is considered. PMID:24608810

  19. Macrophage-derived reactive oxygen species protects against autoimmune priming with a defined polymeric adjuvant.

    PubMed

    Shakya, Akhilesh Kumar; Kumar, Ashok; Holmdahl, Rikard; Nandakumar, Kutty Selva

    2016-01-01

    Understanding the nature of adjuvants and the immune priming events in autoimmune diseases, such as rheumatoid arthritis, is a key challenge to identify their aetiology. Adjuvants are, however, complex structures with inflammatory and immune priming properties. Synthetic polymers provide a possibility to separate these functions and allow studies of the priming mechanisms in vivo. A well-balanced polymer, poly-N-isopropyl acrylamide (PNiPAAm) mixed with collagen type II (CII) induced relatively stronger autoimmunity and arthritis compared with more hydrophilic (polyacrylamide) or hydrophobic (poly-N-isopropylacrylamide-co-poly-N-tertbutylacrylamide and poly-N-tertbutylacrylamide) polymers. Clearly, all the synthesized polymers except the more hydrophobic poly-N-tertbutylacrylamide induced arthritis, especially in Ncf1-deficient mice, which are deficient in reactive oxygen species (ROS) production. We identified macrophages as the major infiltrating cells present at PNiPAAm-CII injection sites and demonstrate that ROS produced by the macrophages attenuated the immune response and the development of arthritis. Our results reveal that thermo-responsive polymers with high immune priming capacity could trigger an autoimmune response to CII and the subsequent arthritis development, in particular in the absence of NOX2 derived ROS. Importantly, ROS from macrophages protected against the autoimmune priming, demonstrating a critical regulatory role of macrophages in immune priming events. PMID:26455429

  20. Macrophage-secreted factors impair human adipogenesis: involvement of proinflammatory state in preadipocytes.

    PubMed

    Lacasa, Danièle; Taleb, Soraya; Keophiphath, Mayoura; Miranville, Alexandra; Clement, Karine

    2007-02-01

    Obesity is considered a chronic low-grade inflammatory state. The white adipose tissue produces a variety of inflammation-related proteins whose expression is increased in obese subjects. The nonadipose cell fraction, which includes infiltrated macrophages, is a determinant source of inflammation-related molecules within the adipose tissue. Our working hypothesis is that macrophage infiltration affects fat expansion through a paracrine action on adipocyte differentiation. Human primary preadipocytes were then differentiated in the presence of conditioned media obtained from macrophages differentiated from blood monocytes. Preadipocytes treated by macrophage-conditioned medium displayed marked reduction of adipogenesis as assessed by decreased cellular lipid accumulation and reduced gene expression of adipogenic and lipogenic markers. In addition to this effect, the activation of macrophages by lipopolysaccharides stimulated nuclear factor kappaB signaling, increased gene expression and release of proinflammatory cytokines and chemokines, and induced preadipocyte proliferation. This phenomenon was associated with increased cyclin D1 gene expression and maintenance of the fibronectin-rich matrix. Anti-TNFalpha neutralizing antibody inhibits the inflammatory state of preadipocytes positioning TNFalpha as an important mediator of inflammation in preadipocytes. Strikingly, conditioned media produced by macrophages isolated from human adipose tissue exerted comparable effects with activated macrophages, i.e. decreased adipogenesis and increased inflammatory state in the preadipocytes. These data show that macrophage-secreted factors inhibit the formation of mature adipocytes, suggesting possible role in limiting adipose tissue expansion in humans. PMID:17082259

  1. Glycyrrhizic Acid Promotes M1 Macrophage Polarization in Murine Bone Marrow-Derived Macrophages Associated with the Activation of JNK and NF-κB

    PubMed Central

    Mao, Yulong; Wang, Baikui; Xu, Xin; Du, Wei; Li, Weifen; Wang, Youming

    2015-01-01

    The roots and rhizomes of Glycyrrhiza species (licorice) have been widely used as natural sweeteners and herbal medicines. The aim of this study is to investigate the effect of glycyrrhizic acid (GA) from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GA were assessed. Our results showed that GA obviously increased the cell surface expression of CD80, CD86, and MHCII molecules. Meanwhile, GA upregulated the expression of CCR7 and the production of TNF-α, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Ym1, and Arg1 (the markers of alternatively activated (M2) macrophage). The functional tests showed that GA dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation are required for GA-induced NO and M1-related cytokines production, while ERK1/2 pathway exhibits a regulatory effect via induction of IL-10. Together, these findings indicated that GA promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GA in macrophage polarization. PMID:26664149

  2. A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)*

    PubMed Central

    Bai, Fengwei; Asojo, Oluwatoyin A.; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A.; Leng, Lin; Koski, Raymond A.; Powell, Thomas J.; Bucala, Richard; Anthony, Karen G.

    2012-01-01

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases. PMID:22782901

  3. Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

    PubMed Central

    Mawhinney, Leona; Armstrong, Michelle E; O’ Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2014-01-01

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems. PMID:25826675

  4. Macrophage migration inhibitory factor, a role in COPD.

    PubMed

    Husebø, Gunnar R; Bakke, Per S; Grønseth, Rune; Hardie, Jon A; Ueland, Thor; Aukrust, Pål; Eagan, Tomas M L

    2016-07-01

    Macrophage migration inhibitor factor (MIF) is a pluripotent cytokine associated with several different inflammatory conditions, but its role within lung inflammation and chronic obstructive pulmonary disease (COPD) is unclear. This study aimed to examine MIF in both stable COPD and during acute exacerbations (AECOPD). The study included 433 patients with COPD aged 41-76 and 325 individuals from the Bergen COPD cohort study who served as controls. All patients had an FEV1 of <80% predicted, FEV1/FVC ratio of <0.7, and a smoking history >10 pack-years. Serum levels of MIF were compared between the two groups at baseline, and for 149 patients, measurements were also carried out during AECOPD. Linear regression models were fitted with MIF as the outcome variable and adjusted for sex, age, body composition, smoking, and Charlson Comorbidity Score (CCS). Median MIF (interquartile range) in patients with COPD was 20.1 ng/ml (13.5-30.9) compared with 14.9 ng/ml (11.1-21.6) in controls (P < 0.01). MIF was bivariately associated with sex, body composition, and CCS (P < 0.05 for all). In the regression analyses, MIF was significantly higher in patients with COPD, coefficient 1.32 (P < 0.01) and 1.30 (P < 0.01) unadjusted and adjusted, respectively. In addition, in 149 patients during episodes of AECOPD, MIF was significantly elevated, with a median of 23.2 ng/ml (14.1-42.3) compared with measurements at stable disease of 19.3 ng/ml (12.4-31.3, P < 0.01). Serum levels of MIF were significantly higher in patients with COPD compared with controls. We also identified an additional increase in MIF levels during episodes of AECOPD. PMID:27190066

  5. The Kmif (Kveim-induced macrophage migration inhibition factor) test in sarcoidosis

    PubMed Central

    Williams, W. Jones; Pioli, E.; Jones, D. J.; Dighero, M.

    1972-01-01

    Circulating lymphocytes from 30 patients with sarcoidosis when stimulated in vitro with Kveim-induced macrophage migration factor, the Kmif test, produced a guinea-pig macrophage migration inhibition factor in 21 of 30 cases (70%). In those patients not on steroids the results showed a good correlation with the cutaneous Kveim test. One positive test was found in 16 normal subjects. Our results suggest that the Kmif test may prove a useful rapid alternative to the Kveim test. PMID:4675181

  6. Interferon-alpha inhibits murine macrophage transforming growth factor-beta mRNA expression.

    PubMed

    Dhanani, S; Huang, M; Wang, J; Dubinett, S M

    1994-06-01

    Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide is produced by a wide variety of cells and regulates a broad array of physiological and pathological functions. TGF-beta appears to play a central role in pulmonary fibrosis and may contribute to tumor-associated immunosuppression. Alveolar macrophages are a rich source of TGF-beta and are intimately involved in lung inflammation. We therefore chose to study TGF-beta regulation in murine alveolar macrophages as well as an immortalized peritoneal macrophage cell line (IC-21). Murine macrophages were incubated with cytokines to evaluate their role in regulating TGF-beta mRNA expression. We conclude that IFN-alpha downregulates TGF-beta mRNA expression in murine macrophages. PMID:8088926

  7. Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression.

    PubMed

    Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

    2016-08-01

    Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP‑1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP‑1 cells were differentiated to macrophages by phorbol 12‑myristate 13‑acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon‑γ (IFN‑γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription‑quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme‑linked immunosorbent assay. IRF5 protein and nuclei co‑localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN‑γ stimulation‑induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels. PMID:27277156

  8. Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression

    PubMed Central

    Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

    2016-01-01

    Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP-1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP-1 cells were differentiated to macrophages by phorbol 12-myristate 13-acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription-quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme-linked immunosorbent assay. IRF5 protein and nuclei co-localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN-γ stimulation-induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels. PMID:27277156

  9. Identifying panaxynol, a natural activator of nuclear factor erythroid-2 related factor 2 (Nrf2) from American ginseng as a suppressor of inflamed macrophage-induced cardiomyocyte hypertrophy

    PubMed Central

    Qu, Chen; Li, Bin; Lai, Yimu; Li, Hechu; Windust, Anthony; Hofseth, Lorne J.; Nagarkatti, Mitzi; Nagarkatti, Prakash; Wang, Xing Li; Tang, Dongqi; Janicki, Joseph S.; Tian, Xingsong; Cui, Taixing

    2015-01-01

    Ethnopharmacological relevance American ginseng is capable of ameliorating cardiac dysfunction and activating Nrf2, a master regulator of antioxidant defense, in the heart. This study was designed to isolate compounds from American ginseng and to determine those responsible for the Nrf2-mediated resolution of inflamed macrophage-induced cardiomyocyte hypertrophy. Materials and methods A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. A bioassay-based fractionization of American ginseng was performed to identify the putative substances which could activate Nrf2-mediated suppression of pro-inflammatory cytokine expression in macrophages and macrophage-mediated pro-hypertrophic growth in cardiomyocytes. Results A hexane fraction of an anti-inflammatory crude extract of American ginseng was found to be most effective in suppressing the inflammatory responses in macrophages. Preparative, reverse-phase HPLC and a comparative analysis by analytical scale LC–UV/MS revealed the hexane fraction contains predominantly C17 polyacetylenes and linolenic acid. Panaxynol, one of the major polyacetylenes, was found to be a potent Nrf2 activator. Panaxynol posttranscriptionally activated Nrf2 by inhibiting Kelch-like ECH-associated protein (Keap) 1-mediated degradation without affecting the binding of Keap1 and Nrf2. Moreover, panaxynol suppressed a selected set of cytokine expression via the activation of Nrf2 while minimally regulating nuclear factor-kappa B (NF-κB)-mediated cytokine expression in macrophages. It also dramatically inhibited the inflamed macrophage-mediated cardiomyocyte death and hypertrophy by activating Nrf2 in macrophages. Conclusions These results demonstrate that American ginseng-derived panaxynol is a specific Nrf2 activator and panaxynol-activated Nrf2 signaling is at least partly responsible for American ginseng-induced health benefit in the heart. PMID

  10. [The modulation of low-level laser on polarization of mouse bone marrow-derived macrophages].

    PubMed

    Dai, Chen; Song, Jiwei; Liang, Zhuowen; Zhang, Qian; Zhang, Kun; Wang, Zhe; Hu, Xueyu

    2016-08-01

    Objective To investigate the influence of 810 nm low-level laser of different energy on the polarization of macrophages. Methods The macrophages were isolated from the bone borrow of BALB/c mice and cultured in macrophage colony stimulating factor (M-CSF) conditioned cultural medium. The expression of F4/80 was examined by flow cytometry for identification. After lipopolysaccharide-γ interferon (LPS-IFN-γ) induced polarization status in the macrophages, the mRNA expressions of inducible nitric oxide synthase (iNOS), arginase 1 (Arg1) and CD86 were detected by reverse transcription PCR, and the protein expressions of iNOS and Arg1 were tested by Western blotting. Thereafter, the M1 macrophages were exposed to 810 nm low-level laser of (1, 2, 3, 4) J/cm(2), and then the cell viability was evaluated by MTT assay; the expressions of iNOS and Arg1 were observed by immunofluorescent cytochemical staining; the mRNA and protein levels of iNOS and Arg1 were studied by reverse transcription PCR and Western blotting. Results Flow cytometry showed that the percentage of F4/80 positive cells cultured with M-CSF conditioned medium was 99.9%. The mRNA and protein levels of iNOS and CD86 in macrophages were both significantly raised after induction by LPS-IFN-γ. Compared with the control cells, the viability of M1 cells significantly decreased when the energy of the low-level laser exposure was 4 J/cm(2), while the viability remained unchanged when the energy was 1, 2 or 3 J/cm(2). Immunocytochemistry revealed that the percentage of Arg1 positive cells that represent M2 macrophages was not significantly different from the control group when the irradiation dose was 1 or 2 J/cm(2), however, the Arg1 positive cells significantly increased and the iNOS positive cells that represent M1 macrophages significantly decreased when the irradiation dose was 3 or 4 J/cm(2). When the irradiation dose was 1 or 2 J/cm(2), the mRNA and protein levels of iNOS and Arg1 remained unchanged

  11. Human alveolar macrophages synthesize factor VII in vitro. Possible role in interstitial lung disease.

    PubMed Central

    Chapman, H A; Allen, C L; Stone, O L; Fair, D S

    1985-01-01

    Both fibrin and tissue macrophages are prominent in the histopathology of chronic inflammatory pulmonary disease. We therefore examined the procoagulant activity of freshly lavaged human alveolar macrophages in vitro. Intact macrophages (5 X 10(5) cells) from 13 healthy volunteers promoted clotting of whole plasma in a mean of 65 s. Macrophage procoagulant activity was at least partially independent of exogenous Factor VII as judged by a mean clotting time of 99 s in Factor VII-deficient plasma and by neutralization of procoagulant activity by an antibody to Factor VII. Immunoprecipitation of extracts of macrophages metabolically labeled with [35S]methionine by Factor VII antibody and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a labeled protein consistent in size with the known molecular weight of blood Factor VII, 48,000. The addition of 50 micrograms of unlabeled, purified Factor VII blocked recovery of the 48,000-mol wt protein. In addition, supernatants of cultured macrophages from six normal volunteers had Factor X-activating activity that was suppressed an average of 71% after culture in the presence of 50 microM coumadin or entirely by the Factor VII antibody indicating that Factor VII synthesized by the cell was biologically active. Endotoxin in vitro induced increases in cellular tissue factor but had no consistent effect on macrophage Factor VII activity. We also examined the tissue factor and Factor VII activities of freshly lavaged alveolar cells from nine subjects with clinical and/or histologic evidence of sarcoidosis. Four of the nine subjects expressed increased tissue factor and seven of nine had increased Factor VII activity over the normal range (P less than 0.01). We estimate the mean Factor VII associated with the cells of sarcoid patients to be 4.7 ng/10(6) cells (range 0.4-20) as compared to a mean of 0.74 ng/10(6) cells (range 0.2-2) for that of normal subjects. Along with previous data showing synthesis

  12. Pharmacologic reduction in tumor necrosis factor activity of pulmonary alveolar macrophages.

    PubMed

    Leeper-Woodford, S K; Fisher, B J; Sugerman, H J; Fowler, A A

    1993-02-01

    Tumor necrosis factor-alpha (TNF), an inflammatory cytokine released by macrophages, may be a mediator of lung injury during septicemia. We previously reported that the cyclooxygenase inhibitor ibuprofen and histamine receptor antagonists cimetidine (H2 antagonist) and diphenhydramine (H1 antagonist) attenuate lung injury and reduce circulating TNF surges during porcine sepsis. Since pulmonary alveolar macrophages (PAM) may participate in early sepsis by producing TNF, we hypothesized that the TNF activity of PAM is reduced by ibuprofen, cimetidine, and diphenhydramine. To test this, we examined changes in PAM-derived TNF bioactivity and cell viability of freshly isolated porcine PAM during exposure to bacterial endotoxin (LPS), ibuprofen, cimetidine, and diphenhydramine. The TNF activity (% L929 cytotoxicity of PAM conditioned medium) was elevated in LPS-stimulated PAM cultures (15 to 25% increase at 1 to 6 h and 40 to 43% increase at 6 to 48 h, compared with non-LPS-stimulated cultures), and ibuprofen (150 micrograms/ml) added with LPS decreased the TNF activity for 24 h (20 to 28% reduction at 1 to 24 h). Ibuprofen added 1 h after LPS was less effective in reducing the PAM-derived TNF activity (20 to 22% reduction at 2 to 6 h). Cimetidine (112 micrograms/ml) reduced the TNF activity of LPS-stimulated PAM cultures during the first 4 h of LPS exposure (15 to 24% decrease at 1 to 4 h). Diphenhydramine (150 micrograms/ml) attenuated the PAM-derived TNF activity but also decreased viability of PAM, indicating a toxic effect of this agent on PAM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8093999

  13. Transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages

    PubMed Central

    Gibbings, Sophie L.; Goyal, Rajni; Desch, A. Nicole; Leach, Sonia M.; Prabagar, Miglena; Atif, Shaikh M.; Bratton, Donna L.; Janssen, William

    2015-01-01

    Alveolar macrophages (AMs) reside on the luminal surfaces of the airways and alveoli where they maintain host defense and promote alveolar homeostasis by ingesting inhaled particulates and regulating inflammatory responses. Recent studies have demonstrated that AMs populate the lungs during embryogenesis and self-renew throughout life with minimal replacement by circulating monocytes, except under extreme conditions of depletion or radiation injury. Here we demonstrate that on a global scale, environment appears to dictate AM development and function. Indeed, transcriptome analysis of embryonic host-derived and postnatal donor-derived AMs coexisting within the same mouse demonstrated >98% correlation and overall functional analyses were similar. However, we also identified several genes whose expression was dictated by origin rather than environment. The most differentially expressed gene not altered by environment was Marco, a gene recently demonstrated to have enhancer activity in embryonic-derived but not postnatal-derived tissue macrophages. Overall, we show that under homeostatic conditions, the environment largely dictates the programming and function of AMs, whereas the expression of a small number of genes remains linked to the origin of the cell. PMID:26232173

  14. Functional Relationship between Tumor-Associated Macrophages and Macrophage Colony-Stimulating Factor as Contributors to Cancer Progression

    PubMed Central

    Laoui, Damya; Van Overmeire, Eva; De Baetselier, Patrick; Van Ginderachter, Jo A.; Raes, Geert

    2014-01-01

    The current review article describes the functional relationship between tumor-associated macrophages (TAM) as key cellular contributors to cancer malignancy on the one hand and macrophage-colony-stimulating factor (M-CSF or CSF-1) as an important molecular contributor on the other. We recapitulate the available data on expression of M-CSF and the M-CSF receptor (M-CSFR) in human tumor tissue as constituents of a stromal macrophage signature and on the limits of the predictive and prognostic value of plasma M-CSF levels. After providing an update on current insights into the nature of TAM heterogeneity at the level of M1/M2 phenotype and TAM subsets, we give an overview of experimental evidence, based on genetic, antibody-mediated, and pharmacological disruption of M-CSF/M-CSFR signaling, for the extent to which M-CSFR signaling can not only determine the TAM quantity, but can also contribute to shaping the phenotype and heterogeneity of TAM and other related tumor-infiltrating myeloid cells (TIM). Finally, we review the accumulating information on the – sometimes conflicting – effects blocking M-CSFR signaling may have on various aspects of cancer progression such as tumor growth, invasion, angiogenesis, metastasis, and resistance to therapy and we thereby discuss in how far these different effects actually reflect a contribution of TAM. PMID:25339957

  15. Macrophage activation by factors released from acetaminophen-injured hepatocytes: Potential role of HMGB1

    SciTech Connect

    Dragomir, Ana-Cristina; Laskin, Jeffrey D.; Laskin, Debra L.

    2011-06-15

    Toxic doses of acetaminophen (AA) cause hepatocellular necrosis. Evidence suggests that activated macrophages contribute to the pathogenic process; however, the factors that activate these cells are unknown. In these studies, we assessed the role of mediators released from AA-injured hepatocytes in macrophage activation. Treatment of macrophages with conditioned medium (CM) collected 24 hr after treatment of mouse hepatocytes with 5 mM AA (CM-AA) resulted in increased production of reactive oxygen species (ROS). Macrophage expression of heme oxygenase-1 (HO-1) and catalase mRNA was also upregulated by CM-AA, as well as cyclooxygenase (COX)-2 and 12/15-lipoxygenase (LOX). CM-AA also upregulated expression of the proinflammatory chemokines, MIP-1{alpha} and MIP-2. The effects of CM-AA on expression of COX-2, MIP-1{alpha} and MIP-2 were inhibited by blockade of p44/42 MAP kinase, suggesting a biochemical mechanism mediating macrophage activation. Hepatocytes injured by AA were found to release HMGB1, a potent macrophage activator. This was inhibited by pretreatment of hepatocytes with ethyl pyruvate (EP), which blocks HMGB1 release. EP also blocked CM-AA induced ROS production and antioxidant expression, and reduced expression of COX-2, but not MIP-1{alpha} or MIP-2. These findings suggest that HMGB1 released by AA-injured hepatocytes contributes to macrophage activation. This is supported by our observation that expression of the HMGB1 receptor RAGE is upregulated in macrophages in response to CM-AA. These data indicate that AA-injured hepatocytes contribute to the inflammatory environment in the liver through the release of mediators such as HMGB1. Blocking HMGB1/RAGE may be a useful approach to limiting classical macrophage activation and AA-induced hepatotoxicity. - Research Highlights: > These studies analyze macrophage activation by mediators released from acetaminophen-damaged hepatocytes. > Factors released from acetaminophen-injured hepatocytes induce

  16. Hyperbaric oxygen enhances neutrophil apoptosis and their clearance by monocyte-derived macrophages.

    PubMed

    Almzaiel, Anwar J; Billington, Richard; Smerdon, Gary; Moody, A John

    2015-08-01

    Neutrophil apoptosis and clearance by macrophages are essential for wound healing. Evidence suggests that hyperbaric oxygen (HBO) exposure may enhance neutrophil apoptosis, but HBO effects leading to neutrophil clearance by macrophages are still unclear. In the current study, bovine neutrophils and monocyte-derived macrophages (MDMΦ) were co-cultured under HBO (97.9% O2, 2.1% CO2 at 2.4 atm absolute (ATA)) (1 atm = 101.325 kPa), hyperbaric normoxia (8.8% O2 at 2.4 ATA), normobaric hyperoxia (95% O2, 5% CO2), normoxia (air), and normobaric hypoxia (5% O2, 5% CO2). Phagocytosis of fresh and 22 h aged neutrophils by MDMΦ was increased after HBO pre-treatment, assessed using flow cytometry and light microscopy. Enhanced clearance of neutrophils was accompanied by an increase in H2O2 levels following HBO pre-treatment with upregulation of IL-10 (anti-inflammatory cytokine) mRNA expression in LPS-stimulated MDMΦ that had ingested aged neutrophils. TNF-α (pro-inflammatory cytokine) gene expression did not change in LPS-stimulated MDMΦ that had ingested fresh or aged neutrophils after HBO, pressure, and hyperoxia. These findings suggest that HBO-activated MDMΦ participate in the clearance of apoptotic cells. Uptake of neutrophils by MDMΦ exposed to HBO may contribute to resolution of inflammation, because HBO induced up-regulation of IL-10 mRNA expression. PMID:26194051

  17. Peroxynitrite, a potent macrophage-derived oxidizing cytotoxin to combat invading pathogens

    PubMed Central

    Prolo, Carolina; Álvarez, María Noel; Radi, Rafael

    2013-01-01

    Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill microorganisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage-derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens. PMID:24281946

  18. Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages

    PubMed Central

    Hollenbaugh, Joseph A.; Montero, Catherine; Schinazi, Raymond F.; Munger, Joshua; Kim, Baek

    2016-01-01

    We evaluated cellular metabolism profiles of HIV-1 and HIV-2 infected primary human monocyte-derived macrophages (MDMs). First, HIV-2 GL-AN displays faster production kinetics and greater amounts of virus as compared to HIV-1s: YU-2, 89.6 and JR-CSF. Second, quantitative LC–MS/MS metabolomics analysis demonstrates very similar metabolic profiles in glycolysis and TCA cycle metabolic intermediates between HIV-1 and HIV-2 infected macrophages, with a few notable exceptions. The most striking metabolic change in MDMs infected with HIV-2 relative to HIV-1-infected MDMs was the increased levels of quinolinate, a metabolite in the tryptophan catabolism pathway that has been linked to HIV/AIDS pathogenesis. Third, both HIV-1 and HIV-2 infected MDMs showed elevated levels of ribose-5-phosphate, a key metabolic component in nucleotide biosynthesis. Finally, HIV-2 infected MDMs display increased dNTP concentrations as predicted by Vpx-mediated SAMHD1 degradation. Collectively, these data show differential metabolic changes during HIV-1 and HIV-2 infection of macrophages. PMID:26895248

  19. Macrophage-derived lipid agonists of PPAR-α as intrinsic controllers of inflammation.

    PubMed

    Pontis, Silvia; Ribeiro, Alison; Sasso, Oscar; Piomelli, Daniele

    2016-01-01

    Macrophages are multi-faceted phagocytic effector cells that derive from circulating monocytes and undergo differentiation in target tissues to regulate key aspects of the inflammatory process. Macrophages produce and degrade a variety of lipid mediators that stimulate or suppress pain and inflammation. Among the analgesic and anti-inflammatory lipids released from these cells are the fatty acid ethanolamides (FAEs), which produce their effects by engaging nuclear peroxisome proliferator activated receptor-α (PPAR-α). Two members of this lipid family, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), have recently emerged as important intrinsic regulators of nociception and inflammation. These substances are released from the membrane precursor, N-acylphosphatidylethanolamine (NAPE), by the action of a NAPE-specific phospholipase D (NAPE-PLD), and in macrophage are primarily deactivated by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). NAPE-PLD and NAAA regulate FAE levels, exerting a tight control over the ability of these lipid mediators to recruit PPAR-α and attenuate the inflammatory response. This review summarizes recent findings on the contribution of the FAE-PPAR-α signaling complex in inflammation, and on NAAA inhibition as a novel mechanistic approach to treat chronic inflammatory disorders. PMID:26585314

  20. Vascular endothelial growth factor promotes macrophage apoptosis through stimulation of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT).

    PubMed

    Petreaca, Melissa L; Yao, Min; Ware, Carl; Martins-Green, Manuela M

    2008-01-01

    Resolution of inflammation is critical for normal wound healing. Inflammation is prolonged and fails to resolve properly in chronic wounds. We used in vivo and in vitro approaches to show that vascular endothelial growth factor (VEGF) induces macrophage apoptosis and to delineate mechanisms involved in this process. VEGF inhibition during wound healing leads to an increased number of macrophages remaining in wounds, suggesting the involvement of VEGF in removal of these cells from the wound. If this effect has physiological relevance, it likely occurs via apoptosis. We show that VEGF increases apoptosis of macrophages in vitro using Annexin V-FITC staining and caspase activation. Microarray analysis, reverse transcription-polymerase chain reaction, and immunoblotting showed that VEGF increases the expression of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) in macrophages. We also show that in macrophages LIGHT promotes apoptosis through the lymphotoxin beta receptor. Moreover, inhibition of LIGHT prevents VEGF-induced death, suggesting that LIGHT mediates VEGF-induced macrophage apoptosis. Taken together, our results identify a novel role for VEGF and for LIGHT in macrophage apoptosis during wound healing, an event critical in the resolution of inflammation. This finding may lead to the development of new strategies to improve resolution of inflammation in problematic wounds. PMID:19128255

  1. Protective role of macrophage-derived ceruloplasmin in inflammatory bowel disease

    PubMed Central

    Bakhautdin, Bakytzhan; Febbraio, Maria; Goksoy, Esen; de la Motte, Carol A; Gulen, Muhammet F; Childers, Erin Patricia; Hazen, Stanley L; Li, Xiaoxia; Fox, Paul L

    2013-01-01

    Objective Intestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase with bactericidal activities. The goal is to understand the role of ceruloplasmin in colitis progression in a genetically modified murine model. Design Experimental colitis was induced in ceruloplasmin null (Cp−/−) and wild-type (WT) mice by dextran sulphate sodium administration. The role of ceruloplasmin was further evaluated by transplantation of WT macrophages into Cp−/− mice. Results Cp−/− mice rapidly lost weight and were moribund by day 14, while WT mice survived at least 30 days. Colon culture supernatants from Cp−/− mice exhibited elevated levels of TNFα, KC and MCP-1, indicative of increased inflammation and neutrophil and macrophage infiltration. Elevated leucocytes and severe histopathology were observed in Cp−/− mice. Elevated protein carbonyl content was detected in colons from Cp−/− mice suggesting ceruloplasmin antioxidant activity might contribute to its protective function. Unexpectedly, intraperitoneal administration of human ceruloplasmin into Cp−/− mice did not afford protection. Bone marrow transplantation from WT mice or injection of isolated peripheral blood monocytes markedly reduced severity of colitis and morbidity in Cp−/− mice. Conclusion Macrophage-derived ceruloplasmin contributes importantly to protection against inflammation and tissue injury in acute and chronic experimental colitis. The findings suggest that defects in ceruloplasmin expression or processing may influence the onset or progression of inflammatory bowel disease in patients. PMID:22345661

  2. Macrophage migration inhibitory factor drives neutrophil accumulation by facilitating IL-1β production in a murine model of acute gout.

    PubMed

    Galvão, Izabela; Dias, Ana Carolina Fialho; Tavares, Livia Duarte; Rodrigues, Irla Paula Stopa; Queiroz-Junior, Celso Martins; Costa, Vivian Vasconcelos; Reis, Alesandra Corte; Ribeiro Oliveira, Rene Donizeti; Louzada-Junior, Paulo; Souza, Daniele Glória; Leng, Lin; Bucala, Richard; Sousa, Lirlândia Pires; Bozza, Marcelo Torres; Teixeira, Mauro Martins; Amaral, Flávio Almeida

    2016-06-01

    This study evaluated the role of macrophage migration inhibitory factor in inflammation caused by monosodium urate crystals. The concentration of macrophage migration inhibitory factor was increased in synovial fluid of patients with acute gout, and there was a positive correlation between intra-articular macrophage migration inhibitory factor and IL-1β concentrations. In mice, the injection of monosodium urate crystals into the knee joint increased the levels of macrophage migration inhibitory factor in macrophages and in inflamed tissue. The injection of recombinant macrophage migration inhibitory factor into the joint of mice reproduced the inflammatory response observed in acute gout, including histologic changes, the recruitment of neutrophils, and increased levels of IL-1β and CXCL1. Importantly, the accumulation of neutrophils and the amount IL-1β in the joints were reduced in macrophage migration inhibitory factor-deficient mice when injected with monosodium urate crystals. We observed a similar effect when we blocked macrophage migration inhibitory factor with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid or anti-macrophage migration inhibitory factor. In addition, the blockade of IL-1R and CXCR2 reduced recombinant macrophage migration inhibitory factor-induced neutrophil recruitment. Mechanistically, recombinant macrophage migration inhibitory factor is important for the synthesis of il1β mRNA in vivo and in isolated macrophages. Altogether, macrophage migration inhibitory factor promotes neutrophil accumulation and is important for IL-1β production, which are 2 crucial events contributing to the pathogenesis of acute gout. PMID:26868525

  3. Mononuclear phagocyte accumulates a stearic acid derivative during differentiation into macrophages. Effects of stearic acid on macrophage differentiation and Mycobacterium tuberculosis control.

    PubMed

    Mosquera-Restrepo, Sergio Fabián; Caro, Ana Cecilia; Peláez-Jaramillo, Carlos Alberto; Rojas, Mauricio

    2016-05-01

    The fatty acid composition of monocytes changes substantially during differentiation into macrophages, increasing the proportion of saturated fatty acids. These changes prompted us to investigate whether fatty acid accumulation in the extracellular milieu could affect the differentiation of bystander mononuclear phagocytes. An esterified fatty acid derivative, stearate, was the only fatty acid that significantly increased in macrophage supernatants, and there were higher levels when cells differentiated in the presence of Mycobacterium tuberculosis H37Rv or purified protein derivative (PPD). Exogenous stearic acid enhanced the expression of HLA-DR and CD64; there was also accumulation of IL-12, TNF-α, IL-6, MIP-1 α and β and a reduction in MCP-1 and the bacterial load. These results suggested that during differentiation, a derivative of stearic acid, which promotes the process as well as the effector mechanisms of phagocytes against the mycobacterium, accumulates in the cell supernatants. PMID:26932544

  4. Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis.

    PubMed

    Gambelli, Federica; Di, Peter; Niu, Xiaomei; Friedman, Mitchell; Hammond, Timothy; Riches, David W H; Ortiz, Luis A

    2004-01-16

    Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure. PMID:14570868

  5. Functional significance of macrophage-derived exosomes in inflammation and pain

    PubMed Central

    McDonald, Marguerite K.; Tian, Yuzhen; Qureshi, Rehman A.; Gormley, Michael; Ertel, Adam; Gao, Ruby; Lopez, Enrique Aradillas; Alexander, Guillermo M.; Sacan, Ahmet; Fortina, Paolo; Ajit, Seena K.

    2014-01-01

    Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary depending on the source and the physiological conditions of cells and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome-mediated information transfer in vitro, in a rodent model of inflammatory pain and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides (LPS) secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from LPS-stimulated cells were sufficient to cause NF-kappaB activation in naïve cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a mouse model of inflammatory pain, suggesting an immunoprotective role for macrophage-derived exosomes. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. Macrophage-derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain and should be explored for their therapeutic utility. PMID:24792623

  6. Effect of size of man-made and natural mineral fibers on chemiluminescent response in human monocyte-derived macrophages.

    PubMed Central

    Ohyama, M; Otake, T; Morinaga, K

    2001-01-01

    Fiber size is an important factor in the tumorigenicity of various mineral fibers and asbestos fibers in animal experiments. We examined the time course of the ability to induce lucigenin-dependent chemiluminescence (CL) from human monocyte-derived macrophages exposed to Japan Fibrous Material standard reference samples (glass wool, rock wool, micro glass fiber, two types of refractory ceramic fiber, refractory mullite fiber, potassium titanium whisker, silicon carbide whisker, titanium oxide whisker, and wollastonite). We determined how fiber length or width might modify the response of cells. We found that the patterns of time-dependent increase of CL (sigmoid type) were similar for each sample except wollastonite. We observed a strong correlation between geometric-mean length and ability to induce CL in seven samples > 6 microm in length over the time course (largest r(2) = 0.9760). Although we also observed a close positive correlation between geometric-mean width and the ability to induce CL in eight samples < 1.8 microm in width at 15 min (r(2) = 0.8760), a sample of 2.4 microm in width had a low ability to induce CL. Moreover, the relationship between width and the rate of increase in ability to induce CL had a negative correlation at 30-60 min (largest r(2) = 0.7473). Our findings suggest that the release of superoxide from macrophages occurs nonspecifically for various types of mineral fibers depending on fiber length. PMID:11675268

  7. The major plant-derived cannabinoid Δ(9)-tetrahydrocannabinol promotes hypertrophy and macrophage infiltration in adipose tissue.

    PubMed

    Wong, A; Gunasekaran, N; Hancock, D P; Denyer, G S; Meng, L; Radford, J L; McGregor, I S; Arnold, J C

    2012-02-01

    Synthetic cannabinoid receptor agonists activate lipoprotein lipase and the formation of lipid droplets in cultured adipocytes. Here we extend this work by examining whether Δ(9)-tetrahydrocannabinol (THC), a major plant-derived cannabinoid, increases adipocyte size in vivo. Further, possibly as a consequence of hypertrophy, we hypothesize that THC exposure promotes macrophage infiltration into adipose tissue, an inflammatory state observed in obese individuals. Rats repeatedly exposed to THC in vivo had reduced body weight, fat pad weight, and ingested less food over the drug injection period. However, THC promoted adipocyte hypertrophy that was accompanied by a significant increase in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) expression, an enzyme important in packaging triglycerides. We also showed that THC induced macrophage infiltration and increased expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) in adipose tissue but did not induce apoptosis as measured by TUNEL staining. That THC increased adipocyte cell size in the absence of greater food intake, body weight and fat provides a unique model to explore mechanisms underlying changes in adipocyte size associated with a mild inflammatory state in fat tissue. PMID:22189757

  8. The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages

    PubMed Central

    2014-01-01

    Background Immune system dysregulation is well-recognized in autism and thought to be part of the etiology of this disorder. The endocannabinoid system is a key regulator of the immune system via the cannabinoid receptor type 2 (CB2R) which is highly expressed on macrophages and microglial cells. We have previously published significant differences in peripheral blood mononuclear cell CB2R gene expression in the autism population. The use of the Gc protein-derived Macrophage Activating Factor (GcMAF), an endogenous glycosylated vitamin D binding protein responsible for macrophage cell activation has demonstrated positive effects in the treatment of autistic children. In this current study, we investigated the in vitro effects of GcMAF treatment on the endocannabinoid system gene expression, as well as cellular activation in blood monocyte-derived macrophages (BMDMs) from autistic patients compared to age-matched healthy developing controls. Methods To achieve these goals, we used biomolecular, biochemical and immunocytochemical methods. Results GcMAF treatment was able to normalize the observed differences in dysregulated gene expression of the endocannabinoid system of the autism group. GcMAF also down-regulated the over-activation of BMDMs from autistic children. Conclusions This study presents the first observations of GcMAF effects on the transcriptionomics of the endocannabinoid system and expression of CB2R protein. These data point to a potential nexus between endocannabinoids, vitamin D and its transporter proteins, and the immune dysregulations observed with autism. PMID:24739187

  9. Tissue factor activity. A marker of alveolar macrophage maturation in rabbits. Effects of granulomatous pneumonitis.

    PubMed Central

    Rothberger, H; McGee, M P; Lee, T K

    1984-01-01

    Experiments were carried out to examine relationships between alveolar macrophage maturity and amounts of tissue factor (Clotting Factor III) in these cells under physiologic conditions and during immunologically induced pneumonitis. Using discontinuous density gradient centrifugation, alveolar macrophages from healthy rabbits were rapidly isolated into five subpopulations at different stages of maturation, as demonstrated by morphologic and morphometric evaluation. Very large amounts of tissue factor activity were found in fully mature cells that were purified in the lowest density subpopulation and assayed without preliminary in vitro stimulation or culture. In the remaining four subpopulations of increasing density, amounts of tissue factor were found to progressively diminish in direct correlation with declines of cell maturity. These differences at mean levels were as great as 35-fold. In addition, blood monocytes had less than 1/219 and less than 1/6 of the activity of the fully mature and the least mature subpopulations, respectively. After 16 h culture of the five isolated subpopulations in the absence of lymphokines or of significant numbers of lymphocytes, tissue factor activity increased in inverse correlation with the preincubation stage of cell maturity (2,387 and 109% in the least mature and most mature subpopulations, respectively). These increases required protein synthesis and were accompanied by morphologic and morphometric changes which indicated cellular maturation during the period of tissue factor activity generation in vitro, thus further demonstrating relationships between macrophage maturity and tissue factor content. In additional experiments, direct correlations between cell maturity and tissue factor activity content were also found in activated alveolar macrophage populations from rabbits with Bacillus Calmette Guering (BCG)-induced granulomatous pneumonitis. However, as compared with controls, the BCG populations had increased total

  10. Mycobacterium leprae upregulates IRGM expression in monocytes and monocyte-derived macrophages.

    PubMed

    Yang, Degang; Chen, Jia; Zhang, Linglin; Cha, Zhanshan; Han, Song; Shi, Weiwei; Ding, Ru; Ma, Lan; Xiao, Hong; Shi, Chao; Jing, Zhichun; Song, Ningjing

    2014-08-01

    Leprosy is caused by the infection of Mycobacterium leprae, which evokes a strong inflammatory response and leads to nerve damage. Immunity-related GTPase family M protein (IRGM) plays critical roles in controlling inflammation. The objective of the study was to investigate whether IRGM is involved in the infection of M. leprae. Levels of IRGM were assessed in M. leprae-infected CD4(+) T cells, monocytes, and monocyte-derived macrophages. Data revealed that both protein and mRNA levels of IRGM were increased in monocytes after M. leprae infection. Interestingly, monocyte-derived macrophages showed more prominent IRGM expression with M. leprae infection, whereas the bacteria did not affect IRGM in CD4(+) T cells. Furthermore, we assessed levels of IRGM in CD4(+) T cells and monocytes from 78 leprosy patients and 40 healthy controls, and observed upregulated protein level of IRGM in the monocytes from leprosy patients. Also, IRGM expression was inversely correlated with the severity of the disease. These findings suggested a close involvement of IRGM in M. leprae infection and indicated a potential mechanism of defending M. leprae infection. PMID:24469081

  11. A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

    PubMed Central

    Lima, Djalma S.; Zamboni, Dario S.

    2010-01-01

    The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells. PMID:21179419

  12. Protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis.

    PubMed

    Heinrichs, Daniel; Berres, Marie-Luise; Coeuru, Melanie; Knauel, Meike; Nellen, Andreas; Fischer, Petra; Philippeit, Claudia; Bucala, Richard; Trautwein, Christian; Wasmuth, Hermann E; Bernhagen, Jürgen

    2014-12-01

    MIF is an inflammatory cytokine but is hepatoprotective in models of hepatotoxin-induced liver fibrosis. Hepatic fibrosis can also develop from metabolic liver disease, such as nonalcoholic fatty liver disease (NASH). We investigated the role of MIF in high-fat or methionine- and choline-deficient diet mouse models of NASH. Mif(-/-) mice showed elevated liver triglyceride levels (WT, 53±14 mg/g liver; Mif(-/-), 103±7 mg/g liver; P<0.05) and a 2-3-fold increased expression of lipogenic genes. Increased fatty degeneration in the livers of Mif(-/-) mice was associated with increased hepatic inflammatory cells (1.6-fold increase in F4/80(+) macrophages) and proinflammatory cytokines (e.g., 2.3-fold increase in Tnf-α and 2-fold increase in Il-6 expression). However, inflammatory cells and cytokines were decreased by 50-90% in white adipose tissue (WAT) of Mif(-/-) mice. Subset analysis showed that macrophage phenotypes in livers of Mif(-/-) mice were skewed toward M2 (e.g., 1.7-fold and 2.5-fold increase in Arg1 and Il-13, respectively, and 2.5-fold decrease in iNos), whereas macrophages were generally reduced in WAT of these mice (70% reduction in mRNA expression of F4/80(+) macrophages). The protective MIF effect was scrutinized in isolated hepatocytes. MIF reversed inflammation-induced triglyceride accumulation in Hepa1-6 cells and primary hepatocytes and also attenuated oleic acid-elicited triglyceride increase in 3T3-L1 adipocytes. Protection from fatty hepatocyte degeneration was paralleled by a 2- to 3-fold reduction by MIF of hepatocyte proinflammatory cytokine production. Blockade of MIF receptor cluster of differentiation 74 (CD74) but not of CXCR2 or CXCR4 fully reverted the protective effect of MIF, comparable to AMPK inhibition. In summary, we demonstrate that MIF mediates hepatoprotection through the CD74/AMPK pathway in hepatocytes in metabolic models of liver injury. PMID:25122558

  13. Activating transcription factor 4 promotes angiogenesis of breast cancer through enhanced macrophage recruitment.

    PubMed

    Liu, Chen; Li, Zongjin; Wang, Lina; Tong, Lingling; He, Ningning; Chen, Yanan; Liu, Yanhua; Wu, Zhongjun; Sun, Peiqing; Xiang, Rong; Ren, Guosheng; Su, Weijun

    2015-01-01

    Angiogenesis plays an important role in the progression of tumor. Besides being regulated by tumor cells per se, tumor angiogenesis is also influenced by stromal cells in tumor microenvironment (TME), for example, tumor associated macrophages (TAMs). Activating transcription factor 4 (ATF4), a member of the ATF/CREB family, has been reported to be related to tumor angiogenesis. In this study, we found that exogenous overexpression of ATF4 in mouse breast cancer cells promotes tumor growth via increasing tumor microvascular density. However, ATF4 overexpression failed to increase the expression level of a series of proangiogenic factors including vascular endothelial growth factor A (VEGFA) in tumor cells in this model. Thus, we further investigated the infiltration of proangiogenic macrophages in tumor tissues and found that ATF4-overexpressing tumors could recruit more macrophages via secretion of macrophage colony stimulating factor (M-CSF). Overall, we concluded that exogenous overexpression of ATF4 in breast cancer cells may facilitate the recruitment of macrophages into tumor tissues and promote tumor angiogenesis and tumor growth indirectly. PMID:25883982

  14. Effect of Mycobacterium tuberculosis-Specific 10-Kilodalton Antigen on Macrophage Release of Tumor Necrosis Factor Alpha and Nitric Oxide

    PubMed Central

    Trajkovic, Vladimir; Singh, Gyanesh; Singh, Balwan; Singh, Sarman; Sharma, Pawan

    2002-01-01

    Secreted proteins of Mycobacterium tuberculosis are major targets of the specific immunity in tuberculosis and constitute promising candidates for the development of more efficient vaccines and diagnostic tests. We show here that M. tuberculosis-specific antigen 10 (MTSA-10, originally designated CFP-10) can bind to the surface of mouse J774 macrophage-like cells and stimulate the secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). MTSA-10 also synergized with gamma interferon (IFN-γ) for the induction of the microbicidal free radical nitric oxide (NO) in J774 cells, as well as in bone marrow-derived and peritoneal macrophages. On the other hand, pretreatment of J774 cells with MTSA-10 markedly reduced NO but not TNF-α or interleukin 10 (IL-10) release upon subsequent stimulation with lipopolysaccharide or the cell lysate of M. tuberculosis. The presence of IFN-γ during stimulation with M. tuberculosis lysate antagonized the desensitizing effect of MTSA-10 pretreatment on macrophage NO production. The activation of protein tyrosine kinases (PTK) and the serine/threonine kinases p38 MAPK and ERK was apparently required for MTSA-10 induction of TNF-α and NO release, as revealed by specific kinase inhibitors. However, only p38 MAPK activity, not PTK or ERK activity, was partly responsible for MTSA-10-mediated macrophage desensitization. The modulation of macrophage function by MTSA-10 suggests a novel mechanism for its involvement in immunopathogenesis of tuberculosis and might have implications for the prevention, diagnosis, and therapy of this disease. PMID:12438325

  15. Endotoxin-induced myocardial dysfunction: effects of macrophage migration inhibitory factor neutralization.

    PubMed

    Chagnon, Frederic; Metz, Christine N; Bucala, Richard; Lesur, Olivier

    2005-05-27

    The pathophysiology of sepsis-induced myocardial dysfunction still remains controversial. Macrophage migration inhibitory factor (MIF) has recently been identified as a cardiac-derived myocardial depressant factor in septic shock. Putative mechanisms by which MIF affects cardiac function are unknown. In an investigation of possible mechanisms of action, a rat model of endotoxin toxicity was designed using intraperitoneal (I/P) injection of lipopolysaccharides (LPS) with or without coinfusion of neutralizing anti-MIF or isotypic-matched antibodies. Echocardiographic evaluation revealed that MIF neutralization reversed endotoxin-induced myocardial dysfunction at 24 hours after injection. RNase protection assay (RPA) and Western blot established that MIF neutralization prevented LPS-induced mRNA expression and production of heart-derived inflammatory paracrine and autocrine cytokines such as IL-1s and IL-6. Moreover, MIF immunoneutralization increased heart Bcl-2/Bax protein ratio and suppressed endotoxin-induced release of mitochondrial cytochrome-c, as demonstrated by Western blotting. Inhibition of mitochondrial loss of cytochrome-c decreased in heart caspase-3 activity at 6 and 24 hours after injection. MIF neutralization also restored the LPS-induced deficient nuclear translocation of phospho-Akt and consequently the expression of the heart survival nuclear factor GATA-4. The restoration of the translocation/expression of survival factors by MIF inhibition resulted in lowered endotoxin-induced DNA fragmentation at 24 hours, a hallmark of downstream cardiomyocyte apoptosis. Our data indicate that early inactivation of MIF significantly reverses the imbalance of proapoptotic to prosurvival pathways and reduces acute inflammation of the heart thereby improving myocardial dysfunction induced by endotoxin. PMID:15879312

  16. Hypoxia-inducible factors 1 and 2 are important transcriptional effectors in primary macrophages experiencing hypoxia.

    PubMed

    Fang, Hsin-Yu; Hughes, Russell; Murdoch, Craig; Coffelt, Seth B; Biswas, Subhra K; Harris, Adrian L; Johnson, Randall S; Imityaz, Hongxia Z; Simon, M Celeste; Fredlund, Erik; Greten, Florian R; Rius, Jordi; Lewis, Claire E

    2009-07-23

    Ischemia exists in many diseased tissues, including arthritic joints, atherosclerotic plaques, and malignant tumors. Macrophages accumulate in these sites and up-regulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18 hours. For example, they were seen to up-regulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, vascular endothelial growth factor A, interleukin (IL)-1beta and IL-8, adrenomedullin, CXCR4, and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the nuclear factor-kappaB (NF-kappaB) signaling pathway. We then used both genetic and pharmacologic methods to manipulate the levels of HIFs-1alpha and 2alpha or NF-kappaB in primary macrophages to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIF-1 and -2, but not NF-kappaB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues, such as malignant tumors. PMID:19454749

  17. The roles of blood-derived macrophages and resident microglia in the neuroinflammatory response to implanted Intracortical microelectrodes

    PubMed Central

    Ravikumar, Madhumitha; Sunil, Smrithi; Black, James; Barkauskas, Deborah S.; Haung, Alex Y.; Miller, Robert H.; Selkirk, Stephen M.; Capadona, Jeffrey R.

    2014-01-01

    Resident microglia and blood-borne macrophages have both been implicated to play a dominant role in mediating the neuroinflammatory response affecting implanted intracortical microelectrodes. However, the distinction between each cell type has not been demonstrated due to a lack of discriminating cellular markers. Understanding the subtle differences of each cell population in mediating neuroinflammation can aid in determining the appropriate therapeutic approaches to improve microelectrode performance. Therefore, the goal of this study is to characterize the role of infiltrating blood-derived cells, specifically macrophages, in mediating neuroinflammation following intracortical microelectrode implantation. Interestingly, we found no correlation between microglia and neuron populations at the microelectrode-tissue interface. On the other hand, blood-borne macrophages consistently dominated the infiltrating cell population following microelectrode implantation. Most importantly, we found a correlation between increased populations of blood-derived cells (including the total macrophage population) and neuron loss at the microelectrode-tissue interface. Specifically, the total macrophage population was greatest at two and sixteen weeks post implantation, at the same time points when we observed the lowest densities of neuronal survival in closest proximity to the implant. Together, our results suggest a dominant role of infiltrating macrophages, and not resident microglia, in mediating neurodegeneration following microelectrode implantation. PMID:24973296

  18. The roles of blood-derived macrophages and resident microglia in the neuroinflammatory response to implanted intracortical microelectrodes.

    PubMed

    Ravikumar, Madhumitha; Sunil, Smrithi; Black, James; Barkauskas, Deborah S; Haung, Alex Y; Miller, Robert H; Selkirk, Stephen M; Capadona, Jeffrey R

    2014-09-01

    Resident microglia and blood-borne macrophages have both been implicated to play a dominant role in mediating the neuroinflammatory response affecting implanted intracortical microelectrodes. However, the distinction between each cell type has not been demonstrated due to a lack of discriminating cellular markers. Understanding the subtle differences of each cell population in mediating neuroinflammation can aid in determining the appropriate therapeutic approaches to improve microelectrode performance. Therefore, the goal of this study is to characterize the role of infiltrating blood-derived cells, specifically macrophages, in mediating neuroinflammation following intracortical microelectrode implantation. Interestingly, we found no correlation between microglia and neuron populations at the microelectrode-tissue interface. On the other hand, blood-borne macrophages consistently dominated the infiltrating cell population following microelectrode implantation. Most importantly, we found a correlation between increased populations of blood-derived cells (including the total macrophage population) and neuron loss at the microelectrode-tissue interface. Specifically, the total macrophage population was greatest at two and sixteen weeks post implantation, at the same time points when we observed the lowest densities of neuronal survival in closest proximity to the implant. Together, our results suggest a dominant role of infiltrating macrophages, and not resident microglia, in mediating neurodegeneration following microelectrode implantation. PMID:24973296

  19. Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel

    PubMed Central

    Ohradanova-Repic, Anna; Machacek, Christian; Fischer, Michael B; Stockinger, Hannes

    2016-01-01

    The mononuclear phagocyte system, consisting of monocytes, macrophages and dendritic cells (DCs), has an important role in tissue homeostasis as well as in eliciting immune responses against invading pathogens. Blood monocytes have been viewed for decades as precursors of tissue macrophages. Although the newest data show that in the steady state resident macrophages of many organs are monocyte independent, blood monocytes critically contribute to tissue macrophage and DC pools upon inflammation. To better understand the relationship between these populations and their phenotype, we isolated and differentiated human blood CD14+ monocytes in vitro into immature and mature monocyte-derived dendritic cells (MoDCs) as well as into seven different monocyte-derived macrophage subsets. We used the panel of 70 monoclonal antibodies (mAbs) submitted to the 10th Human Leukocyte Differentiation Antigen Workshop to determine the expression profiles of these 10 populations by flow cytometry. We now can compile subpanels of mAbs to differentiate the 10 monocyte/macrophage/MoDC subsets, providing the basis for novel diagnostic and therapeutic tools. PMID:26900469

  20. A novel role of breast cancer-derived hyaluronan on inducement of M2-like tumor-associated macrophages formation.

    PubMed

    Zhang, Guoliang; Guo, Lin; Yang, Cuixia; Liu, Yiwen; He, Yiqing; Du, Yan; Wang, Wenjuan; Gao, Feng

    2016-06-01

    Microenvironmental signals determine the differentiation types and distinct functions of macrophages. Tumor-associated macrophages (TAM) constitute major infiltrates around solid tumor cells and accelerate tumor progression due to their immunosuppressive functions. However, the mechanisms through which tumor microenvironment modulates macrophages transition are not completely elucidated. Hyaluronan (HA), a prominent component in tumor microenvironment, is a notable immunoregulator and its high level is often related to poor prognosis. Herein, we found that the number of M2 macrophages was highly correlated with HA expression in tumor tissues from breast cancer patients. Experimental data showed that breast cancer-derived HA stimulated M2-like TAM formation in a mouse model and had multiple effects on macrophages transformation in vitro, including upregulating CD204, CD206, IL-10 and TGF-β, activating STAT3 signal, and suppressing killing capacity. These data indicate that HA derived from breast cancer activates macrophages in an alternative manner. Further mechanism study revealed that HA-CD44-ERK1/2-STAT3 pathway served as an important regulator in M2-like TAM formation. Therefore, targeting TAM by abrogating HA-CD44 interaction may be a potential strategy for breast cancer immunotherapy. PMID:27471651

  1. Elastin-Derived Peptides Promote Abdominal Aortic Aneurysm Formation by Modulating M1/M2 Macrophage Polarization.

    PubMed

    Dale, Matthew A; Xiong, Wanfen; Carson, Jeffrey S; Suh, Melissa K; Karpisek, Andrew D; Meisinger, Trevor M; Casale, George P; Baxter, B Timothy

    2016-06-01

    Abdominal aortic aneurysm is a dynamic vascular disease characterized by inflammatory cell invasion and extracellular matrix degradation. Damage to elastin in the extracellular matrix results in release of elastin-derived peptides (EDPs), which are chemotactic for inflammatory cells such as monocytes. Their effect on macrophage polarization is less well known. Proinflammatory M1 macrophages initially are recruited to sites of injury, but, if their effects are prolonged, they can lead to chronic inflammation that prevents normal tissue repair. Conversely, anti-inflammatory M2 macrophages reduce inflammation and aid in wound healing. Thus, a proper M1/M2 ratio is vital for tissue homeostasis. Abdominal aortic aneurysm tissue reveals a high M1/M2 ratio in which proinflammatory cells and their associated markers dominate. In the current study, in vitro treatment of bone marrow-derived macrophages with EDPs induced M1 macrophage polarization. By using C57BL/6 mice, Ab-mediated neutralization of EDPs reduced aortic dilation, matrix metalloproteinase activity, and proinflammatory cytokine expression at early and late time points after aneurysm induction. Furthermore, direct manipulation of the M1/M2 balance altered aortic dilation. Injection of M2-polarized macrophages reduced aortic dilation after aneurysm induction. EDPs promoted a proinflammatory environment in aortic tissue by inducing M1 polarization, and neutralization of EDPs attenuated aortic dilation. The M1/M2 imbalance is vital to aneurysm formation. PMID:27183603

  2. Unilamellar liposomes modulate secretion of tumor necrosis factor by lipopolysaccharide-stimulated macrophages.

    PubMed Central

    Brisseau, G F; Kresta, A; Schouten, D; Bohnen, J M; Shek, P N; Fok, E; Rotstein, O D

    1994-01-01

    Liposomal encapsulation of antimicrobial agents has been used to improve drug delivery, particularly against intracellular pathogens. The effect of unilamellar liposomes on macrophage activation in response to Escherichia coli lipopolysaccharide was examined. Liposomes caused a dose- and time-dependent inhibition of tumor necrosis factor release by lipopolysaccharide-treated cells. The accumulation of tumor necrosis factor mRNA transcripts was unaffected, suggesting a posttranscriptional mechanism for this effect. However, induction of macrophage procoagulant activity was unaffected by liposomes, indicating a selective rather than a global inhibition. These data suggest that liposomes used for drug delivery may modulate the host response to infection. Images PMID:7872768

  3. Bjcul, a snake venom lectin, modulates monocyte-derived macrophages to a pro-inflammatory profile in vitro.

    PubMed

    Dias-Netipanyj, M F; Boldrini-Leite, L M; Trindade, E S; Moreno-Amaral, A N; Elifio-Esposito, S

    2016-06-01

    Macrophages are cells of high plasticity and can act in different ways to ensure that the appropriate immune response remains controlled. This study shows the effects of the C-type Bothrops jararacussu venom lectin (BJcuL) on the activation of human macrophages derived from the U937 cell line. BJcuL binds on the cell surface, and this event is inhibited by its specific carbohydrate. It induced phagocytosis and production of H2O2, and expression of antigen presentation molecules. It also enhanced the production of TNF-α, GM-CSF and IL-6 by macrophages and indirectly induced T cells to an increased production of TNF-α, IFN-γ and IL-6 in the presence of LPS. Our results suggest that BJcuL can modulate macrophage functional activation towards an M1 state. PMID:26944802

  4. Class I and class II major histocompatibility molecules play a role in bone marrow-derived macrophage development

    NASA Technical Reports Server (NTRS)

    Armstrong, J. W.; Simske, S. J.; Beharka, A. A.; Balch, S.; Luttges, M. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Class I and class II major histocompatibility complex (MHC) molecules play significant roles in T cell development and immune function. We show that MHCI- and MHCII-deficient mice have low numbers of macrophage precursors and circulating monocytes, as well as abnormal bone marrow cell colony-stimulating factor type 1 secretion and bone composition. We suggest that MHCI and MHCII molecules play a significant role in macrophage development.

  5. Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    PubMed Central

    Lei, Guang-Sheng; Zhang, Chen

    2014-01-01

    Myeloid-derived suppressor cells (MDSCs) were recently found to accumulate in the lungs during Pneumocystis pneumonia (PcP). Adoptive transfer of these cells caused lung damage in recipient mice, suggesting that MDSC accumulation is a mechanism of pathogenesis in PcP. In this study, the phagocytic activity of alveolar macrophages (AMs) was found to decrease by 40% when they were incubated with MDSCs from Pneumocystis-infected mice compared to those incubated with Gr-1+ cells from the bone marrow of uninfected mice. The expression of the PU.1 gene in AMs incubated with MDSCs also was decreased. This PU.1 downregulation was due mainly to decreased histone 3 acetylation and increased DNA methylation caused by MDSCs. MDSCs were found to express high levels of PD-L1, and alveolar macrophages (AMs) were found to express high levels of PD-1 during PcP. Furthermore, PD-1 expression in AMs from uninfected mice was increased by 18-fold when they were incubated with MDSCs compared to those incubated with Gr-1+ cells from the bone marrow of uninfected mice. The adverse effects of MDSCs on AMs were diminished when the MDSCs were pretreated with anti-PD-L1 antibody, suggesting that MDSCs disable AMs through PD-1/PD-L1 ligation during PcP. PMID:25404033

  6. Inhibition of macrophage-derived foam cell formation by ezetimibe via the caveolin-1/MAPK pathway.

    PubMed

    Qin, Li; Yang, Yun-Bo; Yang, Yi-Xin; Zhu, Neng; Liu, Zheng; Ni, Ya-Guang; Li, Shun-Xiang; Zheng, Xi-Long; Liao, Duan-Fang

    2016-02-01

    Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, effectively reduces plasma cholesterol, but its effect on atherosclerosis is unclear. Foam cell formation has been implicated as a key mediator during the development of atherosclerosis. The purpose of this study was to investigate the effects of ezetimibe on foam cell formation and explore the underlying mechanism. The results presented here show that ezetimibe reduces atherosclerotic lesions in apolipoprotein E deficient (apoE-/-) mice by lowering cholesterol levels. Treatment of macrophages with Chol:MβCD resulted in foam cell formation, which was concentration-dependently inhibited by the presence of ezetimibe. Mechanically, ezetimibe treatment downregulated the expression of CD36 and scavenger receptor class B1 (SR-B1), but upregulated the expression of apoE and caveolin-1 in macrophage-derived foam cells, which kept consistent with our microarray results. Moreover, treatment with ezetimibe abrogated the increase of phospho-extracellular signal regulated kinase (ERK) 1/2 and their nuclear accumulation in foam cells. Inhibition of the MAPK pathway by the MEK inhibitor PD98059 attenuated the inhibitory effect of ezetimibe on the expression of p-ERK1/2 and caveolin-1. Taken together, these results showed that ezetimibe suppressed foam cell formation via the caveolin-1/MAPK signalling pathway, suggesting that inhibition of foam cell formation might be a novel mechanism underlying the anti-atherosclerotic effect of ezetimibe. PMID:26666965

  7. Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear.

    PubMed

    Bank, Lisa M; Bianchi, Lynne M; Ebisu, Fumi; Lerman-Sinkoff, Dov; Smiley, Elizabeth C; Shen, Yu-chi; Ramamurthy, Poornapriya; Thompson, Deborah L; Roth, Therese M; Beck, Christine R; Flynn, Matthew; Teller, Ryan S; Feng, Luming; Llewellyn, G Nicholas; Holmes, Brandon; Sharples, Cyrrene; Coutinho-Budd, Jaeda; Linn, Stephanie A; Chervenak, Andrew P; Dolan, David F; Benson, Jennifer; Kanicki, Ariane; Martin, Catherine A; Altschuler, Richard; Koch, Alisa E; Koch, Alicia E; Jewett, Ethan M; Germiller, John A; Barald, Kate F

    2012-12-01

    This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development. PMID:23172918

  8. 5-Azacytidine modulates interferon regulatory factor 1 in macrophages to exert a cardioprotective effect.

    PubMed

    Jeong, Hye-yun; Kang, Wan Seok; Hong, Moon Hwa; Jeong, Hae Chang; Shin, Myun-Geun; Jeong, Myung Ho; Kim, Yong Sook; Ahn, Youngkeun

    2015-01-01

    Macrophages are actively involved in inflammatory responses during the progression of cardiac injury, including myocardial infarction (MI). A previous study showed that 5-azacytidine (5AZ), a DNA methylation inhibitor, can ameliorate cardiac injury by shifting macrophages toward an anti-inflammatory phenotype via iNOS inhibition. Here, we show that the beneficial effect of 5AZ is associated with sumoylation of interferon regulatory factor-1 (IRF1) in macrophages. IRF1 is a critical transcription factor for iNOS induction and is antagonized by IRF2. In the stimulated macrophages, IRF1 accumulated in the nucleus without degradation by 5AZ treatment. In animal study, 5AZ administration resulted in significant improvements in cardiac function and fibrosis. IRF1-expressing macrophages were more abundant in the 5AZ-treated MI group than in the PBS-treated MI group. Because sumoylated IRF1 is known to mimic IRF2, we examined the IRF1 sumoylation. Sumoylated IRF1 was resistant to degradation and significantly increased in the 5AZ-treated MI group. Collectively, 5AZ had a protective effect after MI by potentiation of IRF1 sumoylation and is suggested as a novel therapeutic intervention for cardiac repair. PMID:26510961

  9. 5-Azacytidine modulates interferon regulatory factor 1 in macrophages to exert a cardioprotective effect

    PubMed Central

    Jeong, Hye-yun; Kang, Wan Seok; Hong, Moon Hwa; Jeong, Hae Chang; Shin, Myun-Geun; Jeong, Myung Ho; Kim, Yong Sook; Ahn, Youngkeun

    2015-01-01

    Macrophages are actively involved in inflammatory responses during the progression of cardiac injury, including myocardial infarction (MI). A previous study showed that 5-azacytidine (5AZ), a DNA methylation inhibitor, can ameliorate cardiac injury by shifting macrophages toward an anti-inflammatory phenotype via iNOS inhibition. Here, we show that the beneficial effect of 5AZ is associated with sumoylation of interferon regulatory factor-1 (IRF1) in macrophages. IRF1 is a critical transcription factor for iNOS induction and is antagonized by IRF2. In the stimulated macrophages, IRF1 accumulated in the nucleus without degradation by 5AZ treatment. In animal study, 5AZ administration resulted in significant improvements in cardiac function and fibrosis. IRF1-expressing macrophages were more abundant in the 5AZ-treated MI group than in the PBS-treated MI group. Because sumoylated IRF1 is known to mimic IRF2, we examined the IRF1 sumoylation. Sumoylated IRF1 was resistant to degradation and significantly increased in the 5AZ-treated MI group. Collectively, 5AZ had a protective effect after MI by potentiation of IRF1 sumoylation and is suggested as a novel therapeutic intervention for cardiac repair. PMID:26510961

  10. Viral and host factors induce macrophage activation and loss of Toll Like Receptor tolerance in chronic HCV infection

    PubMed Central

    Dolganiuc, Angela; Norkina, Oxana; Kodys, Karen; Catalano, Donna; Bakis, Gennadiy; Marshall, Christopher; Mandrekar, Pranoti; Szabo, Gyongyi

    2007-01-01

    Background&Aims Persistent inflammation contributes to progression of liver damage in chronic HCV (cHCV) infection. Repeated exposure to Toll like receptor (TLR) ligands results in tolerance, a protective mechanism aimed at limiting inflammation. Methods Monocytes/macrophages were repeatedly stimulated via pro-inflammatory cytokine-inducing TLRs and evaluated for activation markers. Results Unlike monocytes (Mo) of controls or patients with non-alcoholic steatohepatitis, the Mo of cHCV patients were hyper-responsive and failed to show homo- or hetero-tolerance to TLR ligands, manifested by elevated TNFα production. Serum levels of IFNγ, endotoxin (TLR4 ligand) and HCV core protein (TLR2 ligand) were elevated in cHCV patients suggesting potential mechanisms for in vivo monocyte pre-activation. Treatment of normal monocytes with IFNγ resulted in loss of tolerance to LPS or HCV core protein. Further, we found increased levels of MyD88-IRAK1 complexes and NFκB activity both in monocytes of cHCV patients and in normal monocytes that lost TLR tolerance after IFNγ+LPS pretreatment. In vitro differentiation of TLR tolerant cHCV monocytes into macrophages restored their capacity to exhibit TLR tolerance to LPS and HCV core protein and this could be reversed by administration of IFNγ. cHCV patients exhibited increased TNFα in the circulation and in the liver. In cHCV livers we found Kupffer cell/macrophage activation indicated by increased CD163 and CD33 expression. Conclusions We identified that host-derived factors (IFNγ and endotoxin) and viral factors (HCV core protein) act in tandem to induce and maintain monocyte/macrophage activation, thus favoring persistent inflammation in patients with cHCV infection. PMID:17916356

  11. Macrophages commit postnatal endothelium-derived progenitors to angiogenesis and restrict endothelial to mesenchymal transition during muscle regeneration

    PubMed Central

    Zordan, P; Rigamonti, E; Freudenberg, K; Conti, V; Azzoni, E; Rovere-Querini, P; Brunelli, S

    2014-01-01

    The damage of the skeletal muscle prompts a complex and coordinated response that involves the interactions of many different cell populations and promotes inflammation, vascular remodeling and finally muscle regeneration. Muscle disorders exist in which the irreversible loss of tissue integrity and function is linked to defective neo-angiogenesis with persistence of tissue necrosis and inflammation. Here we show that macrophages (MPs) are necessary for efficient vascular remodeling in the injured muscle. In particular, MPs sustain the differentiation of endothelial-derived progenitors to contribute to neo-capillary formation, by secreting pro-angiogenic growth factors. When phagocyte infiltration is compromised endothelial-derived progenitors undergo a significant endothelial to mesenchymal transition (EndoMT), possibly triggered by the activation of transforming growth factor-β/bone morphogenetic protein signaling, collagen accumulates and the muscle is replaced by fibrotic tissue. Our findings provide new insights in EndoMT in the adult skeletal muscle, and suggest that endothelial cells in the skeletal muscle may represent a new target for therapeutic intervention in fibrotic diseases. PMID:24481445

  12. uPAR Induces Expression of Transforming Growth Factor β and Interleukin-4 in Cancer Cells to Promote Tumor-Permissive Conditioning of Macrophages

    PubMed Central

    Hu, Jingjing; Jo, Minji; Eastman, Boryana M.; Gilder, Andrew S.; Bui, Jack D.; Gonias, Steven L.

    2015-01-01

    Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell–like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow–derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression. PMID:25310970

  13. Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

    PubMed

    Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

    2015-08-01

    In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. PMID:26129676

  14. Equine monocyte-derived macrophage cultures and their applications for infectivity and neutralization studies of equine infectious anemia virus.

    PubMed

    Raabe, M R; Issel, C J; Montelaro, R C

    1998-03-01

    Equine infectious anemia virus (EIAV) has been shown to infect cells of monocyte/macrophage lineage. These primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. As a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. We describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. The in vitro differentiation of these cells into mature equine macrophage was verified using various cytochemical staining methods. The equine monocyte-derived macrophage (MDM) cultures were found to replicate cell-adapted and field strains of EIAV more efficiently than cultures of fully differentiated equine splenic macrophage. Having established reproducible and fully differentiated cultures of equine macrophage, in vitro assays of virus infectivity and serum neutralization were developed using the in vivo target cell of EIAV. These procedures, while developed for the EIAV system, should be equally useful for in vitro cultures of other macrophage-tropic pathogens of horses. PMID:9628225

  15. Distinct immunoregulatory properties of macrophage migration inhibitory factors encoded by Eimeria parasites and their chicken host

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in host defense against a variety of microorganisms including protozoan parasites. Interestingly, some microbial pathogens also express a MIF-like protein, although its role in disease pathogenesi...

  16. Macrophage Migration Inhibitory Factor (MIF): A Key Player in Protozoan Infections

    PubMed Central

    de Dios Rosado, Juan; Rodriguez-Sosa, Miriam

    2011-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine produced by the pituitary gland and multiple cell types, including macrophages (Mø), dendritic cells (DC) and T-cells. Upon releases MIF modulates the expression of several inflammatory molecules, such as TNF-α, nitric oxide and cyclooxygenase 2 (COX-2). These important MIF characteristics have prompted investigators to study its role in parasite infections. Several reports have demonstrated that MIF plays either a protective or deleterious role in the immune response to different pathogens. Here, we review the role of MIF in the host defense response to some important protozoan infections. PMID:22110378

  17. Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.

    PubMed Central

    Nishizawa, M; Nagata, S

    1990-01-01

    Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene. Images PMID:1691438

  18. Hypoxia inducible factors 1 and 2 are important transcriptional effectors in primary macrophages experiencing hypoxia

    PubMed Central

    Fang, Hsin-Yu; Hughes, Russell; Murdoch, Craig; Coffelt, Seth; Biswas, Subhra K.; Harris, Adrian L.; Johnson, Randall S.; Imityaz, Hongxia Z.; Simon, M. Celeste; Fredlund, Erik; Greten, Florian; Rius, Jordi; Lewis, Claire E.

    2010-01-01

    Ischemia exists in many diseased tissues including arthritic joints, atherosclerotic plaques and malignant tumors. Macrophages accumulate in these sites and upregulate hypoxia-inducible transcription factors (HIFs) 1 and 2 in response to the hypoxia present. Here we show that the gene expression profile in primary human and murine macrophages changes markedly when they are exposed to hypoxia for 18h. For example, they were seen to upregulate the cell surface receptors, CXCR4 and GLUT1, and the potent, tumor-promoting cytokines, VEGFA, interleukins 1β and 8, adrenomedullin, CXCR4 and angiopoietin-2. Hypoxia also stimulated their expression and/or phosphorylation of various proteins in the NF-κB signalling pathway. We then used both genetic and pharmacological methods to manipulate the levels of HIFs 1α and 2α or NF-κB in primary macrophages in order to elucidate their role in the hypoxic induction of many of these key genes. These studies showed that both HIFs 1 and 2, but not NF-κB, are important transcriptional effectors regulating the responses of macrophages to such a period of hypoxia. Further studies using experimental mouse models are now warranted to investigate the role of such macrophage responses in the progression of various diseased tissues like malignant tumors. PMID:19454749

  19. Granulocyte-macrophage colony-stimulating factor: pleiotropic cytokine with potential clinical usefulness.

    PubMed

    Ruef, C; Coleman, D L

    1990-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 23-kDa glycoprotein with remarkably diverse effects on immune and nonimmune cells. GM-CSF induces differentiation of granulocyte, macrophage, and eosinophil precursor cells. Proliferation of monocyte-macrophages, T lymphocytes, keratinocytes, and endothelial cells is also stimulated by GM-CSF. In addition, GM-CSF alters the functional properties of mature granulocytes, macrophages, eosinophils, and basophils. GM-CSF is produced by T lymphocytes, macrophages, and several cell types in extramedullary sites, where it may act in a paracrine manner to regulate the local response to antigenic challenge. Clinical trials of GM-CSF have been conducted in patients with AIDS, aplastic anemia, myelodysplastic syndromes, and sarcoma and following bone marrow transplantation and accidental radiation exposure. GM-CSF significantly increased circulating numbers of several myeloid cells and produced dose-dependent toxicity consisting primarily of myalgias, fever, fluid retention, and serosal effusions. Additional studies are needed to define the role of GM-CSF in treatment of patients with qualitative and quantitative dysfunction of immune cells. PMID:2405468

  20. Adiponectin Regulates Vascular Endothelial Growth Factor-C Expression in Macrophages via Syk-ERK Pathway

    PubMed Central

    Hu, Di; Fukuhara, Atsunori; Miyata, Yugo; Yokoyama, Chieko; Otsuki, Michio; Kihara, Shinji; Shimomura, Iichiro

    2013-01-01

    Adiponectin is exclusively expressed in adipose tissues and exhibits protective effects against cardiovascular and metabolic diseases. It enhances AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα) signaling in the liver and skeletal muscles, however, its signaling pathways in macrophages remain to be elucidated. Here, we show that adiponectin upregulated the expression of vascular endothelial growth factor (VEGF)-C, and induced phosphorylation of extracellular signal-regulated kinase (ERK) in macrophages. Inhibition of Syk abrogated adiponectin-induced VEGF-C expression and ERK phosphorylation. Furthermore, inhibition of ERK blocked the induction of VEGF-C gene. Inhibition of Syk, but not that of ERK, abrogated adiponectin-induced expression of cyclooxygenase (COX)-2, tissue inhibitor of metalloproteinase (TIMP)-1, and interleukin (IL)-6. These results indicate that adiponectin regulates VEGF-C expression via Syk-ERK pathway in macrophages. PMID:23424645

  1. iPS-cell derived dendritic cells and macrophages for cancer therapy.

    PubMed

    Senju, Satoru

    2016-08-01

    Antibody-based anti-cancer immunotherapy was recently recognized as one of the truly effective therapies for cancer patients. Antibodies against cell surface cancer antigens, such as CD20, and also those against immune-inhibitory molecules called "immune checkpoint blockers", such as CTLA4 or PD1, have emerged. Large-scale clinical trials have confirmed that, in some cases, antibody-based drugs are superior to conventional chemotherapeutic agents. These antibody-based drugs are now being manufactured employing a mass-production system by pharmaceutical companies. Anti-cancer therapy by immune cells, i.e. cell-based immunotherapy, is expected to be more effective than antibody therapy, because immune cells can recognize, infiltrate, and act in cancer tissues more directly than antibodies. In order to achieve cell-based anti-cancer immunotherapy, it is necessary to develop manufacturing systems for mass-production of immune cells. Our group has been studying immunotherapy with myeloid cells derived from ES cells or iPS cells. These pluripotent stem cells can be readily propagated under constant culture conditions, with expansion into a large quantity. We consider these stem cells to be the most suitable cellular source for mass-production of immune cells. This review introduces our studies on anti-cancer therapy with iPS cell-derived dendritic cells and iPS cell-derived macrophages. PMID:27599426

  2. Macrophage Phenotype as a Predictor of Constructive Remodeling following the Implantation of Biologically Derived Surgical Mesh Materials

    PubMed Central

    Brown, Bryan N.; Londono, Ricardo; Tottey, Stephen; Zhang, Li; Kukla, Kathryn A.; Wolf, Matthew T.; Daly, Kerry A.; Reing, Janet E.; Badylak, Stephen F.

    2015-01-01

    Macrophages have been classified as having plastic phenotypes which exist within a spectrum between M1 (classically activated; pro-inflammatory) and M2 (alternatively activated; regulatory, homeostatic). To date, the effects of polarization towards a predominantly M1 or M2 phenotype have been studied largely in the context of response to pathogen or cancer. Recently, M1 and M2 macrophages have been shown to play distinct roles in tissue remodeling following injury. In the present study, the M1/M2 paradigm was utilized to examine the role of macrophages in the remodeling process following implantation of 14 biologically derived surgical mesh materials in the rat abdominal wall. In situ polarization of macrophages responding to the materials was examined and correlated to a quantitative measure of the observed tissue remodeling response to determine whether macrophage polarization is an accurate predictor of the ability of a biologic scaffold to promote constructive tissue remodeling. Additionally the ability of M1 and M2 macrophages to differentially recruit progenitor-like cells in vitro, which are commonly observed to participate in the remodeling of those ECM scaffolds which have a positive clinical outcome, was examined as a possible mechanism underlying the differences in the observed remodeling responses. The results of the present study show that there is a strong correlation between the early macrophage response to implanted materials and the outcome of tissue remodeling. Increased numbers of M2 macrophages and higher ratios of M2:M1 macrophages within the site of remodeling at 14 days were associated with more positive remodeling outcomes (r2=0.525–0.686, p<0.05). Further, the results of the present study suggest that the constructive remodeling outcome may be due to the recruitment and survival of different cell populations to the sites of remodeling associated with materials that elicit an M1 versus M2 response. Both M2 and M0 macrophage conditioned

  3. Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation.

    PubMed

    Hagemeyer, Nora; Kierdorf, Katrin; Frenzel, Kathrin; Xue, Jia; Ringelhan, Marc; Abdullah, Zeinab; Godin, Isabelle; Wieghofer, Peter; Costa Jordão, Marta Joana; Ulas, Thomas; Yorgancioglu, Gülden; Rosenbauer, Frank; Knolle, Percy A; Heikenwalder, Mathias; Schultze, Joachim L; Prinz, Marco

    2016-08-15

    Recent studies have shown that tissue macrophages (MΦ) arise from embryonic progenitors of the yolk sac (YS) and fetal liver and colonize tissues before birth. Further studies have proposed that developmentally distinct tissue MΦ can be identified based on the differential expression of F4/80 and CD11b, but whether a characteristic transcriptional profile exists is largely unknown. Here, we took advantage of an inducible fate-mapping system that facilitated the identification of CD45(+)c-kit(-)CX3CR1(+)F4/80(+) (A2) progenitors of the YS as the source of F4/80(hi) but not CD11b(hi) MΦ. Large-scale transcriptional profiling of MΦ precursors from the YS stage to adulthood allowed for building computational models for F4/80(hi) tissue macrophages being direct descendants of A2 progenitors. We further identified a distinct molecular signature of F4/80(hi) and CD11b(hi) MΦ and found that Irf8 was vital for MΦ maturation. Our data provide new cellular and molecular insights into the origin and developmental pathways of tissue MΦ. PMID:27412700

  4. Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities.

    PubMed

    Bain, Calum C; Hawley, Catherine A; Garner, Hannah; Scott, Charlotte L; Schridde, Anika; Steers, Nicholas J; Mack, Matthias; Joshi, Anagha; Guilliams, Martin; Mowat, Allan Mc I; Geissmann, Frederic; Jenkins, Stephen J

    2016-01-01

    Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80(hi)GATA6(+) macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C(+) monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80(lo)MHCII(+) cells that act, in part, as precursors of F4/80(hi)GATA6(+) macrophages. Notably, monocyte-derived F4/80(hi) macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age. PMID:27292029

  5. Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities

    PubMed Central

    Bain, Calum C.; Hawley, Catherine A.; Garner, Hannah; Scott, Charlotte L.; Schridde, Anika; Steers, Nicholas J.; Mack, Matthias; Joshi, Anagha; Guilliams, Martin; Mowat, Allan Mc I.; Geissmann, Frederic; Jenkins, Stephen J.

    2016-01-01

    Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80hiGATA6+ macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C+ monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80loMHCII+ cells that act, in part, as precursors of F4/80hiGATA6+ macrophages. Notably, monocyte-derived F4/80hi macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age. PMID:27292029

  6. Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes

    PubMed Central

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M.; Ochoa, Sara A.; Gónzalez-Pedrajo, Bertha; Espinosa, Norma; Eslava, Carlos; Hernández-Chiñas, Ulises; Mendoza-Hernández, Guillermo; Rodríguez-Leviz, Alejandra; Valencia-Mayoral, Pedro; Sadowinski-Pine, Stanislaw; Hernández-Castro, Rigoberto; Estrada-García, Iris; Muñoz-Hernández, Onofre; Rosas, Irma; Xicohtencatl-Cortes, Juan

    2012-01-01

    Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria. PMID:23284883

  7. Induction of Monocyte Chemoattractant Proteins in Macrophages via the Production of Granulocyte/Macrophage Colony-Stimulating Factor by Breast Cancer Cells

    PubMed Central

    Yoshimura, Teizo; Imamichi, Tomozumi; Weiss, Jonathan M.; Sato, Miwa; Li, Liangzhu; Matsukawa, Akihiro; Wang, Ji Ming

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1)/CCL2 plays an important role in the initiation and progression of cancer. We previously reported that in 4T1 murine breast cancer, non-tumor stromal cells, including macrophages, were the major source of MCP-1. In the present study, we analyzed the potential mechanisms by which MCP-1 is upregulated in macrophages infiltrating 4T1 tumors. We found that cell-free culture supernatants of 4T1 cells (4T1-sup) markedly upregulated MCP-1 production by peritoneal inflammatory macrophages. 4T1-sup also upregulated other MCPs, such as MCP-3/CCL7 and MCP-5/CCL12, but modestly upregulated neutrophil chemotactic chemokines, such as KC/CXCL1 or MIP-2/CXCL2. Physicochemical analysis indicated that an approximately 2–3 kDa 4T1 cell product was responsible for the capacity of 4T1-sup to upregulate MCP-1 expression by macrophages. A neutralizing antibody against granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage CSF, almost completely abrogated MCP-1-inducing activity of 4T1-sup, and recombinant GM-CSF potently upregulated MCP-1 production by macrophages. The expression levels of GM-CSF in 4T1 tumors in vivo were higher than other tumors, such as Lewis lung carcinoma. Treatment of mice with anti-GM-CSF antibody significantly reduced the growth of 4T1 tumors at the injection sites but did not reduce MCP-1 production or lung metastasis in tumor-bearing mice. These results indicate that 4T1 cells have the capacity to directly upregulate MCP-1 production by macrophages by releasing GM-CSF; however, other mechanisms are also involved in increased MCP-1 levels in the 4T1 tumor microenvironment. PMID:26834744

  8. Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

    PubMed Central

    Hsuan, S. L.; Kannan, M. S.; Jeyaseelan, S.; Prakash, Y. S.; Sieck, G. C.; Maheswaran, S. K.

    1998-01-01

    Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The

  9. Characterisation of a Novel Fc Conjugate of Macrophage Colony-stimulating Factor

    PubMed Central

    Gow, Deborah J; Sauter, Kristin A; Pridans, Clare; Moffat, Lindsey; Sehgal, Anuj; Stutchfield, Ben M; Raza, Sobia; Beard, Philippa M; Tsai, Yi Ting; Bainbridge, Graeme; Boner, Pamela L; Fici, Greg; Garcia-Tapia, David; Martin, Roger A; Oliphant, Theodore; Shelly, John A; Tiwari, Raksha; Wilson, Thomas L; Smith, Lee B; Mabbott, Neil A; Hume, David A

    2014-01-01

    We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs. The impact of CSF1-Fc was examined using the Csf1r-enhanced green fluorescent protein (EGFP) reporter gene in MacGreen mice. Administration of CSF1-Fc to mice drove extensive infiltration of all tissues by Csf1r-EGFP positive macrophages. The main consequence was hepatosplenomegaly, associated with proliferation of hepatocytes. Expression profiles of the liver indicated that infiltrating macrophages produced candidate mediators of hepatocyte proliferation including urokinase, tumor necrosis factor, and interleukin 6. CSF1-Fc also promoted osteoclastogenesis and produced pleiotropic effects on other organ systems, notably the testis, where CSF1-dependent macrophages have been implicated in homeostasis. However, it did not affect other putative CSF1 targets, notably intestine, where Paneth cell numbers and villus architecture were unchanged. CSF1 has therapeutic potential in regenerative medicine in multiple organs. We suggest that the CSF1-Fc conjugate retains this potential, and may permit daily delivery by injection rather than continuous infusion required for the core molecule. PMID:24962162

  10. Synthesis of antimony complexes of yeast mannan and mannan derivatives and their effect on Leishmania-infected macrophages.

    PubMed Central

    Cantos, G; Barbieri, C L; Iacomini, M; Gorin, P A; Travassos, L R

    1993-01-01

    Antimony(Sb)-yeast mannan complexes were synthesized as a strategy to introduce Sb into macrophages infected with Leishmania amastigotes. The complexes were taken up by endocytosis after specific recognition by alpha-D-mannosyl receptors on the macrophage membrane. About 90% of the intracellular parasites were destroyed by Sb-mannan in vitro, whereas the corresponding Sb concentration used as the pentavalent antimonial drug glucantime destroyed about 60% of the amastigotes. None of the Sb complexes prepared with mannan acid or basic derivatives was as effective as the simple Sb-mannan complex in clearing macrophage infection by Leishmania (L) amazonensis. The leishmanicidal effect of Sb-mannan was also demonstrated in vivo with infected hamsters. The alternative use of Sb-mannan complex in the treatment of human leishmaniasis is envisaged on the basis of parasite-killing efficiency and the use of a low antimony dose. Images Figure 1 PMID:8424752

  11. Active spice-derived components can inhibit inflammatory responses of adipose tissue in obesity by suppressing inflammatory actions of macrophages and release of monocyte chemoattractant protein-1 from adipocytes.

    PubMed

    Woo, Hae-Mi; Kang, Ji-Hye; Kawada, Teruo; Yoo, Hoon; Sung, Mi-Kyung; Yu, Rina

    2007-02-13

    Inflammation plays a key role in obesity-related pathologies such as cardiovascular disease, type II diabetes, and several types of cancer. Obesity-induced inflammation entails the enhancement of the recruitment of macrophages into adipose tissue and the release of various proinflammatory proteins from fat tissue. Therefore, the modulation of inflammatory responses in obesity may be useful for preventing or ameliorating obesity-related pathologies. Some spice-derived components, which are naturally occurring phytochemicals, elicit antiobesity and antiinflammatory properties. In this study, we investigated whether active spice-derived components can be applied to the suppression of obesity-induced inflammatory responses. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet and cultured to prepare an adipose tissue-conditioned medium. Raw 264.7 macrophages were treated with the adipose tissue-conditioned medium with or without active spice-derived components (i.e., diallyl disulfide, allyl isothiocyanate, piperine, zingerone and curcumin). Chemotaxis assay was performed to measure the degree of macrophage migration. Macrophage activation was estimated by measuring tumor necrosis factor-alpha (TNF-alpha), nitric oxide, and monocyte chemoattractant protein-1 (MCP-1) concentrations. The active spice-derived components markedly suppressed the migration of macrophages induced by the mesenteric adipose tissue-conditioned medium in a dose-dependent manner. Among the active spice-derived components studied, allyl isothiocyanate, zingerone, and curcumin significantly inhibited the cellular production of proinflammatory mediators such as TNF-alpha and nitric oxide, and significantly inhibited the release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components can suppress obesity-induced inflammatory responses by suppressing adipose tissue macrophage accumulation or activation and inhibiting MCP-1 release from adipocytes

  12. Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1

    SciTech Connect

    Mizoguchi, Izuru; Ooe, Yoshihiro; Hoshino, Shigeki; Shimura, Mari; Kasahara, Tadashi; Kano, Shigeyuki; Ohta, Toshiko; Takaku, Fumimaro; Nakayama, Yasuhide; Ishizaka, Yukihito . E-mail: zakay@ri.imcj.go.jp

    2005-12-23

    Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application.

  13. Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages.

    PubMed Central

    Reddy, V Y; Zhang, Q Y; Weiss, S J

    1995-01-01

    Human macrophages are believed to damage host tissues in chronic inflammatory disease states, but these cells have been reported to express only modest degradative activity in vitro. However, while examining the ability of human monocytes to degrade the extracellular matrix component elastin, we identified culture conditions under which the cells matured into a macrophage population that displayed a degradative phenotype hundreds of times more destructive than that previously ascribed to any other cell population. The monocyte-derived macrophages synthesized elastinolytic matrix metalloproteinases (i.e., gelatinase B and matrilysin) as well as cysteine proteinases (i.e., cathepsins B, L, and S), but only the cathepsins were detected in the extracellular milieu as fully processed, mature enzymes by either vital fluorescence or active-site labeling. Consistent with these observations, macrophage-mediated elastinolytic activity was not affected by matrix metalloproteinase inhibitors but could be almost completely abrogated by inhibiting cathepsins L and S. These data demonstrate that human macrophages mobilize cysteine proteinases to arm themselves with a powerful effector mechanism that can participate in the pathophysiologic remodeling of the extracellular matrix. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7731994

  14. CD45 Phosphatase Inhibits STAT3 Transcription Factor Activity in Myeloid Cells and Promotes Tumor-Associated Macrophage Differentiation.

    PubMed

    Kumar, Vinit; Cheng, Pingyan; Condamine, Thomas; Mony, Sridevi; Languino, Lucia R; McCaffrey, Judith C; Hockstein, Neil; Guarino, Michael; Masters, Gregory; Penman, Emily; Denstman, Fred; Xu, Xiaowei; Altieri, Dario C; Du, Hong; Yan, Cong; Gabrilovich, Dmitry I

    2016-02-16

    Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy. PMID:26885857

  15. Immunocytochemical Localization of Latent Transforming Growth Factor-B1 Activation by Stimulated Macrophages

    SciTech Connect

    Chong, Hyonkyong; Vodovotz, Yoram; Cox, G.W.; Barcellos-Hoff, M.H.

    1998-09-22

    Transforming growth factor-{beta}1 (TGF-{beta}) is secreted in a latent form consisting of mature TGF-{beta} noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-{beta} from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-{beta} action. We have identified two events associated with latent TGF-{beta} (LTGF-{beta}) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-{beta} concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-{gamma} and lipopolysaccharide reportedly activate LTGF-{beta} via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-{beta} activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-{beta} epitopes. The induction of TGF-{beta} immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-{beta} activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-{beta} and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-{beta} activation provides an important tool for studies of its regulation.

  16. Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

    NASA Technical Reports Server (NTRS)

    Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1999-01-01

    Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

  17. Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion.

    PubMed

    Pamir, Nathalie; Liu, Ning-Chun; Irwin, Angela; Becker, Lev; Peng, YuFeng; Ronsein, Graziella E; Bornfeldt, Karin E; Duffield, Jeremy S; Heinecke, Jay W

    2015-06-01

    The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2(-/-)) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2(-/-) mice had 75% fewer CD45(+)Cd11b(+)Cd11c(+)MHCII(+) F4/80(-) DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2(-/-) mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2(-/-) mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45(+)Cd11b(+)Cd11c(+)MHCII(+)F4/80(-) DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion. PMID:25931125

  18. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.

    PubMed

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-01-01

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  19. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway

    PubMed Central

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-01-01

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release ‘messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial ‘apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50–120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  20. Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

    PubMed Central

    Huynh-Thu, Vân Anh; Irrthum, Alexandre; Smeets, Hubert J. M.; Gustafsson, Jan-Åke; Steffensen, Knut R.; Mulder, Monique; Stinissen, Piet; Hellings, Niels; Hendriks, Jerome J. A.

    2012-01-01

    Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis. PMID:22984598

  1. Myelin-derived lipids modulate macrophage activity by liver X receptor activation.

    PubMed

    Bogie, Jeroen F J; Timmermans, Silke; Huynh-Thu, Vân Anh; Irrthum, Alexandre; Smeets, Hubert J M; Gustafsson, Jan-Åke; Steffensen, Knut R; Mulder, Monique; Stinissen, Piet; Hellings, Niels; Hendriks, Jerome J A

    2012-01-01

    Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis. PMID:22984598

  2. Macrophage-derived BAFF induces AID expression through the p38MAPK/CREB and JNK/AP-1 pathways.

    PubMed

    Kim, Hyun-A; Seo, Goo-Young; Kim, Pyeung-Hyeun

    2011-03-01

    BAFF is expressed primarily by macrophages and DCs. BAFF stimulates the differentiation and survival of B cells and induces Ig production. We have demonstrated previously that murine macrophages treated with TGF-β1 or IFN-γ express membrane-bound and soluble forms of BAFF. The ability of these two forms of BAFF to induce expression of AID, which plays a critical role in Ig CSR in B cells, was investigated. Both forms of BAFF, derived from macrophages activated by IFN-γ or TGF-β1, can increase AID expression. Subsequent analysis of BAFF signaling suggested that BAFF induces AID through BCMA, a BAFF-receptor, and p38MAPK and CREB act as intermediates in AID expression. In addition, JNK and AP-1 have similar activities. Our findings suggest that macrophage-derived BAFF stimulates B cells to express AID through BCMA and at least two different pathways, including the p38MAPK/CREB and the JNK/AP-1 pathways. PMID:21169521

  3. Macrophage migration inhibitory factor: a potential therapeutic target for rheumatoid arthritis

    PubMed Central

    Kim, Kyoung-Woon; Kim, Hae-Rim

    2016-01-01

    Macrophage migration inhibitory factor (MIF) is originally identified in the culture medium of activated T lymphocytes as a soluble factor that inhibits the random migration of macrophages. MIF is now recognized as a multipotent cytokine involved in the regulation of immune and inf lammatory responses. In rheumatoid arthritis (RA), MIF promotes inf lammatory responses by inducing proinflammatory cytokines and tissue-degrading molecules, promoting the proliferation and survival of synovial fibroblasts, stimulating neutrophil chemotaxis, and regulating angiogenesis and osteoclast differentiation. Expression of MIF in synovial tissue and synovial fluid levels of MIF are elevated in RA patients. Specifically, MIF levels correlate with RA disease activity and high levels are associated with bone erosion. In animal models of RA, the genetic and therapeutic inhibition of MIF has been shown to control inflammation and bone destruction. Based on the role of MIF in RA pathogenesis, small molecular inhibitors targeting it or its receptor pathways could provide a new therapeutic option for RA patients. PMID:27169879

  4. Macrophage migration inhibitory factor: a potential therapeutic target for rheumatoid arthritis.

    PubMed

    Kim, Kyoung-Woon; Kim, Hae-Rim

    2016-07-01

    Macrophage migration inhibitory factor (MIF) is originally identified in the culture medium of activated T lymphocytes as a soluble factor that inhibits the random migration of macrophages. MIF is now recognized as a multipotent cytokine involved in the regulation of immune and inf lammatory responses. In rheumatoid arthritis (RA), MIF promotes inf lammatory responses by inducing proinflammatory cytokines and tissue-degrading molecules, promoting the proliferation and survival of synovial fibroblasts, stimulating neutrophil chemotaxis, and regulating angiogenesis and osteoclast differentiation. Expression of MIF in synovial tissue and synovial fluid levels of MIF are elevated in RA patients. Specifically, MIF levels correlate with RA disease activity and high levels are associated with bone erosion. In animal models of RA, the genetic and therapeutic inhibition of MIF has been shown to control inflammation and bone destruction. Based on the role of MIF in RA pathogenesis, small molecular inhibitors targeting it or its receptor pathways could provide a new therapeutic option for RA patients. PMID:27169879

  5. Immune Enhancing Activity of β-(1,3)-Glucan Isolated from Genus Agrobacterium in Bone-Marrow Derived Macrophages and Mice Splenocytes.

    PubMed

    Byun, Eui-Baek; Jang, Beom-Su; Byun, Eui-Hong; Sung, Nak-Yun

    2016-01-01

    An effective method for activating macrophages and deriving a Th1 immune response could be used to improve the defenses of hosts. In this study, we investigated the immunomodulation effect and the related signaling mechanism of [Formula: see text]-(1,3)-glucan, isolated from the Agrobacterium species. Here, we found that [Formula: see text]-(1,3)-glucan predominantly induced the tumor necrosis factor (TNF)-[Formula: see text], interleukin (IL)-1[Formula: see text], IL-6, IL-12p70, and nitric oxide, which was dependent on mitogen-activated protein kinases (MAPK) and nuclear factor (NF)-[Formula: see text]B signaling. Additionally, [Formula: see text]-(1,3)-glucan treatment significantly up-regulated the expression of the co-stimulatory molecules CD80 and CD86, and also significantly increased the expression of iNOS and Dectin-1, which is a transmembrane protein that binds [Formula: see text]-glucan and associates with macrophage activation. Importantly, the splenic T cells co-cultured with [Formula: see text]-(1,3)-glucan-treated macrophages produced the a Th1 cytokine profile that includes high levels of IFN-[Formula: see text], but not IL-4 (Th2 cytokine), indicating that [Formula: see text]-(1,3)-glucan contributes to Th1 polarization of the immune response. Taken together, our results suggest that [Formula: see text]-(1,3)-glucan isolated from Agrobacterium species can induce macrophage activation through the MAPK and NF-[Formula: see text]B signaling pathway, as well as Th1 polarization. PMID:27430908

  6. Microglia and monocyte-derived macrophages: functionally distinct populations that act in concert in CNS plasticity and repair

    PubMed Central

    London, Anat; Cohen, Merav; Schwartz, Michal

    2013-01-01

    Functional macrophage heterogeneity is recognized outside the central nervous system (CNS), where alternatively activated macrophages can perform immune-resolving functions. Such functional heterogeneity was largely ignored in the CNS, with respect to the resident microglia and the myeloid-derived cells recruited from the blood following injury or disease, previously defined as blood-derived microglia; both were indistinguishably perceived detrimental. Our studies have led us to view the myeloid-derived infiltrating cells as functionally distinct from the resident microglia, and accordingly, to name them monocyte-derived macrophages (mo-MΦ). Although microglia perform various maintenance and protective roles, under certain conditions when they can no longer provide protection, mo-MΦ are recruited to the damaged CNS; there, they act not as microglial replacements but rather assistant cells, providing activities that cannot be timely performed by the resident cells. Here, we focus on the functional heterogeneity of microglia/mo-MΦ, emphasizing that, as opposed to the mo-MΦ, microglia often fail to timely acquire the phenotype essential for CNS repair. PMID:23596391

  7. The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation.

    PubMed

    Qian, Feng; Deng, Jing; Lee, Yong Gyu; Zhu, Jimmy; Karpurapu, Manjula; Chung, Sangwoon; Zheng, Jun-Nian; Xiao, Lei; Park, Gye Young; Christman, John W

    2015-12-01

    The transcription factor PU.1 is involved in regulation of macrophage differentiation and maturation. However, the role of PU.1 in alternatively activated macrophage (AAM) and asthmatic inflammation has yet been investigated. Here we report that PU.1 serves as a critical regulator of AAM polarization and promotes the pathological progress of asthmatic airway inflammation. In response to the challenge of DRA (dust mite, ragweed, and Aspergillus) allergens, conditional PU.1-deficient (PU/ER(T)(+/-)) mice displayed attenuated allergic airway inflammation, including decreased alveolar eosinophil infiltration and reduced production of IgE, which were associated with decreased mucous glands and goblet cell hyperplasia. The reduced asthmatic inflammation in PU/ER(T)(+/-) mice was restored by adoptive transfer of IL-4-induced wild-type (WT) macrophages. Moreover, after treating PU/ER(T)(+/-) mice with tamoxifen to rescue PU.1 function, the allergic asthmatic inflammation was significantly restored. In vitro studies demonstrate that treatment of PU.1-deficient macrophages with IL-4 attenuated the expression of chitinase 3-like 3 (Ym-1) and resistin-like molecule alpha 1 (Fizz-1), two specific markers of AAM polarization. In addition, PU.1 expression in macrophages was inducible in response to IL-4 challenge, which was associated with phosphorylation of signal transducer and activator of transcription 6 (STAT6). Furthermore, DRA challenge in sensitized mice almost abrogated gene expression of Ym-1 and Fizz-1 in lung tissues of PU/ER(T)(+/-) mice compared with WT mice. These data, all together, indicate that PU.1 plays a critical role in AAM polarization and asthmatic inflammation. PMID:26101328

  8. Dual transcriptome sequencing reveals resistance of TLR4 ligand-activated bone marrow-derived macrophages to inflammation mediated by the BET inhibitor JQ1

    PubMed Central

    Das, Amitabh; Chai, Jin Choul; Yang, Chul-su; Lee, Young Seek; Das, Nando Dulal; Jung, Kyoung Hwa; Chai, Young Gyu

    2015-01-01

    Persistent macrophage activation is associated with the expression of various pro-inflammatory genes, cytokines and chemokines, which may initiate or amplify inflammatory disorders. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities in macrophages. However, a genome-wide search for JQ1 molecular targets has not been undertaken. The present study aimed at evaluating the anti-inflammatory function and underlying genes that are targeted by JQ1 in LPS-stimulated primary bone marrow-derived macrophages (BMDMs) using global transcriptomic RNA sequencing and quantitative real-time PCR. Among the annotated genes, transcriptional sequencing of BMDMs that were treated with JQ1 revealed a selective effect on LPS-induced gene expression in which the induction of cytokines/chemokines, interferon-stimulated genes, and prominent (transcription factors) TFs was suppressed. Additionally, we found that JQ1 reduced the expression of previously unidentified genes that are important in inflammation. Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced cytokines/chemokines in the supernatants of LPS treated BMDMs. Moreover, the biological pathways and gene ontology of the differentially expressed genes were determined in the JQ1 treatment of BMDMs. These unprecedented results suggest that the BET inhibitor JQ1 is a candidate for the prevention or therapeutic treatment of inflammatory disorders. PMID:26582142

  9. Infection of porcine bone marrow-derived macrophages by porcine respiratory and reproductive syndrome virus impairs phagosomal maturation.

    PubMed

    Chaudhuri, Sibapriya; McKenna, Neil; Balce, Dale R; Yates, Robin M

    2016-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense, ssRNA virus of the genus Arterivirus, is a devastating disease of swine worldwide. Key early targets of PRRSV infection in pigs include professional phagocytes in the lung, such as alveolar and interstitial macrophages and dendritic cells, the dysfunction of which is believed to be responsible for much of the associated mortality. In order to study the effect of virus infection on phagocyte function, the development of a robust, reproducible model would be advantageous. Given the limitations of current models, we set out to develop a porcine bone marrow-derived macrophage (PBMMΦ) cell model to study phagosomal maturation and function during PRRSV infection. Derivation of PBMMΦs from marrow using cultured L929 fibroblast supernatant produced a homogeneous population of cells that exhibited macrophage-like morphology and proficiency in Fc-receptor-mediated phagocytosis and phagosomal maturation. PBMMΦs were permissive to PRRSV infection, resulting in a productive infection that peaked at 24 h. Assessment of the effect of PRRSV infection on the properties of phagosomal maturation in PBMMΦs revealed a significant decrease in phagosomal proteolysis and lowered production of reactive oxygen species, but no change in PBMMΦ viability, phagocytosis or the ability of phagosomes to acidify. In this study, we present a new model to investigate PRRSV infection of phagocytes, which demonstrates a significant effect on phagosomal maturation with the associated implications on proper macrophage function. This model can also be used to study the effect on the phagosomal microenvironment of infection by other viruses targeting porcine macrophages. PMID:26702996

  10. Mycobacterium tuberculosis senses host-derived carbon monoxide during macrophage infection

    PubMed Central

    Shiloh, Michael U.; Manzanillo, Paolo; Cox, Jeffery S.

    2010-01-01

    Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistance in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment. PMID:18474359

  11. Effect of cortisol and/or DHEA on THP1-derived macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Bongiovanni, Bettina; Mata-Espinosa, Dulce; D'Attilio, Luciano; Leon-Contreras, Juan Carlos; Marquez-Velasco, Ricardo; Bottasso, Oscar; Hernandez-Pando, Rogelio; Bay, María Luisa

    2015-09-01

    Tuberculosis (TB) is a major health problem requiring an appropriate cell immune response to be controlled. Macrophages play a central role in the response against Mycobacterium tuberculosis (Mtb). Given our prior studies in which adrenal steroids were found to modify the cellular immune responses from TB patients, it was sensible to analyze the immunomodulatory capability of cortisol and DHEA on macrophages infected with Mtb. The human macrophage-like THP-1 cells were infected with the H37Rv strain of Mtb and treated with Cortisol and DHEA at different doses. We monitored phagocytosis, intracellular-bacterial growth, autophagosoma formation, as well as cytokine gene expression and production. Cultures exposed to cortisol showed a decreased production of IL-1β, TNF-α, with DHEA being unable to modify the pattern of cytokine production or to reverse the cortisol inhibitory effects. Interestingly the intra-macrophagic bacterial burden was found reduced by DHEA treatment. While this effect was not related to a different cytokine pattern, in terms their production or mRNA expression, DHEA treatment did promote autophagy in Mtb-infected macrophages, irrespective of Cortisol presence. In essence, the better control of Mtb load by DHEA-treated macrophages seems to be dependent on an autophagic mechanism. The present results are relevant for two reasons as autophagy is not only important for clearance of mycobacteria but also for the prevention of tissue damage. PMID:26099547

  12. Peroxisome Proliferator-activated Receptor γ Induces Apoptosis and Inhibits Autophagy of Human Monocyte-derived Macrophages via Induction of Cathepsin L

    PubMed Central

    Mahmood, Dler Faieeq Darweesh; Jguirim-Souissi, Imene; Khadija, El-Hadri; Blondeau, Nicolas; Diderot, Vimala; Amrani, Souliman; Slimane, Mohamed-Naceur; Syrovets, Tatiana; Simmet, Thomas; Rouis, Mustapha

    2011-01-01

    Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) γ, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPARγ might affect macrophage function in the context of chronic inflammation such as atherogenesis. In the present study, we examined the effect of PPARγ activation on the expression of CatL in human monocyte-derived macrophages (HMDM). Activation of PPARγ by the specific agonist GW929 concentration-dependently increased the levels of CatL mRNA and protein in HMDM. By promoter analysis, we identified a functional PPAR response element-like sequence that positively regulates CatL expression. In addition, we found that PPARγ-induced CatL promotes the degradation of Bcl2 without affecting Bax protein levels. Consistently, degradation of Bcl2 could be prevented by a specific CatL inhibitor, confirming the causative role of CatL. PPARγ-induced CatL was found to decrease autophagy through reduction of beclin 1 and LC3 protein levels. The reduction of these proteins involved in autophagic cell death was antagonized either by the CatL inhibitor or by CatL knockdown. In conclusion, our data show that PPARγ can specifically induce CatL, a proatherogenic protease, in HMDM. In turn, CatL inhibits autophagy and induces apoptosis. Thus, the proatherogenic effect of CatL could be neutralized by apoptosis, a beneficial phenomenon, at least in the early stages of atherosclerosis. PMID:21700710

  13. Interleukin-12 gene expression in human monocyte-derived macrophages stimulated with Mycobacterium bovis BCG: cytokine regulation and effect of NK cells.

    PubMed Central

    Matsumoto, H; Suzuki, K; Tsuyuguchi, K; Tanaka, E; Amitani, R; Maeda, A; Yamamoto, K; Sasada, M; Kuze, F

    1997-01-01

    Macrophage-derived interleukin-12 (IL-12) is essential for the activation of a protective immune response against intracellular pathogens. In this study, we examined the regulation of IL-12 mRNA expression by monocyte-derived macrophages (MDM) in response to Mycobacterium bovis BCG stimulation. A reverse transcription-PCR assay detected p40 mRNA of IL-12 at 3 h and showed a peak at 6 to 12 h with a subsequent decline. Semiquantitation of mRNA levels by competitive PCR revealed that pretreatment with gamma interferon (IFN-gamma) amplified the expression approximately 100-fold, while pretreatment with tumor necrosis factor alpha (TNF-alpha) or granulocyte-macrophage colony-stimulating factor augmented this expression about 10-fold. In contrast, pretreatment with IL-10 and IL-4 inhibited IL-12 mRNA expression. These results were further confirmed by measuring the p70 bioactive protein level in each conditioned medium by an enzyme-linked immunosorbent assay. Since IL-12 mRNA expression was weak without cytokine pretreatment and IFN-gamma strongly augmented production, we speculated that IFN-gamma might have a role in BCG stimulation of IL-12 mRNA expression. Unexpectedly, the addition of three different kinds of anti-IFN-gamma antibodies and anti-IFN-gamma receptor antibody and the coaddition of anti-TNF-alpha antibody with anti-IFN-gamma receptor antibody all failed to inhibit IL-12 mRNA expression. However, the MiniMACS method used to remove NK cells from a mononuclear cell suspension inhibited the expression of p40 mRNA but not the expression of mRNA of TNF-alpha or IL-1beta. We concluded that the coexistence of NK cells was essential for the induction of IL-12 in MDM stimulated with BCG rather than through the secretion of IFN-gamma. PMID:9353012

  14. Enhanced invasion of lung adenocarcinoma cells after co-culture with THP-1-derived macrophages via the induction of EMT by IL-6.

    PubMed

    Dehai, Che; Bo, Pan; Qiang, Tian; Lihua, Shang; Fang, Liu; Shi, Jin; Jingyan, Cao; Yan, Yu; Guangbin, Wang; Zhenjun, Yuan

    2014-07-01

    Lung cancer is the leading cause of cancer mortality worldwide, and the cause of death is metastasis. The epithelial-to-mesenchymal transition (EMT) plays a key role in the process of metastasis. Macrophages within the lung cancer microenvironment release cytokines, such as interleukin-6 (IL-6), and promote lung cancer cell invasion and metastasis. However, the interaction between macrophages and lung cancer cells and the effect of this interaction on the expression of IL-6, EMT, and the invasiveness of lung cancer cells remain unclear. Therefore, we established an in vitro co-culture model of human lung adenocarcinoma A549 or H1299 cells with THP-1-derived macrophages to illuminate the important role of macrophages in the invasion of lung cancer. In this study, we demonstrated that the concentrations of IL-6 in the co-culture supernatants were significantly increased compared with controls. Thus, a complex chemical cross-talk is induced by the indirect cell-to-cell contact between lung cancer cells and THP-1-derived macrophages. THP-1-derived macrophages appeared to play an important initiator role in the process. The analysis of the mRNA expression profiles of the sorted cells from the co-culture system revealed that the co-cultured lung cancer cells are the main source of the observed increase in IL-6 secretion. In addition, the interactions between lung cancer cells and THP-1-derived macrophages are bidirectional. The THP-1-derived macrophages underwent differentiation towards the M2-macrophage phenotype during the co-culture process. The expression of IL-6 was correlated with the induction of EMT, which contributed to a significant increase in the invasiveness of the A549 and H1299 cells in vitro. In addition, the addition of an anti-IL-6 antibody reversed these changes. In summary, we demonstrated that the in vitro co-culture of A549 or H1299 cells with THP-1-derived macrophages upregulates IL-6 expression, which increases the invasion ability of the A549 and

  15. Euphorbia supina inhibits inflammatory mediators in mouse bone marrow-derived mast cells and macrophages.

    PubMed

    Chae, Hee-Sung; Song, Hyuk-Hwan; Kim, Young-Mi; Lee, Hyeong-Kyu; Oh, Sei-Ryang; Chin, Young-Won

    2015-12-01

    Euphorbia supina has been traditionally used for the treatment of furuncle and bloody diarrhea relevant to the inflammatory process. It has been proven to have a variety of pharmacological efficacies including antiarthritic, detoxification, hemostatic, and diuretic activities. RAW 264.7 macrophages and bone marrow-derived mast cells (BMMCs) were used to determine the anti-inflammatory and anti-allergic effects of E. supina (ES). NO production was assayed by measuring the nitrite content of the supernatants of cultured RAW 264.7 cells. β-hexosaminidase, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis. ELISA was used for the analysis of interleukin-6 expression, and Western blotting was used to analyze 5-LOX, iNOS, and MAPK activation. The relevant gene expression upon ES treatment was measured by RT-PCR. ES inhibited inducible nitric oxide synthase (iNOS) in RAW 264.7 cells, and IL-6 and LTC4 production in PMA- and A23187-induced BMMCs along with the downregulation of 5-LOX gene expression. Furthermore, in the present study, a decrease in p-ERK, p-JNK, and p-P38 expression, as well as the suppression of degranulation, were observed by treatment with ES. Further in vivo study revealed that ES treatment also remarkably inhibited xylene-induced mouse ear edema and MPO levels in mice ears. This study demonstrates that ES has a potential regulatory effect on the expression of inflammatory mediators through the inhibition of both the phosphorylation of MAPK signaling and the activation of degranulation. PMID:26386544

  16. Granulocyte-macrophage colony-stimulating factor and pulmonary surfactant homeostasis.

    PubMed

    Reed, J A; Whitsett, J A

    1998-01-01

    Pulmonary surfactant lining the alveolus of the lung is critical to postnatal adaptation to air breathing. Precise concentrations of surfactant proteins and lipids are maintained in the alveolar space by a careful balance among synthesis, recycling, and catabolism. Pulmonary alveolar proteinosis is a rare pulmonary disease associated with accumulation of surfactant lipids and proteins in the alveolar spaces. Recent work with transgenic mice demonstrated that disruption of the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) or the common beta-subunit of the GM-CSF receptor caused alveolar proteinosis that was histologically similar to that seen in human patients. The defect in surfactant homeostasis is caused by decreased surfactant clearance, mediated (at least in part) by dysfunction of the alveolar macrophage. Local production of GM-CSF corrects the alveolar proteinosis in the GM-CSF knockout mouse. Likewise, transplantation of wild-type bone marrow cells expressing the common beta-chain of the GM-CSF receptor restores surfactant homeostasis in the GM-CSF receptor knockout mouse. These studies demonstrate the previously unanticipated role of GM-CSF signaling in surfactant homeostasis, mediated (at least in part) by its actions on the clearance of surfactant lipids and proteins by the alveolar macrophage. These findings may have important implications for the diagnosis and treatment of pulmonary alveolar proteinosis syndromes in humans. PMID:9686680

  17. Effect of anti-macrophage migration inhibitory factor antibody on lipopolysaccharide-induced pulmonary neutrophil accumulation.

    PubMed

    Makita, H; Nishimura, M; Miyamoto, K; Nakano, T; Tanino, Y; Hirokawa, J; Nishihira, J; Kawakami, Y

    1998-08-01

    Macrophage migration inhibitory factor (MIF) is a recently rediscovered pro-inflammatory cytokine that has the unique potential to override the anti-inflammatory action of glucocorticoids. Since recent reports suggest the pivotal role of MIF in acute lung injury, we examined the protective effect of anti-MIF antibody on lipopolysaccharide (LPS)-induced acute lung injury in rats. Rats were injected with LPS (7 mg/kg) intraperitoneally with or without pretreatment with anti-MIF antibody. The anti-MIF antibody significantly attenuated LPS-induced migration of neutrophils to the lungs at 4 and 24 h as demonstrated by observation of the number of neutrophils per alveolus, the activity of myeloperoxidase of the lung tissue, and cell differentiation of neutrophils in bronchoalveolar lavage (BAL) fluid. The increased level of macrophage inflammatory protein-2, a powerful neutrophil chemokine, in BAL fluid was also significantly attenuated by pretreatment with the anti-MIF antibody as compared with the control group. Additionally, positive immunostaining for MIF was observed in bronchial epithelial cells and alveolar macrophages, and Northern blot analysis of lung tissues demonstrated increased MIF mRNA 24 h after LPS injection. These data suggest that the anti-MIF antibody has therapeutic potential for the treatment of acute lung injury by suppressing the level of neutrophil chemokine in the lungs. PMID:9700137

  18. Synovial macrophage-derived IL-1β regulates the calcitonin receptor in osteoarthritic mice.

    PubMed

    Takano, S; Uchida, K; Miyagi, M; Inoue, G; Aikawa, J; Fujimaki, H; Minatani, A; Sato, M; Iwabuchi, K; Takaso, M

    2016-01-01

    Recent studies have reported that calcitonin gene-related peptide (CGRP) contributes to joint pain. However, regulation of the CGRP/CGRP receptor signalling in osteoarthritis (OA) is not fully understood. To investigate the regulation of CGRP/CGRP receptor signalling by macrophages in the synovial tissue (ST) of OA joints, we characterized the gene expression profiles of CGRP and CGRP receptors in the ST of OA mice (STR/Ort). In addition, we examined whether macrophage depletion by the systemic injection of clodronate-laden liposomes affected the expression of CGRP and CGRP receptors in ST. CD11c(+) macrophages in the ST of STR/Ort and C57BL/6J mice were analysed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to evaluate the expression of interleukin (IL)-1β, CGRP, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in F4/80(+) and F4/80(-) cells. The effects of IL-1β on the expression of CGRP and CLR by cultured synovial cells were also examined. The percentage of CD11c(+) macrophages in the ST of STR/Ort was higher than that in C57/BL6J mice. Notably, the F4/80(+) cell fraction expressed IL-1β highly, whereas the F4/80(-) cell fraction expressed CGRP, CLR, and RAMP1 highly. In addition, expression of the IL-1β and CLR genes was increased in ST, but was decreased upon macrophage depletion, and the IL-1β treatment of cultured synovial cells up-regulated CLR. Taken together, the present findings suggest that synovial macrophages are the major producers of IL-1β and regulators of CLR in OA mice. Therefore, macrophages and IL-1β may be suitable therapeutic targets for treating OA pain. PMID:26400621

  19. Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion*

    PubMed Central

    Pamir, Nathalie; Liu, Ning-Chun; Irwin, Angela; Becker, Lev; Peng, YuFeng; Ronsein, Graziella E.; Bornfeldt, Karin E.; Duffield, Jeremy S.; Heinecke, Jay W.

    2015-01-01

    The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2−/−) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2−/− mice had 75% fewer CD45+Cd11b+Cd11c+MHCII+ F4/80− DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2−/− mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2−/− mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45+Cd11b+Cd11c+MHCII+F4/80− DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion. PMID:25931125

  20. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

    PubMed Central

    Jensen, Kirsty; Anderson, Jennifer A.; Glass, Elizabeth J.

    2014-01-01

    The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA. PMID:24598124

  1. Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor alpha required as a costimulatory factor for interferon gamma-induced activation.

    PubMed Central

    Oswald, I P; Wynn, T A; Sher, A; James, S L

    1992-01-01

    Interleukin 10 (IL-10) inhibits interferon gamma-induced macrophage activation for cytotoxicity against larvae of the human parasite Schistosoma mansoni by suppressing production of the toxic effector molecule nitric oxide (NO). In this study, the mechanism of IL-10 action was identified as inhibition of endogenous tumor necrosis factor alpha (TNF-alpha) production by interferon gamma-activated macrophages. TNF-alpha appears to serve as a cofactor for interferon gamma-mediated activation, since both schistosomulum killing and NO production were inhibited by anti-TNF-alpha antibody, whereas TNF-alpha alone was unable to stimulate these macrophage functions. IL-10 blocked TNF-alpha production by interferon gamma-treated macrophages at the levels of both protein and mRNA synthesis. Addition of exogenous TNF-alpha reversed IL-10-mediated suppression of macrophage cytotoxic activity as well as NO production. Likewise, addition of a macrophage-triggering agent (bacterial lipopolysaccharide or muramyl dipeptide), which induced the production of TNF-alpha, also reversed the suppressive effect of IL-10 on cytotoxic function. In contrast to IL-10, two other cytokines, IL-4 and transforming growth factor beta, which also inhibit macrophage activation for schistosomulum killing and NO production, did not substantially suppress endogenous TNF-alpha production. These results, therefore, describe a separate pathway by which macrophage microbicidal function is inhibited by the down-regulatory cytokine IL-10. Images PMID:1528880

  2. Macrophage Migration Inhibitory Factor (MIF) Supports Homing of Osteoclast Precursors to Peripheral Osteolytic Lesions.

    PubMed

    Movila, Alexandru; Ishii, Takenobu; Albassam, Abdullah; Wisitrasameewong, Wichaya; Howait, Mohammed; Yamaguchi, Tsuguno; Ruiz-Torruella, Montserrat; Bahammam, Laila; Nishimura, Kazuaki; Van Dyke, Thomas; Kawai, Toshihisa

    2016-09-01

    By binding to its chemokine receptor CXCR4 on osteoclast precursor cells (OCPs), it is well known that stromal cell-derived factor-1 (SDF-1) promotes the chemotactic recruitment of circulating OCPs to the homeostatic bone remodeling site. However, the engagement of circulating OCPs in pathogenic bone resorption remains to be elucidated. The present study investigated a possible chemoattractant role of macrophage migration inhibitory factor (MIF), another ligand for C-X-C chemokine receptor type 4 (CXCR4), in the recruitment of circulating OCPs to the bone lytic lesion. To accomplish this, we used Csf1r-eGFP-knock-in (KI) mice to establish an animal model of polymethylmethacrylate (PMMA) particle-induced calvarial osteolysis. In the circulating Csf1r-eGFP+ cells of healthy Csf1r-eGFP-KI mice, Csf1r+/CD11b+ cells showed a greater degree of RANKL-induced osteoclastogenesis compared to a subset of Csf1r+/RANK+ cells in vitro. Therefore, Csf1r-eGFP+/CD11b+ cells were targeted as functionally relevant OCPs in the present study. Although expression of the two cognate receptors for MIF, CXCR2 and CXCR4, was elevated on Csf1r+/CD11b+ cells, transmigration of OCPs toward recombinant MIF in vitro was facilitated by ligation with CXCR4, but not CXCR2. Meanwhile, the level of PMMA-induced bone resorption in calvaria was markedly greater in wild-type (WT) mice compared to that detected in MIF-knockout (KO) mice. Interestingly, in contrast to the elevated MIF, diminished SDF-1 was detected in a particle-induced bone lytic lesion of WT mice in conjunction with an increased number of infiltrating CXCR4+ OCPs. However, such diminished SDF-1 was not found in the PMMA-injected calvaria of MIF-KO mice. Furthermore, stimulation of osteoblasts with MIF in vitro suppressed their production of SDF-1, suggesting that MIF can downmodulate SDF-1 production in bone tissue. Systemically administered anti-MIF neutralizing monoclonal antibody (mAb) inhibited the homing of CXCR4+ OCPs, as well as

  3. Fate mapping reveals that microglia and recruited monocyte-derived macrophages are definitively distinguishable by phenotype in the retina

    PubMed Central

    O’Koren, E. G.; Mathew, R.; Saban, D. R.

    2016-01-01

    The recent paradigm shift that microglia are yolk sac-derived, not hematopoietic-derived, is reshaping our knowledge about the isolated role of microglia in CNS diseases, including degenerative conditions of the retina. However, unraveling microglial-specific functions has been hindered by phenotypic overlap of microglia with monocyte-derived macrophages. The latter are differentiated from recruited monocytes in neuroinflammation, including retina. Here we demonstrate the use of fate mapping wherein microglia and monocyte-derived cells are endogenously labeled with different fluorescent reporters. Combining this method with 12-color flow cytometry, we show that these two populations are definitively distinguishable by phenotype in retina. We prove that retinal microglia have a unique CD45lo CD11clo F4/80lo I-A/I-E− signature, conserved in the steady state and during retinal injury. The latter was observed in the widely used light-induced retinal degeneration model and corroborated in other models, including whole-body irradiation/bone-marrow transplantation. The literature contains conflicting observations about whether microglia, including in the retina, increase expression of these markers in neuroinflammation. We show that monocyte-derived macrophages have elevated expression of these surface markers, not microglia. Our resolution of such phenotypic differences may serve as a robust way to help characterize isolated roles of these cells in retinal neuroinflammation and possibly elsewhere in CNS. PMID:26856416

  4. Fate mapping reveals that microglia and recruited monocyte-derived macrophages are definitively distinguishable by phenotype in the retina.

    PubMed

    O'Koren, E G; Mathew, R; Saban, D R

    2016-01-01

    The recent paradigm shift that microglia are yolk sac-derived, not hematopoietic-derived, is reshaping our knowledge about the isolated role of microglia in CNS diseases, including degenerative conditions of the retina. However, unraveling microglial-specific functions has been hindered by phenotypic overlap of microglia with monocyte-derived macrophages. The latter are differentiated from recruited monocytes in neuroinflammation, including retina. Here we demonstrate the use of fate mapping wherein microglia and monocyte-derived cells are endogenously labeled with different fluorescent reporters. Combining this method with 12-color flow cytometry, we show that these two populations are definitively distinguishable by phenotype in retina. We prove that retinal microglia have a unique CD45(lo) CD11c(lo) F4/80(lo) I-A/I-E(-) signature, conserved in the steady state and during retinal injury. The latter was observed in the widely used light-induced retinal degeneration model and corroborated in other models, including whole-body irradiation/bone-marrow transplantation. The literature contains conflicting observations about whether microglia, including in the retina, increase expression of these markers in neuroinflammation. We show that monocyte-derived macrophages have elevated expression of these surface markers, not microglia. Our resolution of such phenotypic differences may serve as a robust way to help characterize isolated roles of these cells in retinal neuroinflammation and possibly elsewhere in CNS. PMID:26856416

  5. Killing of Escherichia coli by Crohn's Disease Monocyte-derived Macrophages and Its Enhancement by Hydroxychloroquine and Vitamin D

    PubMed Central

    Flanagan, Paul K.; Chiewchengchol, Direkrit; Wright, Helen L.; Edwards, Steven W.; Alswied, Abdullah; Satsangi, Jack; Subramanian, Sreedhar; Rhodes, Jonathan M.

    2015-01-01

    Background: Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly adherent and invasive Escherichia coli that replicate inside macrophage phagolysosomes. We compared CD and healthy control (HC) macrophages for their abilities to kill E. coli and generate neutrophil chemoattractants and also assessed the effects of hydroxychloroquine (HCQ) and vitamin D on killing of phagocytosed E. coli. Methods: Peripheral blood monocyte-derived macrophages from CD and HC were compared for bacterial killing and generation of neutrophil chemoattractants in response to CD-derived E. coli. Escherichia coli replication was also assessed in the presence and absence of HCQ, alone and with antibiotics, and vitamin D. Results: Monocyte-derived macrophages from patients with CD were similar to HC in allowing replication of phagocytosed CD-derived E. coli: HM605 {CD: N = 10, mean fold replication in 3 hr = 1.08 (95% confidence interval [CI], 0.39–1.78); HC: N = 9, 1.50 (95% CI, 1.02–1.97); P = 0.15} and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD = 2.55 [95% CI, 2.31–2.80]; HC = 2.65 [95% CI, 2.46–2.85], P = 0.42). HCQ and 1,25 OH2-vitamin D3 both caused dose-dependent inhibition of intramacrophage E. coli replication 3-hour postinfection; HCQ: 73.9% inhibition (P < 0.001) at 1 μg/mL, accompanied by raised intraphagosomal pH, and 1,25 OH2-vitamin D3: 80.7% inhibition (P < 0.05) at 80 nM. HCQ had synergistic effects with doxycycline and ciprofloxacin. Conclusions: CD and HC macrophages perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics. PMID:25839777

  6. Nanoparticle based galectin-1 gene silencing, implications in methamphetamine regulation of HIV-1 infection in monocyte derived macrophages.

    PubMed

    Reynolds, Jessica L; Law, Wing Cheung; Mahajan, Supriya D; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E; Yong, Ken-Tye; Hui, Rui; Prasad, Paras N; Schwartz, Stanley A

    2012-09-01

    Galectin-1, an adhesion molecule, is expressed in macrophages and implicated in human immunodeficiency virus (HIV-1) viral adsorption. In this study, we investigated the effects of methamphetamine on galectin-1 production in human monocyte derived macrophages (MDM) and the role of galectin-1 in methamphetamine potentiation of HIV-1 infection. Herein we show that levels of galectin-1 gene and protein expression are significantly increased by methamphetamine. Furthermore, concomitant incubation of MDM with galectin-1 and methamphetamine facilitates HIV-1 infection compared to galectin-1 alone or methamphetamine alone. We utilized a nanotechnology approach that uses gold nanorod (GNR)-galectin-1 siRNA complexes (nanoplexes) to inhibit gene expression for galectin-1. Nanoplexes significantly silenced gene expression for galectin-1 and reversed the effects of methamphetamine on galectin-1 gene expression. Moreover, the effects of methamphetamine on HIV-1 infection were attenuated in the presence of the nanoplex in MDM. PMID:22689223

  7. Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation

    PubMed Central

    Park, Keon-Young; Li, Weiwei A.; Platt, Manu O.

    2012-01-01

    Patient-to-patient variability in disease progression continues to complicate clinical decisions of treatment regimens for cardiovascular diseases, metastatic cancers and osteoporosis. Here, we investigated if monocytes, circulating white blood cells that enter tissues and contribute to disease progression by differentiating into macrophages or osteoclasts, could be useful in understanding this variability. Monocyte-derived macrophages and osteoclasts produce cysteine cathepsins, powerful extracellular matrix proteases which have been mechanistically linked to accelerated atherosclerotic, osteoporotic, and tumor progression. We hypothesized that multivariate analysis of temporal kinase activation states during monocyte differentiation could predict cathepsin proteolytic responses of monocyte-derived macrophages and osteoclasts in a patient-specific manner. Freshly isolated primary monocytes were differentiated with M-CSF or RANKL into macrophages or osteoclasts, respectively, and phosphorylation of ERK1/2, Akt, p38 MAPK, JNK, c-jun, and IκB-α were measured at days 1, 3, 6, and 9. In parallel, cell diameters and numbers of nuclei were measured, and multiplex cathepsin zymography was used to quantify cathepsins K, L, S, and V activity from cell extracts and conditioned media. There was extensive patient-to-patient variability in temporal kinase activation states, cell morphologies, and cathepsin K, L, S, and V proteolytic activity. Partial least squares regression models trained with temporal kinase activation states successfully predicted patient-specific morphological characteristics (mean cell diameter and number of nuclei) and patient-specific cathepsin proteolytic activity with predictability as high as 95%, even with the challenge of incorporating the complex, unknown cues from individual patients’ unique genetic and biochemical backgrounds. This personalized medicine approach considers patient variability in kinase signals to predict cathepsin activity

  8. Chronic Iron Overload Results in Impaired Bacterial Killing of THP-1 Derived Macrophage through the Inhibition of Lysosomal Acidification

    PubMed Central

    Kao, Jun-Kai; Wang, Shih-Chung; Ho, Li-Wei; Huang, Shi-Wei; Chang, Shu-Hao; Yang, Rei-Cheng; Ke, Yu-Yuan; Wu, Chun-Ying; Wang, Jiu-Yao; Shieh, Jeng-Jer

    2016-01-01

    Iron is essential for living organisms and the disturbance of iron homeostasis is associated with altered immune function. Additionally, bacterial infections can cause major complications in instances of chronic iron overload, such as patients with transfusion-dependent thalassemia. Monocytes and macrophages play important roles in maintaining systemic iron homoeostasis and in defense against invading pathogens. However, the effect of iron overload on the function of monocytes and macrophages is unclear. We elucidated the effects of chronic iron overload on human monocytic cell line (THP-1) and THP-1 derived macrophages (TDM) by continuously exposing them to high levels of iron (100 μM) to create I-THP-1 and I-TDM, respectively. Our results show that iron overload did not affect morphology or granularity of I-THP-1, but increased the granularity of I-TDM. Bactericidal assays for non-pathogenic E. coli DH5α, JM109 and pathogenic P. aeruginosa all revealed decreased efficiency with increasing iron concentration in I-TDM. The impaired P. aeruginosa killing ability of human primary monocyte derived macrophages (hMDM) was also found when cells are cultured in iron contained medium. Further studies on the bactericidal activity of I-TDM revealed lysosomal dysfunction associated with the inhibition of lysosomal acidification resulting in increasing lysosomal pH, the impairment of post-translational processing of cathepsins (especially cathepsin D), and decreased autophagic flux. These findings may explain the impaired innate immunity of thalassemic patients with chronic iron overload, suggesting the manipulation of lysosomal function as a novel therapeutic approach. PMID:27244448

  9. Neuroprotective Activities of Granulocyte-Macrophage Colony Stimulating Factor Following Controlled Cortical Impact

    PubMed Central

    Kelso, Matthew L.; Elliott, Bret R.; Haverland, Nicole A.; Mosley, R. Lee; Gendelman, Howard E.

    2014-01-01

    Neurodegeneration after traumatic brain injury (TBI) is facilitated by innate and adaptive immunity and can be harnessed to effect brain repair. In mice subjected to controlled cortical impact (CCI) we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI. PMID:25468272

  10. Effects of interferon-gamma and tumor necrosis factor-alpha on macrophage enzyme levels

    NASA Technical Reports Server (NTRS)

    Pierangeli, Silvia S.; Sonnenfeld, Gerald

    1989-01-01

    Murine peritoneal macrophages were treated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF). Measurements of changes in acid phosphatase and beta-glucuronidase levels were made as an indication of activation by cytokine treatment. IFN-gamma or TNF-gamma treatment resulted in a significant increase in the activities of both enzymes measured in the cell lysates. This increase was observable after 6 h of incubation, but reached its maximum level after 24 h of incubation. The effect of the treatment of the cell with both cytokines together was additive. No synergistic effect of addition of both cytokines on the enzyme levels was observed.

  11. Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

    PubMed

    Singh, Sonali; Barr, Helen; Liu, Yi-Chia; Robins, Adrian; Heeb, Stephan; Williams, Paul; Fogarty, Andrew; Cámara, Miguel; Martínez-Pomares, Luisa

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages

  12. Reversal of impaired wound healing in irradiated rats by platelet-derived growth factor-BB

    SciTech Connect

    Mustoe, T.A.; Purdy, J.; Gramates, P.; Deuel, T.F.; Thomason, A.; Pierce, G.F. )

    1989-10-01

    This study examined the potential influence of platelet-derived growth factor-BB homodimers (PDGF-BB) on surgical incisions in irradiated animals with depressed wound healing. Rats were irradiated with either 800 rads total body or 2,500 rads surface irradiation. Parallel dorsal skin incisions were made 2 days later, and PDGF-BB was applied topically a single time to one of two incisions. In total body-irradiated rats, bone marrow-derived elements were severely depressed, wound macrophages were virtually eliminated, and PDGF-BB treatment was ineffective. However, in surface-irradiated rats, PDGF-BB treatment recruited macrophages into wounds and partially reversed impaired healing on day 7 (p less than 0.005) and day 12 (p less than 0.001). PDGF-BB-treated wounds were 50 percent stronger than the paired control wounds. The results suggest PDGF requires bone marrow-derived cells, likely wound macrophages, for activity and that it may be useful as a topical agent in postirradiation surgical incisions.

  13. Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence.

    PubMed

    Holowka, Thomas; Castilho, Tiago M; Garcia, Alvaro Baeza; Sun, Tiffany; McMahon-Pratt, Diane; Bucala, Richard

    2016-06-01

    Leishmania major encodes 2 orthologs of the cytokine macrophage migration inhibitory factor (MIF), whose functions in parasite growth or in the host-parasite interaction are unknown. To determine the importance of Leishmania-encoded MIF, both LmMIF genes were removed to produce an mif(-/-) strain of L. major This mutant strain replicated normally in vitro but had a 2-fold increased susceptibility to clearance by macrophages. Mice infected with mif(-/-) L. major, when compared to the wild-type strain, also showed a 3-fold reduction in parasite burden. Microarray and functional analyses revealed a reduced ability of mif(-/-) L. major to activate antigen-presenting cells, resulting in a 2-fold reduction in T-cell priming. In addition, there was a reduction in inflammation and effector CD4 T-cell formation in mif(-/-) L. major-infected mice when compared to mice infected with wild-type L. major Notably, effector CD4 T cells that developed during infection with mif(-/-) L. major demonstrated statistically significant differences in markers of functional exhaustion, including increased expression of IFN-γ and IL-7R, reduced expression of programmed death-1, and decreased apoptosis. These data support a role for LmMIF in promoting parasite persistence by manipulating the host response to increase the exhaustion and depletion of protective CD4 T cells.-Holowka, T., Castilho, T. M., Baeza Garcia, A., Sun, T., McMahon-Pratt, D., Bucala, R. Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence. PMID:26956417

  14. Postbiotic activities of lactobacilli-derived factors.

    PubMed

    Cicenia, Alessia; Scirocco, Annunziata; Carabotti, Marilia; Pallotta, Lucia; Marignani, Massimo; Severi, Carola

    2014-01-01

    Probiotics are alive nonpathogenic microorganisms present in the gut microbiota that confer benefits to the host for his health. They act through molecular and cellular mechanisms that contrast pathogen bacteria adhesion, enhance innate immunity, decrease pathogen-induced inflammation, and promote intestinal epithelial cell survival, barrier function, and protective responses. Some of these beneficial effects result to be determined by secreted probiotic-derived factors that recently have been identified as "postbiotic" mediators. They have been reported for several probiotic strains but most available literature concerns Lactobacilli. In this review, we focus on the reported actions of several secretory products of different Lactobacillus species highlighting the available mechanistic data. The identification of soluble factors mediating the beneficial effects of probiotics may present an opportunity not only to understand their fine mechanisms of action, but also to develop effective pharmacological strategies that could integrate the action of treatments with live bacteria. PMID:25291118

  15. Cell-Autonomous Sex Differences in Gene Expression in Chicken Bone Marrow–Derived Macrophages

    PubMed Central

    Garcia-Morales, Carla; Nandi, Sunil; Zhao, Debiao; Sauter, Kristin A.; Vervelde, Lonneke; McBride, Derek; Sang, Helen M.; Clinton, Mike

    2015-01-01

    We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome–specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN–responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes. PMID:25637020

  16. Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages.

    PubMed

    Garcia-Morales, Carla; Nandi, Sunil; Zhao, Debiao; Sauter, Kristin A; Vervelde, Lonneke; McBride, Derek; Sang, Helen M; Clinton, Mike; Hume, David A

    2015-03-01

    We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome-specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN-responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes. PMID:25637020

  17. Development and characterization of two porcine monocyte-derived macrophage cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell lines Cdelta2+ and Cdelta2- were developed from monocytes obtained from a 10-month-old, crossbred, female pig. These cells morphologically resembled macrophages, stained positively for a-naphthyl esterase and negatively for peroxidase. The cell lines were bactericidal and highly phagocytic. ...

  18. Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation.

    PubMed

    Brook, Matthew; Tomlinson, Gareth H; Miles, Katherine; Smith, Richard W P; Rossi, Adriano G; Hiemstra, Pieter S; van 't Wout, Emily F A; Dean, Jonathan L E; Gray, Nicola K; Lu, Wuyuan; Gray, Mohini

    2016-04-19

    Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection withSalmonella entericaserovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a "molecular brake" on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage. PMID:27044108

  19. Low-Dose Oxygen Enhances Macrophage-Derived Bacterial Clearance following Cigarette Smoke Exposure

    PubMed Central

    Bain, William G.; Tripathi, Ashutosh; Mandke, Pooja; Gans, Jonathan H.; D'Alessio, Franco R.; Sidhaye, Venkataramana K.; Aggarwal, Neil R.

    2016-01-01

    Background. Chronic obstructive pulmonary disease (COPD) is a common, smoking-related lung disease. Patients with COPD frequently suffer disease exacerbations induced by bacterial respiratory infections, suggestive of impaired innate immunity. Low-dose oxygen is a mainstay of therapy during COPD exacerbations; yet we understand little about whether oxygen can modulate the effects of cigarette smoke on lung immunity. Methods. Wild-type mice were exposed to cigarette smoke for 5 weeks, followed by intratracheal instillation of Pseudomonas aeruginosa (PAO1) and 21% or 35–40% oxygen. After two days, lungs were harvested for PAO1 CFUs, and bronchoalveolar fluid was sampled for inflammatory markers. In culture, macrophages were exposed to cigarette smoke and oxygen (40%) for 24 hours and then incubated with PAO1, followed by quantification of bacterial phagocytosis and inflammatory markers. Results. Mice exposed to 35–40% oxygen after cigarette smoke and PAO1 had improved survival and reduced lung CFUs and inflammation. Macrophages from these mice expressed less TNF-α and more scavenger receptors. In culture, macrophages exposed to cigarette smoke and oxygen also demonstrated decreased TNF-α secretion and enhanced phagocytosis of PAO1 bacteria. Conclusions. Our findings demonstrate a novel, protective role for low-dose oxygen following cigarette smoke and bacteria exposure that may be mediated by enhanced macrophage phagocytosis. PMID:27403445

  20. The contribution of macrophages to normal and pathological pregnancies.

    PubMed

    Nagamatsu, Takeshi; Schust, Danny J

    2010-06-01

    Macrophages represent one of the major leukocyte subsets in the uterine decidua. Owing to their remarkable phenotypic plasticity, decidual macrophages can participate in diverse activities during pregnancy. At baseline, decidual macrophages are characterized by an immunosuppressive phenotype and M2 polarization, supporting feto-maternal immune tolerance. In early pregnancy, macrophage-derived pro-angiogenic factors prompt vascular remodeling within the uterine wall to ensure appropriate utero-placental circulation. Upon invasion by pathogens, pattern recognition receptors on decidual macrophages help to alter the characteristics of these malleable cells toward an M1, inflammatory phenotype. Similar inflammatory characteristics are seen in those macrophages that accumulate in the lower segment of the uterus to drive cervical ripening. Disturbances in the tight control that balances macrophage function during pregnancy can trigger the development of pregnancy complications. Here, we discuss the physiologic role of uterine macrophages at different stages of pregnancy and describe their relevance in selected pregnancy disorders. PMID:20163399

  1. Gaucher iPSC-derived macrophages produce elevated levels of inflammatory mediators and serve as a new platform for therapeutic development

    PubMed Central

    Panicker, Leelamma M.; Miller, Diana; Awad, Ola; Bose, Vivek; Lun, Yu; Park, Tea Soon; Zambidis, Elias T.; Sgambato, Judi A.; Feldman, Ricardo A.

    2014-01-01

    Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid beta-glucocerebrosidase (GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the source of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and if their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human iPS cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of TNF alpha, IL-6, and IL-1beta than control cells, and this phenotype was exacerbated by treatment with LPS. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of RBC remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant glucocerebrosidase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients, and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery. PMID:24801745

  2. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages

    PubMed Central

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E.; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-01-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

  3. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

    PubMed

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-04-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

  4. Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

    PubMed

    Shaked, Iftach; Hanna, Richard N; Shaked, Helena; Chodaczek, Grzegorz; Nowyhed, Heba N; Tweet, George; Tacke, Robert; Basat, Alp Bugra; Mikulski, Zbigniew; Togher, Susan; Miller, Jacqueline; Blatchley, Amy; Salek-Ardakani, Shahram; Darvas, Martin; Kaikkonen, Minna U; Thomas, Graham D; Lai-Wing-Sun, Sonia; Rezk, Ayman; Bar-Or, Amit; Glass, Christopher K; Bandukwala, Hozefa; Hedrick, Catherine C

    2015-12-01

    The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages. PMID:26523867

  5. Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage.

    PubMed

    Wang, Naizhen; Gates, Khalilah L; Trejo, Humberto; Favoreto, Silvio; Schleimer, Robert P; Sznajder, Jacob I; Beitel, Greg J; Sporn, Peter H S

    2010-07-01

    Elevated blood and tissue CO(2), or hypercapnia, is common in severe lung disease. Patients with hypercapnia often develop lung infections and have an increased risk of death following pneumonia. To explore whether hypercapnia interferes with host defense, we studied the effects of elevated P(CO2) on macrophage innate immune responses. In differentiated human THP-1 macrophages and human and mouse alveolar macrophages stimulated with lipopolysaccharide (LPS) and other Toll-like receptor ligands, hypercapnia inhibited expression of tumor necrosis factor and interleukin (IL)-6, nuclear factor (NF)-kappaB-dependent cytokines critical for antimicrobial host defense. Inhibition of IL-6 expression by hypercapnia was concentration dependent, rapid, reversible, and independent of extracellular and intracellular acidosis. In contrast, hypercapnia did not down-regulate IL-10 or interferon-beta, which do not require NF-kappaB. Notably, hypercapnia did not affect LPS-induced degradation of IkappaB alpha, nuclear translocation of RelA/p65, or activation of mitogen-activated protein kinases, but it did block IL-6 promoter-driven luciferase activity in mouse RAW 264.7 macrophages. Elevated P(CO2) also decreased phagocytosis of opsonized polystyrene beads and heat-killed bacteria in THP-1 and human alveolar macrophages. By interfering with essential innate immune functions in the macrophage, hypercapnia may cause a previously unrecognized defect in resistance to pulmonary infection in patients with advanced lung disease. PMID:20181940

  6. Derivation of dose conversion factors for tritium

    SciTech Connect

    Killough, G. G.

    1982-03-01

    For a given intake mode (ingestion, inhalation, absorption through the skin), a dose conversion factor (DCF) is the committed dose equivalent to a specified organ of an individual per unit intake of a radionuclide. One also may consider the effective dose commitment per unit intake, which is a weighted average of organ-specific DCFs, with weights proportional to risks associated with stochastic radiation-induced fatal health effects, as defined by Publication 26 of the International Commission on Radiological Protection (ICRP). This report derives and tabulates organ-specific dose conversion factors and the effective dose commitment per unit intake of tritium. These factors are based on a steady-state model of hydrogen in the tissues of ICRP's Reference Man (ICRP Publication 23) and equilibrium of specific activities between body water and other tissues. The results differ by 27 to 33% from the estimate on which ICRP Publication 30 recommendations are based. The report also examines a dynamic model of tritium retention in body water, mineral bone, and two compartments representing organically-bound hydrogen. This model is compared with data from human subjects who were observed for extended periods. The manner of combining the dose conversion factors with measured or model-predicted levels of contamination in man's exposure media (air, drinking water, soil moisture) to estimate dose rate to an individual is briefly discussed.

  7. Cell viability, adhesion and function of RAW 264.7 macrophages on fluorinated xerogel-derived nitric oxide permeable membrane for the application of cellular sensing.

    PubMed

    Kang, Wook Sung; Seo, Bochan; Kim, Ji-Hye; Kim, Ok-Kyun; Shin, Jae Ho; Lee, Gi-Ja; Park, Hun-Kuk

    2014-11-01

    Organically modified xerogels have an advantage over gas sensing applications due to their open, rigid structure and hydrophobicity. Here we evaluated the biocompatibility of xerogel-derived nitric oxide (NO) permeable membranes modified with fluorinated functional groups for application in cellular sensing by growing RAW 264.7 macrophages on them. We examined the cell viability, adhesion and growth of RAW 264.7 macrophages on NO permselective membrane and other cell-adhesive matrices, poly L-lysine and collagen. The surface roughness of each membrane was obtained from topographic atomic force microscopy (AFM) images. In addition, we measured the level of NO release of RAW 264.7 macrophages by lipopolysaccharide (LPS) stimulation using a Griess assay to confirm the function of cells. The fluorinated xerogel-derived membrane had a very smooth surface with rms roughness 2.1 Å and did not show cytotoxic effects in RAW 264.7 macrophages. As a result, the morphology and function of adhering RAW 264.7 macrophage showed no differences from those of other cell-adhesive membranes. Finally, we successfully detected NO release in RAW 264.7 macrophages stimulated by LPS, using a planar-type xerogel-derived NO sensor. Therefore, we suggest that fluorinated xerogel-derived membrane could be used as both a NO permeable and cell-adhesive membrane for cellular sensing applications. PMID:25958535

  8. Polyunsaturated fatty acid relatively decreases cholesterol content in THP-1 macrophage-derived foam cell: partly correlates with expression profile of CIDE and PAT members

    PubMed Central

    2013-01-01

    Background Polyunsaturated fatty acids (PUFAs) have positive effect on the regulation of plasma lipids. But the mechanism for them to modulate lipid homeostasis in macrophage is still unclear. In this study, we employed PUFA to pretreat macrophages and evaluated the variations of lipid droplet (LD) content, lipid composition, and expressions of LD-associated genes in macrophage-derived foam cells. Method THP-1-derived macrophages or human peripheral blood monocyte-derived macrophages were pre-treated with four non-esterified fatty acids (NEFAs) separately: saturated fatty acid (SFA)-palmitic acid (PA), monounsaturated fatty acids (MUFAs)-oleic acid (OA), PUFAs-linoleic acid (LA) and eicosapentaenoic acid (EPA). Intracellular lipid content and cholesterol efflux were analyzed in THP-1 macrophage-derived foam cells. Related gene expressions were detected by quantitative real-time PCR. Results PUFA pre-treatment reduced cholesterol content in foam cells and increased cholesterol efflux to lipid-free apoAI in conditioned medium compared with PA or OA group. Cell death-inducing DFF45 like effector (CIDE) and Perilipin-Adipophilin-TIP47 (PAT) family members, as LD-associated proteins, showed specific gene expression profiles after PUFA pre-treatment. These results may help to explain the process of lipid metabolism within foam cells. Conclusion PUFA (LA or EPA) had a potential protective effect against cholesterol accumulation. The specific expressions of CIDE and PAT genes may provide clues to explore the protective mechanism of PUFA in foam cells. PMID:23879935

  9. CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

    PubMed Central

    Yang, Z; Carter, C D; Miller, M S; Bochsler, P N

    1995-01-01

    The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml). PMID:7528735

  10. Norwalk Virus Does Not Replicate in Human Macrophages or Dendritic Cells Derived from the Peripheral Blood of Susceptible Humans

    PubMed Central

    Lay, Margarita K.; Atmar, Robert L.; Guix, Susana; Bharadwaj, Uddalak; He, Hong; Neill, Frederick H.; Sastry, Jagannadha K.; Yao, Qizhi; Estes, Mary K.

    2010-01-01

    Human noroviruses are difficult to study due to the lack of an efficient in vitro cell culture system or small animal model. Murine norovirus replicates in murine macrophages (MΦ) and dendritic cells (DCs), raising the possibility that human NoVs might replicate in such human cell types. To test this hypothesis, we evaluated DCs and MΦ derived from monocyte subsets and CD11c+ DCs isolated from peripheral blood mononuclear cells of individuals susceptible to Norwalk virus (NV) infection. These cells were exposed to NV and replication was evaluated by immunofluorescence and by quantitative RT-PCR. A few PBMC-derived DCs expressed NV proteins. However, NV RNA did not increase in any of the cells tested. These results demonstrate that NV does not replicate in human CD11c+ DCs, monocyte-derived DCs and MΦ, but abortive infection may occur in a few DCs. These results suggest that NV tropism is distinct from that of murine noroviruses. PMID:20667573

  11. Derivation of the Korean radwaste scaling factor

    SciTech Connect

    Kwang Yong Jee; Hong Joo Ahn; Se Chul Sohn; Sun Ho Han; Ki Seop Choi

    2007-07-01

    The concentrations of several radionuclides in low and intermediate level radioactive waste (LILW) drums have to be determined before shipping to disposal facilities. A notice, by the Ministry of Science and Technology (MOST) of the Korean Government, related to the disposal of LILW drums came into effect at the beginning of 2005, with regards to a radionuclide regulation inside a waste drum. MOST allows for an indirect radionuclide assay using a scaling factor to measure the inventories due to the difficulty of nondestructively measuring the essential {alpha} and {beta}-emitting nuclides inside a drum. That is, a scaling factor calculated through a correlation of the {alpha} or {beta}-emitting nuclide (DTM, Difficult-To-Measure) with a {gamma}-emitting nuclide (ETM, Easy-To-Measure) which has systematically similar properties with DTM nuclides. In this study, radioactive wastes, such as spent resin and dry active waste which were generated at different sites of a PWR and a site of a PHWR type Korean NPP, were partially sampled and analyzed for regulated radionuclides by using radiochemical methods. According to a reactor type and a waste form, the analysis results of each radionuclide were classified. Korean radwaste scaling factor was derived from database of radionuclide concentrations. (authors)

  12. Silica-induced apoptosis in murine macrophage: involvement of tumor necrosis factor-alpha and nuclear factor-kappaB activation.

    PubMed

    Gozal, Evelyne; Ortiz, Luis A; Zou, Xiaoyan; Burow, Matthew E; Lasky, Joseph A; Friedman, Mitchell

    2002-07-01

    Alveolar macrophages play a critical role in silica-induced lung fibrosis. Silica exposure induces tumor necrosis factor (TNF)-alpha release and nuclear factor (NF)-kappaB activation, and apoptotic mechanisms have been implicated in silica-induced pathogenesis. To characterize potential relationships between these signaling events, we studied their induction in two murine macrophage cell lines. The RAW 264.7 macrophage cell line was more sensitive, and the IC-21 macrophage cell line more tolerant to silica exposure (0.2 or 1 mg/ml for 6 h) as evidenced by significantly higher apoptotic responses in RAW 264.7 (P < 0.05). RAW 264.7 macrophages exhibited enhanced TNF-alpha production and NF-kappaB activation in response to silica, whereas IC-21 macrophages did not produce TNF-alpha in response to silica and did not induce NF-kappaB nuclear binding. Inhibition of NF-kappaB in RAW 264.7 cells with BAY11-7082 significantly increased apoptosis while inhibiting TNF-alpha release. In addition, TNF-alpha and NF-kappaB activation, but not apoptosis, were induced by lipopolysaccharide (LPS) in both cell lines, and NF-kappaB inhibition reduced LPS-induced TNF-alpha release. These data suggest that TNF-alpha induction is dependent on NF-kappaB activation in both cell lines. However, silica can induce apoptosis in murine macrophages, independently of TNF-alpha stimulation, as in IC-21 macrophages. Furthermore, NF-kappaB activation in macrophages may play dual roles, both pro- and antiapoptotic during silica injury. PMID:12091251

  13. Role of macrophage migration inhibitory factor in age-related lung disease.

    PubMed

    Sauler, Maor; Bucala, Richard; Lee, Patty J

    2015-07-01

    The prevalence of many common respiratory disorders, including pneumonia, chronic obstructive lung disease, pulmonary fibrosis, and lung cancer, increases with age. Little is known of the host factors that may predispose individuals to such diseases. Macrophage migration inhibitory factor (MIF) is a potent upstream regulator of the immune system. MIF is encoded by variant alleles that occur commonly in the population. In addition to its role as a proinflammatory cytokine, a growing body of literature demonstrates that MIF influences diverse molecular processes important for the maintenance of cellular homeostasis and may influence the incidence or clinical manifestations of a variety of chronic lung diseases. This review highlights the biological properties of MIF and its implication in age-related lung disease. PMID:25957294

  14. Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes

    PubMed Central

    Naphade, Swati; Sharma, Jay; Chevronnay, Héloïse P. Gaide; Shook, Michael A.; Yeagy, Brian A.; Rocca, Celine J.; Ur, Sarah N.; Lau, Athena J.; Courtoy, Pierre J.; Cherqui, Stephanie

    2014-01-01

    Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multi-systemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon co-culture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins. PMID:25186209

  15. Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle.

    PubMed

    Gherardi, R K; Coquet, M; Cherin, P; Belec, L; Moretto, P; Dreyfus, P A; Pellissier, J F; Chariot, P; Authier, F J

    2001-09-01

    Macrophagic myofasciitis (MMF) is an emerging condition of unknown cause, detected in patients with diffuse arthromyalgias and fatigue, and characterized by muscle infiltration by granular periodic acid-Schiff's reagent-positive macrophages and lymphocytes. Intracytoplasmic inclusions have been observed in macrophages of some patients. To assess their significance, electron microscopy was performed in 40 consecutive cases and chemical analysis was done by microanalysis and atomic absorption spectrometry. Inclusions were constantly detected and corresponded to aluminium hydroxide, an immunostimulatory compound frequently used as a vaccine adjuvant. A lymphocytic component was constantly observed in MMF lesions. Serological tests were compatible with exposure to aluminium hydroxide-containing vaccines. History analysis revealed that 50 out of 50 patients had received vaccines against hepatitis B virus (86%), hepatitis A virus (19%) or tetanus toxoid (58%), 3-96 months (median 36 months) before biopsy. Diffuse myalgias were more frequent in patients with than without an MMF lesion at deltoid muscle biopsy (P < 0.0001). Myalgia onset was subsequent to the vaccination (median 11 months) in 94% of patients. MMF lesion was experimentally reproduced in rats. We conclude that the MMF lesion is secondary to intramuscular injection of aluminium hydroxide-containing vaccines, shows both long-term persistence of aluminium hydroxide and an ongoing local immune reaction, and is detected in patients with systemic symptoms which appeared subsequently to vaccination. PMID:11522584

  16. Macrophage immunomodulation by breast cancer-derived exosomes requires Toll-like receptor 2-mediated activation of NF-κB

    PubMed Central

    Chow, Amy; Zhou, Weiying; Liu, Liang; Fong, Miranda Y.; Champer, Jackson; Van Haute, Desiree; Chin, Andrew R.; Ren, Xiubao; Gugiu, Bogdan Gabriel; Meng, Zhipeng; Huang, Wendong; Ngo, Vu; Kortylewski, Marcin; Wang, Shizhen Emily

    2014-01-01

    Growing evidence links tumor progression with chronic inflammatory processes and dysregulated activity of various immune cells. In this study, we demonstrate that various types of macrophages internalize microvesicles, called exosomes, secreted by breast cancer and non-cancerous cell lines. Although both types of exosomes targeted macrophages, only cancer-derived exosomes stimulated NF-κB activation in macrophages resulting in secretion of pro-inflammatory cytokines such as IL-6, TNFα, GCSF, and CCL2. In vivo mouse experiments confirmed that intravenously injected exosomes are efficiently internalized by macrophages in the lung and brain, which correlated with upregulation of inflammatory cytokines. In mice bearing xenografted human breast cancers, tumor-derived exosomes were internalized by macrophages in axillary lymph nodes thereby triggering expression of IL-6. Genetic ablation of Toll-like receptor 2 (TLR2) or MyD88, a critical signaling adaptor in the NF-κB pathway, completely abolished the effect of tumor-derived exosomes. In contrast, inhibition of TLR4 or endosomal TLRs (TLR3/7/8/9) failed to abrogate NF-κB activation by exosomes. We further found that palmitoylated proteins present on the surface of tumor-secreted exosomes contributed to NF-κB activation. Thus, our results highlight a novel mechanism used by breast cancer cells to induce pro-inflammatory activity of distant macrophages through circulating exosomal vesicles secreted during cancer progression. PMID:25034888

  17. Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis

    PubMed Central

    Rodrigues, Michele F; Alves, Caio C S; Figueiredo, Bárbara B M; Rezende, Alice B; Wohlres-Viana, Sabine; da Silva, Vânia Lúcia; Machado, Marco Antônio; Teixeira, Henrique C

    2013-01-01

    Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1−/− mice at day 3 after bacillus Calmette–Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner. PMID:23489296

  18. Hypoxia Inducible Factor 1 (HIF-1) Recruits Macrophage to Activate Pancreatic Stellate Cells in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Li, Na; Li, Yang; Li, Zengxun; Huang, Chongbiao; Yang, Yanhui; Lang, Mingxiao; Cao, Junli; Jiang, Wenna; Xu, Yu; Dong, Jie; Ren, He

    2016-01-01

    Hypoxia inducible factor 1 (HIF-1) is a transcription factor composed of two subunits, namely, HIF-1α and HIF-1β, in which HIF-1β is constitutively expressed. HIF-1 upregulates several hypoxia-responsive proteins, including angiogenesis factors, glycolysis solution enzymes, and cell survival proteins. HIF-1 is also associated with the degree of inflammation in the tumor region, but the exact mechanism remains unclear. This study aims to identify the molecular mechanism of recruiting monocytes/macrophages by HIF-1α in pancreatic ductal adenocarcinoma (PDAC) and the effects of macrophages on pancreatic stellate cells (PSCs). Immunohistochemistry (IHC) was performed for cluster of differentiation 68 (CD68), HIF-1α, and chemical chemokines 2 (CCL2). Western blot, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation assay, and The Cancer Genome Atlas (TCGA) were used to verify the correlation between HIF-1α and CCL2 at protein and nucleic acid levels. Monocytes/macrophages were co-cultured with PSCs to observe their interaction. Samples showed significant correlation between CD68 and HIF-1α (t-test, p < 0.05). HIF-1α recruited monocytes/macrophages by promoting CCL2 secretion. Moreover, macrophages could accelerate the activation of PSCs. HIF-1α might promote inflammation and fibrosis of PDAC through CCL2 secretion, which may provide a novel target to treat PDAC patients. PMID:27271610

  19. Granulocyte-Macrophage Colony-Stimulating Factor in Staphylococcus aureus-Induced Arthritis

    PubMed Central

    Verdrengh, Margareta; Tarkowski, Andrej

    1998-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is able to increase not only the production of phagocytic cells but also their efficacy with respect to, e.g., bactericidal properties. In this study, we wanted to analyze the impact of GM-CSF on experimental Staphylococcus aureus-induced arthritis. For that purpose, mice were administered GM-CSF before and after bacterial inoculation. Although there was an increase in the total number of leukocytes as well as in the granulocyte fraction, there was no favorable effect on the severity of arthritis or on survival rates. There were no obvious differences between the GM-CSF-pretreated animals and controls with regard to growth of staphylococci in joints and kidneys 4 days after the bacterial inoculation. In contrast, mice that had been pretreated with GM-CSF prior to bacterial inoculation showed approximately four times lower numbers of bacteria in their blood 24 h later. These results, along with those of our previous studies, suggest that on the one hand the granulocyte is the main protective cell during the course of S. aureus infection but that on the other hand, upregulation of granulocyte-macrophage production will not exert any additional protective effects with respect to tissue injury. PMID:9453655

  20. The glycosylation and characterization of the candidate Gc macrophage activating factor.

    PubMed

    Ravnsborg, Tina; Olsen, Dorthe T; Thysen, Anna Hammerich; Christiansen, Maja; Houen, Gunnar; Højrup, Peter

    2010-04-01

    The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood. PMID:20079467

  1. Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation.

    PubMed

    Wen, Fengyun; Zheng, Jin; Yu, Jing; Gao, Mingju; Gao, Sumin; Zhou, Yingying; Liu, Jianyu; Yang, Zaiqing

    2016-07-01

    Obesity is documented to be a state of chronic mild inflammation associated with increased macrophage infiltration into adipose tissue and liver and skeletal muscle. As a pleiotropic inflammatory mediator, macrophage migration inhibitory factor (MIF) is associated with metabolic disease, so MIF may signal molecular links between adipocytes and myocytes. MIF expression was modified during myoblast differentiation, but the role of MIF during this process is unclear. C2C12 cells were transfected with MIF to investigate their role during differentiation. MIF expression attenuated C2C12 differentiation. It did not change proliferation, but downregulated cyclin D1 and CDK4, causing cell accumulation in the G1 phase. p21 protein was increased significantly and MyoD, MyoG, and p21 mRNA also increased significantly in the C2C12 cells treated with ISO-1, suggesting that inhibition of MIF promotes differentiation. MIF inhibits the myoblast differentiation by affecting the cell cycle progression, but does not affect proliferation. PMID:26927414

  2. Recovery from severe hematopoietic suppression using recombinant human granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Monroy, R.L.; Skelly, R.R.; Taylor, P.; Dubois, A.; Donahue, R.E.; MacVittie, T.J.

    1988-06-01

    The ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance recovery of a radiation-suppressed hematopoietic system was evaluated in a nonuniform radiation exposure model using the rhesus monkey. Recombinant human GM-CSF treatment for 7 days after a lethal, nonuniform radiation exposure of 800 cGy was sufficient to enhance hematopoietic reconstitution, leading to an earlier recovery. Monkeys were treated with 72,000 U/kg/day of rhGM-CSF delivered continuously through an Alzet miniosmotic pump implanted subcutaneously on day 3. Treated monkeys demonstrated effective granulocyte and platelet levels in the peripheral blood, 4 and 7 days earlier, respectively, than control monkeys. Granulocyte-macrophage colony-forming unit (CFU-GM) activity in the bone marrow was monitored to evaluate the effect of rhGM-CSF on marrow recovery. Treatment with rhGM-CSF led to an early recovery of CFU-GM activity suggesting that rhGM-CSF acted on an earlier stem cell population to generate CFU-GM. Thus, the effect of rhGM-CSF on hematopoietic regeneration, granulocyte recovery, and platelet recovery are evaluated in this paper.

  3. Quince (Cydonia oblonga Miller) peel polyphenols modulate LPS-induced inflammation in human THP-1-derived macrophages through NF-κB, p38MAPK and Akt inhibition.

    PubMed

    Essafi-Benkhadir, Khadija; Refai, Amira; Riahi, Ichrak; Fattouch, Sami; Karoui, Habib; Essafi, Makram

    2012-02-01

    Chronic inflammation is a hallmark of several pathologies, such as rheumatoid arthritis, gastritis, inflammatory bowel disease, atherosclerosis and cancer. A wide range of anti-inflammatory chemicals have been used to treat such diseases while presenting high toxicity and numerous side effects. Here, we report the anti-inflammatory effect of a non-toxic, cost-effective natural agent, polyphenolic extract from the Tunisian quince Cydonia oblonga Miller. Lipopolysaccharide (LPS) treatment of human THP-1-derived macrophages induced the secretion of high levels of the pro-inflammatory cytokine TNF-α and the chemokine IL-8, which was inhibited by quince peel polyphenolic extract in a dose-dependent manner. Concomitantly, quince polyphenols enhanced the level of the anti-inflammatory cytokine IL-10 secreted by LPS-treated macrophages. We further demonstrated that the unexpected increase in IL-6 secretion that occurred when quince polyphenols were associated with LPS treatment was partially responsible for the polyphenols-mediated inhibition of TNF-α secretion. Biochemical analysis showed that quince polyphenols extract inhibited the LPS-mediated activation of three major cellular pro-inflammatory effectors, nuclear factor-kappa B (NF-κB), p38MAPK and Akt. Overall, our data indicate that quince peel polyphenolic extract induces a potent anti-inflammatory effect that may prove useful for the treatment of inflammatory diseases and that a quince-rich regimen may help to prevent and improve the treatment of such diseases. PMID:22252293

  4. Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro.

    PubMed

    Rice, J M; Hunt, J A; Gallagher, J A; Hanarp, P; Sutherland, D S; Gold, J

    2003-11-01

    The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period. PMID:14530077

  5. Structural Interactions Dictate the Kinetics of Macrophage Migration Inhibitory Factor Inhibition by Different Cancer-Preventive Isothiocyanates

    PubMed Central

    Crichlow, Gregg V.; Fan, Chengpeng; Keeler, Camille; Hodsdon, Michael; Lolis, Elias J.

    2012-01-01

    Regulation of cellular processes by dietary nutrients is known to affect the likelihood of cancer development. One class of cancer preventive nutrients, isothiocyanates (ITCs) derived from consumption of cruciferous vegetables, is known to have various effects on cellular biochemistry. One target of ITCs is macrophage migration inhibitory factor (MIF), a widely expressed protein with known inflammatory, pro-tumorigenic, pro-angiogenic, and anti-apoptotic properties. MIF is covalently inhibited by a variety of ITCs, which in part, may explain how they exert their cancer-preventive effects. We report the crystallographic structures of human MIF bound to phenethylisothiocyanate and to L-sulforaphane (dietary isothiocyanates derived from watercress and broccoli, respectively), and correlate structural features of these two isothiocyanates with their second-order rate constants for MIF inactivation. We also characterize changes in the MIF structure using NMR HSQC spectra of these complexes and observe many changes at the subunit interface. While a number of chemical shifts do not change, many of those that change do not have similar features in magnitude or direction for the two isothiocyanates. The difference in the binding modes of these two ITCs provides a means of using structure-activity relationships to reveal insights into MIF biological interactions. The results of this study provide a framework for the development of therapeutics that target MIF. PMID:22931430

  6. Structural interactions dictate the kinetics of macrophage migration inhibitory factor inhibition by different cancer-preventive isothiocyanates.

    PubMed

    Crichlow, Gregg V; Fan, Chengpeng; Keeler, Camille; Hodsdon, Michael; Lolis, Elias J

    2012-09-25

    Regulation of cellular processes by dietary nutrients is known to affect the likelihood of cancer development. One class of cancer-preventive nutrients, isothiocyanates (ITCs), derived from the consumption of cruciferous vegetables, is known to have various effects on cellular biochemistry. One target of ITCs is macrophage migration inhibitory factor (MIF), a widely expressed protein with known inflammatory, pro-tumorigenic, pro-angiogenic, and anti-apoptotic properties. MIF is covalently inhibited by a variety of ITCs, which in part may explain how they exert their cancer-preventive effects. We report the crystallographic structures of human MIF bound to phenethylisothiocyanate and to l-sulforaphane (dietary isothiocyanates derived from watercress and broccoli, respectively) and correlate structural features of these two isothiocyanates with their second-order rate constants for MIF inactivation. We also characterize changes in the MIF structure using nuclear magnetic resonance heteronuclear single-quantum coherence spectra of these complexes and observe many changes at the subunit interface. While a number of chemical shifts do not change, many of those that change do not have features similar in magnitude or direction for the two isothiocyanates. The difference in the binding modes of these two ITCs provides a means of using structure-activity relationships to reveal insights into MIF biological interactions. The results of this study provide a framework for the development of therapeutics that target MIF. PMID:22931430

  7. A novel flow cytometry-based tool for determining the efficiency of human cytomegalovirus infection in THP-1 derived macrophages.

    PubMed

    Li, Huifen; Mao, Genxiang; Carlson, Joshua; Leng, Sean X

    2015-09-01

    Human cytomegalovirus (hCMV) is a ubiquitous pathogen that causes congenital infection and severe infections in immunocompromised patients. Chronic hCMV infection may also play an important role in immunosenescence and adverse health outcomes in older adults. THP-1, a human monocytic cell line and its derived macrophages serve as a useful cell culture model for mechanistic studies of hCMV infection and its underlying biology. A major methodological challenge is the lack of a quick and reliable tool to accurately determine the efficiency of hCMV infection in THP-1 derived macrophages. In this study, we developed a flow cytometry based method using commercially available monoclonal antibody (MAb) against hCMV immediate early (IE) antigen that can accurately determine infection efficiency. We used 0.5% formaldehyde for fixation, 90% methanol for permeabilization, and incubation with FITC conjugated MAb at 37°C. The method was tested by hCMV infection with laboratory Towne strain in the presence or absence of hydrocortisone. It was also compared with the routine flow cytometry protocol using Cytofix/Cytoperm solution and with immunofluorescence. The results indicate that this new method is reliable and time saving for accurate determination of infection efficiency. It may facilitate further investigations into the underlying biological mechanisms of hCMV infection. PMID:25958130

  8. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

    PubMed

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F; Metzner, Karin J

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells. PMID:27067385

  9. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    PubMed Central

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F.; Metzner, Karin J.

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4+ T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4+ T cells were over-represented in gene hotspots identified in CD4+ T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4+ T cells. PMID:27067385

  10. Increased circulating macrophage migration inhibitory factor levels are associated with coronary artery disease

    PubMed Central

    Yüksel, Arif; Bilgir, Ferda; Bilgir, Oktay; Calan, Mehmet; Bozkaya, Giray

    2015-01-01

    BACKGROUND: To evaluate the macrophage migration inhibitory factor and E-selectin levels in patients with acute coronary syndrome. MATERIALS/METHODS: We examined the plasma migration inhibitory factor and E-selectin levels in 87 patients who presented with chest pain at our hospital. The patients were classified into two groups according to their cardiac status. Sixty-five patients had acute myocardial infarction, and 22 patients had non-cardiac chest pain (non-coronary disease). We designated the latter group of patients as the control group. The patients who presented with acute myocardial infarction were further divided into two subgroups: ST-elevated myocardial infarction (n = 30) and non-ST elevated myocardial infarction (n = 35). RESULTS: We found higher plasma migration inhibitory factor levels in both acute myocardial infarction subgroups than in the control group. However, the E-selectin levels were similar between the acute myocardial infarction and control patients. In addition, we did not find a significant difference in the plasma migration inhibitory factor levels between the ST elevated myocardial infarction and NST-elevated myocardial infarction subgroups. DISCUSSION: The circulating concentrations of migration inhibitory factor were significantly increased in acute myocardial infarction patients, whereas the soluble E-selectin levels were similar between acute myocardial infarction patients and control subjects. Our results suggest that migration inhibitory factor may play a role in the atherosclerotic process. PMID:26017646

  11. Scene kinetics mitigation using factor analysis with derivative factors.

    SciTech Connect

    Larson, Kurt W.; Melgaard, David Kennett; Scholand, Andrew Joseph

    2010-07-01

    Line of sight jitter in staring sensor data combined with scene information can obscure critical information for change analysis or target detection. Consequently before the data analysis, the jitter effects must be significantly reduced. Conventional principal component analysis (PCA) has been used to obtain basis vectors for background estimation; however PCA requires image frames that contain the jitter variation that is to be modeled. Since jitter is usually chaotic and asymmetric, a data set containing all the variation without the changes to be detected is typically not available. An alternative approach, Scene Kinetics Mitigation, first obtains an image of the scene. Then it computes derivatives of that image in the horizontal and vertical directions. The basis set for estimation of the background and the jitter consists of the image and its derivative factors. This approach has several advantages including: (1) only a small number of images are required to develop the model, (2) the model can estimate backgrounds with jitter different from the input training images, (3) the method is particularly effective for sub-pixel jitter, and (4) the model can be developed from images before the change detection process. In addition the scores from projecting the factors on the background provide estimates of the jitter magnitude and direction for registration of the images. In this paper we will present a discussion of the theoretical basis for this technique, provide examples of its application, and discuss its limitations.

  12. Tumor-induced myeloid deviation: when myeloid-derived suppressor cells meet tumor-associated macrophages

    PubMed Central

    Ugel, Stefano; De Sanctis, Francesco; Mandruzzato, Susanna; Bronte, Vincenzo

    2015-01-01

    The generation of an inflammatory environment is favorable and often decisive for the growth of both primary tumors and metastases. Tumor cells either express membrane molecules or release tumor-derived soluble factors able to alter myelopoiesis. Tumor-reprogrammed myeloid cells not only create a tolerogenic environment by blocking T cell functions and proliferation, but also directly drive tumor growth by promoting cancer stemness, angiogenesis, stroma deposition, epithelial-to-mesenchymal transition, and metastasis formation. In this Review, we discuss the interplay between immunosuppressive and protumoral myeloid cells and detail their immune-regulatory mechanisms, the molecular pathways involved in their differentiation, as well as their potential role as prognostic and diagnostic biomarkers and prospective targets for innovative approaches to treat tumor-bearing hosts. PMID:26325033

  13. Transforming growth factor beta 1 and gamma interferon provide opposing signals to lipopolysaccharide-activated mouse macrophages.

    PubMed Central

    Hausmann, E H; Hao, S Y; Pace, J L; Parmely, M J

    1994-01-01

    Bacterial lipopolysaccharides (LPS) are potent inducers of macrophage activation, leading to the production of a number of proinflammatory mediators. Although several cytokines that prime macrophages for enhanced LPS-triggered responses have been identified, far less is known regarding the role that cytokines play in down-regulating macrophage responses to LPS. This study was designed to determine the effects of recombinant transforming growth factor beta 1 (rTGF-beta 1) on macrophage activation by LPS. Pretreatment of either mouse peritoneal macrophages or cells of the RAW 264.7 macrophage-like cell line with rTGF-beta 1 inhibited their ability to produce both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) in response to LPS. These inhibitory effects were reversed by increasing the concentration of LPS or by priming cells with optimal concentrations of recombinant gamma interferon (rIFN-gamma). Pretreatment of cells with rTGF-beta 1 had only a modest inhibitory effect on the expression of TNF-alpha mRNA. By contrast, the expression of mRNA for the inducible form of nitric oxide synthase (iNOS), which is responsible for NO production in activated macrophages, was significantly inhibited by rTGF-beta 1 pretreatment. Thus, rTGF-beta 1-dependent suppression of macrophage TNF-alpha biosynthesis was manifest at a posttranscriptional level, whereas the inhibition of NO production correlated with a direct effect on iNOS gene expression. Importantly, both of these suppressive effects of rTGF-beta 1 were reversed by exposing the cells to priming concentrations of rIFN-gamma. As with NO production, immunocytochemical analysis of iNOS expression in LPS-stimulated macrophages revealed that rIFN-gamma and rTGF-beta 1 had antagonistic effects, with the former increasing, and the latter reducing, the number of iNOS-expressing cells induced by LPS. These data suggest that a balance between the priming effects of IFN-gamma and the inhibitory effects of TGF-beta 1 can

  14. Tumor necrosis factor as an autocrine and paracrine signal controlling the macrophage secretory response to Candida albicans.

    PubMed Central

    Blasi, E; Pitzurra, L; Bartoli, A; Puliti, M; Bistoni, F

    1994-01-01

    We have previously demonstrated that the hyphal form of Candida albicans (H-Candida), but not the yeast form (Y-Candida), acts as a macrophage-stimulating agent. The early response (1 to 3 h) of the macrophage cell line ANA-1 to H-Candida results in enhanced tumor necrosis factor (TNF) transcription and production. Here we show that when coincubation times are prolonged (3 to 24 h), Y-Candida also exhibits stimulatory properties. This phenomenon has been ascribed to the occurrence of the dimorphic transition, as demonstrated by microscopic evaluation of the cultures and by experiments in which both killed Y-Candida and the agerminative strain C. albicans PCA-2 failed to induce cytokine production. TNF produced in response to H-Candida acts as an autocrine and paracrine signal controlling the macrophage secretory response to C. albicans. In fact, addition of anti-TNF polyclonal antibodies to the coculture of ANA-1 macrophages and H-Candida results in a marked and time-dependent decrease of TNF transcript levels. Moreover, pretreatment of macrophages with recombinant TNF for 3 h enhances TNF and induces interleukin-1 production in response to both forms of Candida, while pretreatment for 18 h renders macrophages refractory to any stimuli. Interestingly, the kinetics of interleukin-1 transcription and secretion in response to H-Candida are delayed with respect to those of TNF. Overall, these data indicate that TNF, produced by macrophages in response to H-Candida, regulates its own production as well as that of other soluble factors, thus suggesting that this cytokine plays multiple roles in the immune mechanisms involved in Candida infection. Images PMID:8132326

  15. Chemoattraction of macrophages by secretory molecules derived from cells expressing the signal peptide of eosinophil cationic protein

    PubMed Central

    2012-01-01

    Background Eosinophil cationic protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of eosinophil cationic protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-α (TGF-α) and epidermal growth factor receptor (EGFR). Results In the present study, we first generated a DNA microarray dataset, which showed that ECPsp upregulated proinflammatory molecules, including chemokines, interferon-induced molecules, and Toll-like receptors. The levels of mRNAs encoding CCL5, CXCL10, CXCL11, CXCL16, STAT1, and STAT2 were increased in the presence of ECPsp by 2.07-, 4.21-, 7.52-, 2.6-, 3.58-, and 1.67-fold, respectively. We then constructed a functional linkage network by integrating the microarray dataset with the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG). Follow-up analysis revealed that STAT1 and STAT2, important transcriptional factors that regulate cytokine expression and release, served as hubs to connect the pathways of cytokine stimulation (TGF-α and EGFR pathways) and inflammatory responses. Furthermore, integrating TGF-α and EGFR with the functional linkage network indicated that STAT1 and STAT2 served as hubs that connect two functional clusters, including (1) cell proliferation and survival, and (2) inflammation. Finally, we found that conditioned medium in which cells that express ECPsp had been cultured could chemoattract macrophages. Experimentally, we also demonstrated that the migration of macrophage could be inhibited by the individual treatment of siRNAs of STAT1 or STAT2. Therefore, we hypothesize that ECPsp may function as a regulator for enhancing the migration of macrophages through the upregualtion of the transcriptional factors STAT1 and STAT2. Conclusion The increased expression and

  16. Human monocyte-derived macrophages infected with virulent Shigella flexneri in vitro undergo a rapid cytolytic event similar to oncosis but not apoptosis.

    PubMed Central

    Fernandez-Prada, C M; Hoover, D L; Tall, B D; Venkatesan, M M

    1997-01-01

    Infection of human monocyte-derived macrophages in vitro with virulent Shigella flexneri resulted in cell death which involved rupture of the plasma membrane, cell swelling, disintegration of ultrastructure, and generalized karyolysis. These features bore resemblance to oncosis and are in striking contrast to previously described observations of mouse macrophages, where a similar infection by virulent Shigella resulted in cell death by apoptosis. Cell death by oncosis in human macrophages was confirmed by lactate dehydrogenase release, light microscopy, electron microscopy, terminal deoxynucleotidyltransferase end labeling of DNA ends, DNA fragmentation assays, and fluorescence-activated cell sorter analysis of propidium-labeled nuclei. Thus, the phenomena of cell death induced by virulent Shigella in human and mouse macrophages reflect different biochemical pathways. Interleukin-1beta (IL-1beta) was released in culture supernatants of human macrophages infected with virulent bacteria. Inhibition with IL-1beta-converting enzyme inhibitors indicated, however, that this release occurred as a passive event of cell lysis. The patterns of intracellular survival of Shigella strains within human and mouse macrophages reflect differences that exist not only between Shigella serotypes but also between the two different macrophage cell types. PMID:9119491

  17. Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50.

    PubMed

    Baer, M; Dillner, A; Schwartz, R C; Sedon, C; Nedospasov, S; Johnson, P F

    1998-10-01

    Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine. PMID:9742085

  18. Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-κB p50

    PubMed Central

    Baer, Mark; Dillner, Allan; Schwartz, Richard C.; Sedon, Constance; Nedospasov, Sergei; Johnson, Peter F.

    1998-01-01

    Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-α. This activity, termed TNF-α-inhibiting factor (TIF), suppressed the induction of TNF-α expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1β [IL-1β], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-α expression by macrophage conditioned medium was associated with selective induction of the NF-κB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-α promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-α gene. Repression of the TNF-α promoter by TIF required a distal region that includes three NF-κB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-α promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-α expression in activated macrophages. TIF is distinct from the known TNF-α-inhibiting factors IL-4, IL-10, and transforming growth factor β and may represent a novel cytokine. PMID:9742085

  19. Release of tumor necrosis factor alpha by human peritoneal macrophages in response to toxic shock syndrome toxin-1.

    PubMed

    Buyalos, R P; Rutanen, E M; Tsui, E; Halme, J

    1991-08-01

    We examined the release in vitro of tumor necrosis factor-alpha (TNF-alpha) by peritoneal macrophages and peripheral blood monocytes following incubation with toxic shock syndrome toxin-1 (TSST-1). We obtained peritoneal macrophages from 22 women at laparoscopy and peripheral blood monocytes from four healthy women during both the midfollicular and midluteal phases of the menstrual cycle. The samples were incubated for 24 hours at 37 C with 10(-2)-10(4) ng/mL of TSST-1 or 10(4) ng/mL of bacterial endotoxin. Tumor necrosis factor-alpha activity was determined with a bioassay using an actinomycin D-sensitized WEHI-164 murine fibrosarcoma cell line. Twenty-four-hour incubation with TSST-1 resulted in a dose-dependent release of TNF-alpha by both peritoneal macrophages (maximal response 554 +/- 97 U of activity) and peripheral blood monocytes (maximal response 478 +/- 81 U of activity). We observed enhanced TNF-alpha release by peritoneal macrophages from women with endometriosis, compared with those without endometriosis, at a concentration of 10(4) ng/mL of TSST-1 (704 +/- 134 versus 354 +/- 103 U of activity; P less than .05). These data support the theory that the metabolic and physiologic derangements of perimenstrual toxic shock syndrome may be partially mediated by TNF-alpha released by peritoneal macrophages as a result of exposure to TSST-1. PMID:2067760

  20. Transcription factors STAT6 and KLF4 implement macrophage polarization via the dual catalytic powers of MCPIP

    PubMed Central

    Kapoor, Nidhi; Niu, Jianli; Saad, Yasser; Kumar, Sanjay; Sirakova, Tatiana; Becerra, Edilu; Li, Xiaoman; Kolattukudy, Pappachan E.

    2015-01-01

    Macrophage polarization plays a critical role in tissue homeostasis, disease pathogenesis, and inflammation and its resolution. IL-4-induced macrophage polarization involves induction of STAT6 and KLF4 that induce each other and promote M2 polarization. However, how these transcription factors implement M2 polarization is not understood. We report that in murine macrophages MCPIP, induced by KLF4, inhibits M1 polarization by inhibiting NF-κB activation and implements M2 polarization using both its deubiquitinase and RNase activities that cause sequential induction of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress and autophagy required for M2 polarization. MCPIP also induces C/EBPβ and PPARγ that promote M2 polarization. Macrophages from mice with myeloid-targeted overexpression of MCPIP show elevated expression of M2 markers and reduced response to LPS, whereas macrophages from mice with myeloid-specific deletion of MCPIP manifest elevated M1 polarization with enhanced phagocytic activity. Thus, both in vivo and in vitro experiments demonstrate that the transcription factors STAT6 and KLF4 implement IL-4-induced M2 polarization via the dual catalytic activities of MCPIP. PMID:25934862

  1. Inhibition of Macrophage Migration Inhibitory Factor Protects against Inflammation and Matrix Deposition in Kidney Tissues after Injury

    PubMed Central

    Lu, Hong; Bai, Yongyu; Wu, Lianfeng; Hong, Weilong; Liang, Yong; Chen, Bicheng; Bai, Yongheng

    2016-01-01

    Background. Macrophage migration inhibitory factor (MIF) is an important immunoregulatory cytokine involved in inflammation, which may be one important reason resulting in matrix deposition in renal tissues after injury. However, the underlying mechanisms have not yet been elucidated. Methods and Results. We uncovered a crucial role of MIF in inflammation and collagen deposition in vivo and in vitro. In rats, ureteral obstruction induced tubular injury, matrix accumulation, and inflammatory cell infiltration. Additionally, enhanced MIF levels in the obstructed kidneys were closely related to the increasing numbers of CD68-positive macrophages. These obstruction-induced injuries can be relieved by recanalization, consequently resulting in downregulated expression of MIF and its receptor CD74. Similarly, ischemia reperfusion induced renal injury, and it was accompanied by elevated MIF levels and macrophages infiltration. In cultured tubular epithelial cells (TECs), aristolochic acid (AA) promoted matrix production and increased MIF expression, as well as the release of macrophage-related factors. Inhibition of MIF with an antagonist ISO-1 resulted in the abolishment of these genotypes in AA-treated TECs. Conclusion. MIF plays an important role in macrophage-related inflammation and matrix deposition in kidney tissues following injury. MIF as a specific inhibitor may have therapeutic potential for patients with inflammatory and fibrotic kidney diseases. PMID:27313397

  2. Influence of Sae-regulated and Agr-regulated factors on the escape of Staphylococcus aureus from human macrophages.

    PubMed

    Münzenmayer, Lisa; Geiger, Tobias; Daiber, Ellen; Schulte, Berit; Autenrieth, Stella E; Fraunholz, Martin; Wolz, Christiane

    2016-08-01

    Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild-type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol-soluble modulins (PSMs) for phagosomal escape. Thus, Sae-regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis-related caspase 3, 7 or 8 nor to NLRP3-dependent inflammasome activation. PMID:26895738

  3. Macrophage Migration Inhibitory Factor (MIF) as a Chaperone Inhibiting Accumulation of Misfolded SOD1

    PubMed Central

    Israelson, Adrian; Ditsworth, Dara; Sun, Shuying; Song, SungWon; Liang, Jason; Hruska-Plochan, Marian; McAlonis-Downes, Melissa; Abu-Hamad, Salah; Zoltsman, Guy; Shani, Tom; Maldonado, Marcus; Bui, Anh; Navarro, Michael; Zhou, Huilin; Marsala, Martin; Kaspar, Brian K.; Da Cruz, Sandrine; Cleveland, Don W.

    2015-01-01

    Summary Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in non-neuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity. PMID:25801706

  4. TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu1

    PubMed Central

    Sharma, Jyotika; Mishra, Bibhuti B.; Li, Qun; Teale, Judy M.

    2011-01-01

    The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent F. novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains. PMID:21497800

  5. Role of granulocyte‐macrophage colony‐stimulating factor in pulmonary fibrosis following pulmonary alveolar proteinosis

    PubMed Central

    Sha, Joy

    2016-01-01

    Abstract Pulmonary alveolar proteinosis (PAP) is a rare diffuse lung disease characterized by accumulation of lipoproteinacious material in alveoli, with distinct features on high resolution computed tomography and biopsy. Its association with pulmonary fibrosis is infrequently encountered, and a clear understanding of the underlying pathogenesis is yet to be established. We report the case of a 48‐year‐old woman with known autoimmune PAP (aPAP) first diagnosed 20 years ago, who presented with worsening hypoxemia and radiological features consistent with pulmonary fibrosis, after many years of stable disease. We present a review of previously considered mechanisms of causation behind such changes, and in particular, postulate the role of granulocyte‐macrophage colony‐stimulating factor deficiency in pulmonary fibrosis seen in aPAP. PMID:27512562

  6. Macrophage migration inhibitory factor interacts with HBx and inhibits its apoptotic activity

    SciTech Connect

    Zhang Shimeng; Lin Ruxian; Zhou Zhe; Wen Siyuan; Lin Li; Chen Suhong; Shan Yajun; Cong Yuwen; Wang Shengqi . E-mail: sqwang@nic.bmi.ac.cn

    2006-04-07

    HBx, a transcriptional transactivating protein of hepatitis B virus (HBV), is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the precise molecular mechanism remains largely elusive. We used the yeast two-hybrid system to identify that HBx interacts with MIF directly. Macrophage migration inhibitory factor (MIF) is implicated in the regulation of inflammation, cell growth, and even tumor formation. The interaction between HBx and MIF was verified with co-immunoprecipitation, GST pull-down, and cellular colocalization. The expression of MIF was up-regulated in HBV particle producing cell 2.2.15 compared with HepG2 cell. Both HBx and MIF cause HepG2 cell G /G{sub 1} phase arrest, proliferation inhibition, and apoptosis. However, MIF can counteract the apoptotic effect of HBx. These results may provide evidence to explain the link between HBV infection and hepatocellular carcinoma.

  7. Development of ostrich thrombocytes and monocyte-derived macrophages in culture and the control of Toxoplasma gondii reproduction after macrophage activation.

    PubMed

    Miranda, Farlen J B; Damasceno-Sá, João Cláudio; DaMatta, Renato A

    2016-01-01

    Raising ostriches became an important economic activity after their products became commodities. The health of farm animals is of paramount importance, so assessing basic immunological responses is necessary to better understand health problems. We developed a method to obtain ostrich thrombocytes and macrophages. The thrombocytes died by apoptosis after 48 h in culture, and the macrophages expanded in size and increased the number of acidic compartments. Macrophages were activated by chicken interferon-γ, producing high levels of nitric oxide. Toxoplasma gondii was able to infect these macrophages, and activation controlled parasitic reproduction. T. gondii, however, persisted in these cells, and infection reduced the production of nitric oxide. These results are important for the future assessment of the basic cellular and immunobiology of ostriches and demonstrate T. gondii suppression of nitric oxide production. PMID:26794839

  8. Some derivatives of polyvinylpyridine 1-oxides and their effect on the cytotoxicity of quartz in macrophage cultures

    PubMed Central

    Holt, P. F.; Lindsay, H.; Beck, E. G.

    1970-01-01

    1. Poly(2-vinylpyridine 1-oxide) counteracts the pathogenic effects normally produced when quartz is injected into or inhaled by animals and the cytotoxic effects when quartz is added to macrophage cultures. The protective action of this polymer has been attributed variously to the formation of an adsorbed layer on the quartz particles, complex formation with monosilicic acid produced by the dissolution of quartz, and strengthening of the membranes or microstructures of the cells. 2. Stereoregular forms of poly(2-vinylpyridine 1-oxide), some alkyl derivatives of poly(2-vinylpyridine 1-oxide), poly(3-vinylpyridine 1-oxide) and poly(4-vinylpyridine 1-oxide), a copolymer of 2-vinylpyridine 1-oxide and 2-n-propenylpyridine 1-oxide, some poly(1-methyl-2-vinylpyridinium) quaternary salts, and poly(1-methoxy-2-vinylpyridinium iodide), which had previously been synthesized and studied with respect to their viscosities and interaction with silicic acid, were tested for their ability to counteract the cytotoxic effects of quartz in macrophage cultures. The tests were effected both by pretreating the quartz with polymers, and by pretreating the cells. 3. Every polymer proved active in one or other of these conditions, but several were active in one test but inactive in the other. Some polymer quaternary salts, which do not contain the N-oxide group, were found to be active. A remarkable difference in activity was found between the two stereoregular forms of poly(2-vinylpyridine 1-oxide). Pretreatment of the quartz with some of the polymers increased its cytotoxicity significantly. 4. Most of the results could be interpreted on the hypothesis that the polymers form an adsorbed layer on the quartz surface, but it is difficult to apply this explanation to two polymers which are inactive when used to pretreat the macrophages but are active when adsorbed on quartz. PMID:4312930

  9. Macrophage Migration Inhibitory Factor (MIF) of the protozoan parasite Eimeria influences the components of the immune system of its host, the chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Macrophage migration inhibitory factor (MIF) is a soluble factor produced by sensitized T lymphocytes that inhibits the random migration of macrophages. Homologues of MIF from invertebrates have been identified making it an interesting molecule from a functional perspective. In the present study, ...

  10. Colony stimulating factor-1 receptor signaling networks inhibit mouse macrophage inflammatory responses by induction of microRNA-21

    PubMed Central

    Caescu, Cristina I.; Guo, Xingyi; Tesfa, Lydia; Bhagat, Tushar D.; Verma, Amit; Zheng, Deyou

    2015-01-01

    Macrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721–regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721–regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21–regulated messenger RNAs revealed that 80% of the CSF-1–regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R–mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R–regulated miR-21 network that modulates macrophage polarization. PMID:25573988

  11. Macrophages from IBD patients exhibit defective tumour necrosis factor-α secretion but otherwise normal or augmented pro-inflammatory responses to infection.

    PubMed

    Campos, Nair; Magro, Fernando; Castro, Ana Rita; Cabral, Joana; Rodrigues, Pedro; Silva, Ricardo; Appelberg, Rui; Rodrigues, Susana; Lopes, Susana; Macedo, Guilherme; Sarmento, Amélia

    2011-08-01

    Defects in macrophage function have been implicated in the establishment of Crohn's disease (CD). However, the response of macrophages from CD patients to live bacteria, particularly Mycobacterium avium subsp. paratuberculosis (MAP), has not been addressed. Considering MAP has long been associated to CD, our objective was to assess whether macrophages from CD patients showed impaired inflammatory response to infection by MAP comparing to M. avium subsp. avium (MA) and other live intestinal commensal bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, ulcerative colitis (UC) patients and controls. Following in vitro infection with MAP, MA, Escherichia coli or Enterococcus faecalis, cytokine levels and cell surface receptor expression were evaluated at different time points. Macrophages from CD patients showed impaired TNF-α secretion in response to bacterial challenge, but augmented IL-23 secretion and preserved IL-12 secretion and CD-40 expression. In addition, CD macrophages showed low IL-10 secretion. Macrophages from IBD patients showed increased expression of TLR-2 and -4, unaffected by infection. Differences in cytokine secretion observed after bacterial challenge were not MAP-specific, as other bacteria (E. coli and MA) showed similar effects. Macrophages from UC patients showed a less compromised TNF-α synthesis in response to mycobacterial infection than CD macrophages, with increased constitutive IL-12 secretion, and preserved IL-10 secretion. The increased IL-23 levels in response to infection and decreased IL-10 production observed in macrophages from CD patients may contribute to the inflammatory exacerbation observed in those patients. PMID:21269730

  12. Unique genome-wide transcriptome profiles of chicken macrophages exposed to Salmonella-derived endotoxin

    PubMed Central

    2010-01-01

    Background Macrophages play essential roles in both innate and adaptive immune responses. Bacteria require endotoxin, a complex lipopolysaccharide, for outer membrane permeability and the host interprets endotoxin as a signal to initiate an innate immune response. The focus of this study is kinetic and global transcriptional analysis of the chicken macrophage response to in vitro stimulation with endotoxin from Salmonella typhimurium-798. Results The 38535-probeset Affymetrix GeneChip Chicken Genome array was used to profile transcriptional response to endotoxin 1, 2, 4, and 8 hours post stimulation (hps). Using a maximum FDR (False Discovery Rate) of 0.05 to declare genes as differentially expressed (DE), we found 13, 33, 1761 and 61 DE genes between endotoxin-stimulated versus non-stimulated cells at 1, 2, 4 and 8 hps, respectively. QPCR demonstrated that endotoxin exposure significantly affected the mRNA expression of IL1B, IL6, IL8, and TLR15, but not IL10 and IFNG in HD 11 cells. Ingenuity Pathway Analysis showed that 10% of the total DE genes were involved in inflammatory response. Three, 9.7, 96.8, and 11.8% of the total DE inflammatory response genes were significantly differentially expressed with endotoxin stimulation at 1, 2, 4 and 8 hps, respectively. The NFKBIA, IL1B, IL8 and CCL4 genes were consistently induced at all times after endotoxin treatment. NLRC5 (CARD domain containing, NOD-like receptor family, RCJMB04_18i2), an intracellular receptor, was induced in HD11 cells treated with endotoxin. Conclusions As above using an in vitro model of chicken response to endotoxin, our data revealed the kinetics of gene networks involved in host response to endotoxin and extend the known complexity of networks in chicken immune response to Gram-negative bacteria such as Salmonella. The induction of NFKBIA, IL1B, IL8, CCL4 genes is a consistent signature of host response to endotoxin over time. We make the first report of induction of a NOD-like receptor

  13. Lentinan mitigates therarubicin-induced myelosuppression by activating bone marrow-derived macrophages in an MAPK/NF-κB-dependent manner.

    PubMed

    Liu, Qiang; Dong, Lei; Li, Hong; Yuan, Jia; Peng, Yuping; Dai, Shejiao

    2016-07-01

    Bone marrow (BM) suppression (also known as myelosuppression) is the most common and most severe side-effect of therarubicin (THP) and thereby limits the clinical application of this anticancer agent. Lentinan (LNT), a glucan extracted from dried shiitake mushrooms (Lentinula edodes), exhibits a variety of pharmacological activities. The objectives of the present study were to determine the effect of LNT on the myelosuppression of THP-treated mice and to examine the pharmacological mechanism of these effects. In vivo experiments indicated that non-cytotoxic levels of LNT strongly increased blood myeloperoxidase (MPO) activity; improved BM structural injuries; increased the numbers of leukocytes and neutrophils in the blood and BM; elevated the blood levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF); and reduced the self-healing period in THP-treated mice. In vitro experiments indicated that LNT increased the viability of BM-derived macrophages (BMDMs) in a time- and dose-dependent manner without toxic side-effects and markedly increased the release of G-CSF, GM-CSF and M-CSF by BMDMs. Further analyses revealed that LNT activated the NF-κB and MAPK signalling pathways and promoted the nuclear import of p65 and that BAY 11-7082 (a specific inhibitor of NF-κB) suppressed the release of G-CSF, GM-CSF and M-CSF. Furthermore, we found that U0126, SB203580 and SP600125 (specific inhibitors of ERK, p38 and JNK, respectively) markedly inhibited the IKK/IκB/NF-κB-dependent release of G-CSF, GM-CSF and M-CSF. In conclusion, LNT induces the production of G-CSF, GM-CSF and M-CSF by activating the MAPK/NF-κB signalling pathway in BM cells, thereby mitigating THP-induced myelosuppression. PMID:27121155

  14. Mycobacterium leprae-induced Insulin-like Growth Factor I attenuates antimicrobial mechanisms, promoting bacterial survival in macrophages

    PubMed Central

    Batista-Silva, L. R.; Rodrigues, Luciana Silva; Vivarini, Aislan de Carvalho; Costa, Fabrício da Mota Ramalho; Mattos, Katherine Antunes de; Costa, Maria Renata Sales Nogueira; Rosa, Patricia Sammarco; Toledo-Pinto, T. G.; Dias, André Alves; Moura, Danielle Fonseca; Sarno, Euzenir Nunes; Lopes, Ulisses Gazos; Pessolani, Maria Cristina Vidal

    2016-01-01

    Mycobacterium leprae (ML), the etiologic agent of leprosy, can subvert macrophage antimicrobial activity by mechanisms that remain only partially understood. In the present study, the participation of hormone insulin-like growth factor I (IGF-I) in this phenomenum was investigated. Macrophages from the dermal lesions of the disseminated multibacillary lepromatous form (LL) of leprosy expressed higher levels of IGF-I than those from the self-limited paucibacillary tuberculoid form (BT). Higher levels of IGF-I secretion by ML-infected macrophages were confirmed in ex vivo and in vitro studies. Of note, the dampening of IGF-I signaling reverted the capacity of ML-infected human and murine macrophages to produce antimicrobial molecules and promoted bacterial killing. Moreover, IGF-I was shown to inhibit the JAK/STAT1-dependent signaling pathways triggered by both mycobacteria and IFN-γ most probably through its capacity to induce the suppressor of cytokine signaling-3 (SOCS3). Finally, these in vitro findings were corroborated by in vivo observations in which higher SOCS3 expression and lower phosphorylation of STAT1 levels were found in LL versus BT dermal lesions. Altogether, our data strongly suggest that IGF-I contributes to the maintenance of a functional program in infected macrophages that suits ML persistence in the host, reinforcing a key role for IGF-I in leprosy pathogenesis. PMID:27282338

  15. Mycobacterium leprae-induced Insulin-like Growth Factor I attenuates antimicrobial mechanisms, promoting bacterial survival in macrophages.

    PubMed

    Batista-Silva, L R; Rodrigues, Luciana Silva; Vivarini, Aislan de Carvalho; Costa, Fabrício da Mota Ramalho; Mattos, Katherine Antunes de; Costa, Maria Renata Sales Nogueira; Rosa, Patricia Sammarco; Toledo-Pinto, T G; Dias, André Alves; Moura, Danielle Fonseca; Sarno, Euzenir Nunes; Lopes, Ulisses Gazos; Pessolani, Maria Cristina Vidal

    2016-01-01

    Mycobacterium leprae (ML), the etiologic agent of leprosy, can subvert macrophage antimicrobial activity by mechanisms that remain only partially understood. In the present study, the participation of hormone insulin-like growth factor I (IGF-I) in this phenomenum was investigated. Macrophages from the dermal lesions of the disseminated multibacillary lepromatous form (LL) of leprosy expressed higher levels of IGF-I than those from the self-limited paucibacillary tuberculoid form (BT). Higher levels of IGF-I secretion by ML-infected macrophages were confirmed in ex vivo and in vitro studies. Of note, the dampening of IGF-I signaling reverted the capacity of ML-infected human and murine macrophages to produce antimicrobial molecules and promoted bacterial killing. Moreover, IGF-I was shown to inhibit the JAK/STAT1-dependent signaling pathways triggered by both mycobacteria and IFN-γ most probably through its capacity to induce the suppressor of cytokine signaling-3 (SOCS3). Finally, these in vitro findings were corroborated by in vivo observations in which higher SOCS3 expression and lower phosphorylation of STAT1 levels were found in LL versus BT dermal lesions. Altogether, our data strongly suggest that IGF-I contributes to the maintenance of a functional program in infected macrophages that suits ML persistence in the host, reinforcing a key role for IGF-I in leprosy pathogenesis. PMID:27282338

  16. The Lineage-Specific Transcription Factor PU.1 Prevents Polycomb-Mediated Heterochromatin Formation at Macrophage-Specific Genes.

    PubMed

    Tagore, Mohita; McAndrew, Michael J; Gjidoda, Alison; Floer, Monique

    2015-08-01

    Lineage-specific transcription factors (TFs) are important determinants of cellular identity, but their exact mode of action has remained unclear. Here we show using a macrophage differentiation system that the lineage-specific TF PU.1 keeps macrophage-specific genes accessible during differentiation by preventing Polycomb repressive complex 2 (PRC2) binding to transcriptional regulatory elements. We demonstrate that the distal enhancer of a gene becomes bound by PRC2 as cells differentiate in the absence of PU.1 binding and that the gene is wrapped into heterochromatin, which is characterized by increased nucleosome occupancy and H3K27 trimethylation. This renders the gene inaccessible to the transcriptional machinery and prevents induction of the gene in response to an external signal in mature cells. In contrast, if PU.1 is bound at the transcriptional regulatory region of a gene during differentiation, PRC2 is not recruited, nucleosome occupancy is kept low, and the gene can be induced in mature macrophages. Similar results were obtained at the enhancers of other macrophage-specific genes that fail to bind PU.1 as an estrogen receptor fusion (PUER) in this system. These results show that one role of PU.1 is to exclude PRC2 and to prevent heterochromatin formation at macrophage-specific genes. PMID:26012552

  17. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage.

    PubMed

    Ushach, Irina; Zlotnik, Albert

    2016-09-01

    M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved. PMID:27354413

  18. Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes

    SciTech Connect

    Bozza, M.; Gerard, C.; Kolakowski, L.F. Jr.

    1995-06-10

    Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5{prime} untranslated region of the cDNA, a 3{prime} poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. 46 refs., 5 figs., 1 tab.

  19. Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

    SciTech Connect

    Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

    2011-06-01

    We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

  20. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

    PubMed

    Choudhary, Geetika S; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  1. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells

    PubMed Central

    Choudhary, Geetika S.; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A.; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  2. Impaired Macrophage Migration Inhibitory Factor (MIF)-AMPK Activation and Ischemic Recovery in the Senescent Heart

    PubMed Central

    Ma, Heng; Wang, Jingying; Thomas, D Paul; Tong, Chao; Leng, Lin; Wang, Wenkui; Merk, Melanie; Zierow, Swen; Bernhagen, Jürgen; Ren, Jun; Bucala, Richard; Li, Ji

    2010-01-01

    Background Elderly patients are more sensitive to myocardial ischemia, which results in higher mortality. We investigated how aging impacts the cardioprotective AMP-activated protein kinase (AMPK) signaling pathway. Methods and Results Ischemic AMPK activation was impaired in aged compared to young murine hearts. The expression and secretion of the AMPK upstream regulator, macrophage migration inhibitory factor (MIF), were lower in aged compared to young adult hearts. Additionally, the levels of hypoxia-inducible factor 1α (HIF-1α), a known transcriptional activator of MIF, were reduced in aged compared to young hearts. Ischemia-induced AMPK activation in MIF knock-out (MIF KO) mice was blunted, leading to greater contractile dysfunction in MIF-deficient than in wild type (WT) hearts. Furthermore, intra-myocardial injection of adenovirus encoding MIF (Adv-MIF) in aged mice increased MIF expression and ischemic AMPK activation, and reduced infarct size. Conclusions An impaired MIF-AMPK activation response in senescence thus may be attributed to an aging-associated defect in the transcription factor for MIF, HIF-1α. In the clinical setting, impaired cardiac HIF-1α activation and consequent reduced MIF expression may play an important role in the increased susceptibility to myocardial ischemia observed in older cardiac patients. PMID:20606117

  3. Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix

    PubMed Central

    Akkari, Leila; Gocheva, Vasilena; Kester, Jemila C.; Hunter, Karen E.; Quick, Marsha L.; Sevenich, Lisa; Wang, Hao-Wei; Peters, Christoph; Tang, Laura H.; Klimstra, David S.; Reinheckel, Thomas

    2014-01-01

    During the process of tumor progression, cancer cells can produce the requisite growth- and invasion-promoting factors and can also rely on noncancerous cells in the tumor microenvironment as an alternative, cell-extrinsic source. However, whether the cellular source influences the function of such tumor-promoting factors remains an open question. Here, we examined the roles of the cathepsin Z (CtsZ) protease, which is provided by both cancer cells and macrophages in pancreatic neuroendocrine tumors in humans and mice. We found that tumor proliferation was exclusively regulated by cancer cell-intrinsic functions of CtsZ, whereas tumor invasion required contributions from both macrophages and cancer cells. Interestingly, several of the tumor-promoting functions of CtsZ were not dependent on its described catalytic activity but instead were mediated via the Arg–Gly–Asp (RGD) motif in the enzyme prodomain, which regulated interactions with integrins and the extracellular matrix. Together, these results underscore the complexity of interactions within the tumor microenvironment and indicate that cellular source can indeed impact molecular function. PMID:25274726

  4. Deletion of macrophage migration inhibitory factor inhibits murine oral carcinogenesis: Potential role for chronic pro-inflammatory immune mediators.

    PubMed

    Oghumu, Steve; Knobloch, Thomas J; Terrazas, Cesar; Varikuti, Sanjay; Ahn-Jarvis, Jennifer; Bollinger, Claire E; Iwenofu, Hans; Weghorst, Christopher M; Satoskar, Abhay R

    2016-09-15

    Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1β, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer. PMID:27164411

  5. The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.

    PubMed

    Benis, K A; Schneider, G B

    1996-10-15

    Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the

  6. Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1

    PubMed Central

    Singleton, Helen; Graham, Simon P.; Bodman-Smith, Katherine B.; Frossard, Jean-Pierre; Steinbach, Falko

    2016-01-01

    Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection. PMID:27313573

  7. Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate

    SciTech Connect

    Arana, Lide; Gangoiti, Patricia; Ouro, Alberto; Rivera, Io-Guane; Ordonez, Marta; Trueba, Miguel; Lankalapalli, Ravi S.; Bittman, Robert; Gomez-Munoz, Antonio

    2012-02-15

    We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A{sub 2} and protein kinase C-{alpha}, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-{alpha} and cPLA{sub 2}-{alpha} in this pathway. -- Highlights: Black-Right-Pointing-Pointer Ceramide 1-phosphate (C1P) stimulates reactive oxygen species (ROS) formation. Black-Right-Pointing-Pointer The enzyme responsible for ROS generation by C1P in macrophages is NADPH oxidase. Black-Right-Pointing-Pointer NADPH oxidase lies downstream of cPLA{sub 2}-{alpha} and PKC-{alpha} in this pathway. Black-Right-Pointing-Pointer ROS generation is essential for the stimulation of macrophage proliferation by C1P.

  8. Transcriptional mechanisms underlying sensitization of peripheral sensory neurons by Granulocyte-/Granulocyte-macrophage colony stimulating factors

    PubMed Central

    2013-01-01

    Background Cancer-associated pain is a major cause of poor quality of life in cancer patients and is frequently resistant to conventional therapy. Recent studies indicate that some hematopoietic growth factors, namely granulocyte macrophage colony stimulating factor (GMCSF) and granulocyte colony stimulating factor (GCSF), are abundantly released in the tumor microenvironment and play a key role in regulating tumor-nerve interactions and tumor-associated pain by activating receptors on dorsal root ganglion (DRG) neurons. Moreover, these hematopoietic factors have been highly implicated in postsurgical pain, inflammatory pain and osteoarthritic pain. However, the molecular mechanisms via which G-/GMCSF bring about nociceptive sensitization and elicit pain are not known. Results In order to elucidate G-/GMCSF mediated transcriptional changes in the sensory neurons, we performed a comprehensive, genome-wide analysis of changes in the transcriptome of DRG neurons brought about by exposure to GMCSF or GCSF. We present complete information on regulated genes and validated profiling analyses and report novel regulatory networks and interaction maps revealed by detailed bioinformatics analyses. Amongst these, we validate calpain 2, matrix metalloproteinase 9 (MMP9) and a RhoGTPase Rac1 as well as Tumor necrosis factor alpha (TNFα) as transcriptional targets of G-/GMCSF and demonstrate the importance of MMP9 and Rac1 in GMCSF-induced nociceptor sensitization. Conclusion With integrative approach of bioinformatics, in vivo pharmacology and behavioral analyses, our results not only indicate that transcriptional control by G-/GMCSF signaling regulates a variety of established pain modulators, but also uncover a large number of novel targets, paving the way for translational analyses in the context of pain disorders. PMID:24067145

  9. Complement factor H interferes with Mycobacterium bovis BCG entry into macrophages and modulates the pro-inflammatory cytokine response.

    PubMed

    Abdul-Aziz, Munirah; Tsolaki, Anthony G; Kouser, Lubna; Carroll, Maria V; Al-Ahdal, Mohammed N; Sim, Robert B; Kishore, Uday

    2016-09-01

    Mycobacterium tuberculosis is an accomplished intracellular pathogen, particularly within the macrophage and this is of the utmost importance in the host-pathogen stand-off observed in the granuloma during latent tuberculosis. Contact with innate immune molecules is one of the primary interactions that can occur with the pathogen M. tuberculosis once inhaled. Complement proteins may play a role in facilitating M. tuberculosis interactions with macrophages. Here, we demonstrate that factor H, a complement regulatory protein that down-regulates complement alternative pathway activation, binds directly to the model organism M. bovis BCG. Binding of factor H reaches saturation at 5-10μg of factor H/ml, well below the plasma level. C4 binding protein (C4BP) competed with factor H for binding to mycobacteria. Factor H was also found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Real-time qPCR analysis showed stark differential responses of pro- and anti-inflammatory cytokines during the early stages of phagocytosis, as evident from elevated levels of TNF-α, IL-1β and IL-6, and a concomitant decrease in IL-10, TGF-β and IL-12 levels, when THP-1:BCG interaction took place in the presence of factor H. Our results suggest that factor H can interfere with mycobacterial entry into macrophages and modulate inflammatory cytokine responses, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. Our results offer novel insights into complement activation-independent functions of factor H during the host-pathogen interaction in tuberculosis. PMID:27262511

  10. Development and characterization of antiserum to murine granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Mochizuki, D.Y.; Eisenman, J.R.; Conlon, P.J.; Park, L.S.; Urdal, D.L.

    1986-05-15

    The expression in yeast of a cDNA clone encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has made possible the purification of large quantities of this recombinant protein. Rabbits immunized with pure recombinant GM-CSF generated antibodies that were shown to be specific for both recombinant GM-CSF and GM-CSF isolated from natural sources. Other lymphokines, including IL 1..beta.., IL 2, IL 3, and recombinant human GM-CSF did not react with the antiserum. The antiserum together with recombinant GM-CSF that had been radiolabeled with /sup 125/I to high specific activity, formed the foundation for a rapid, sensitive, and quantitative radioimmunoassay specific for murine GM-CSF. Furthermore, the antiserum was found to inhibit the biologic activities of GM-CSF as measured in both a bone marrow proliferation assay and a colony assay, and thus should prove to be a useful reagent for dissecting the complex growth factor activities involved in murine hematopoiesis.

  11. Macrophage phagocytosis: effects of environmental pollutants, alcohol, cigarette smoke, and other external factors

    PubMed Central

    Karavitis, John; Kovacs, Elizabeth J.

    2011-01-01

    The ability of a pathogen to evade host immunity successfully, in contrast to the host's capacity to defend itself against a foreign invader, is a complex struggle, in which eradication of infection is dictated by a robust immunologic response. Often, there are external factors that can alter the outcome by tipping the scale to benefit pathogen establishment rather than resolution by the host's defense system. These external sources, such a cigarettes, alcohol, or environmental pollutants, can negatively influence the effectiveness of the immune system's response to a pathogen. The observed suppression of immune function can be attributed to dysregulated cytokine and chemokine production, the loss of migratory potential, or the inability to phagocytose pathogens by immune cells. This review will focus on the mechanisms involved during the toxin-induced suppression of phagocytosis. The accumulated data support the importance of studying the mechanisms of phagocytosis following exposure to these factors, in that this effect alone cannot only leave the host susceptible to infection but also promote alterations in many other macrophage functions necessary for pathogen clearance and restoration of homeostasis. PMID:21878544

  12. Review: putative roles for the macrophage migratory inhibitory factor at the maternal fetal interface.

    PubMed

    Bevilacqua, E; Paulesu, L; Ferro, E A V; Ietta, F; Faria, M R; Lorenzon, A R; Costa, A F; Martucci, M

    2014-02-01

    Complex and dynamic networks of molecules participate in the essential interactions between maternal organism, placenta and fetus in a healthy and successful pregnancy. Macrophage migratory inhibitory factor (MIF) is one of several molecules produced at implantation sites; MIF is mostly expressed by trophoblast cells. This has led to expectations of MIF's relevance as a partner in the maternal/fetal dialog. MIF is known by its biological interactions and functional roles as an activator of innate immunity, regulating subsequent adaptive responses, which include inhibition of migration of mononuclear cells in vitro, antagonism of glucocorticoids, and regulation of expression of Toll-like receptor 4. Beyond roles in the inflammatory response, MIF can interfere with proliferative activities in different cell types, as well as with cell death pathways. This intriguing factor found at the human, porcine, ovine, bovine and rodent maternal-fetal interfaces is present in a time- and spatially-dependent manner, indicating regulatory roles in the process of embryo implantation, placental development, maintenance of pregnancy and birth. Here, we will review MIF participation in placental physiology, including new evidence for a dialog with uterine cells, and a potential role in protection of uterine decidual cells. PMID:24215782

  13. Macrophage Migration Inhibitory Factor Secretion Is Induced by Ionizing Radiation and Oxidative Stress in Cancer Cells

    PubMed Central

    Du, Weinan; Welford, Scott M.

    2016-01-01

    The macrophage migration inhibitory factor (MIF) has been increasingly implicated in cancer development and progression by promoting inflammation, angiogenesis, tumor cell survival and immune suppression. MIF is overexpressed in a variety of solid tumor types in part due to its responsiveness to hypoxia inducible factor (HIF) driven transcriptional activation. MIF secretion, however, is a poorly understood process owing to the fact that MIF is a leaderless polypeptide that follows a non-classical secretory pathway. Better understanding of MIF processing and release could have therapeutic implications. Here, we have discovered that ionizing radiation (IR) and other DNA damaging stresses can induce robust MIF secretion in several cancer cell lines. MIF secretion by IR appears independent of ABCA1, a cholesterol efflux pump that has been implicated previously in MIF secretion. However, MIF secretion is robustly induced by oxidative stress. Importantly, MIF secretion can be observed both in cell culture models as well as in tumors in mice in vivo. Rapid depletion of MIF from tumor cells observed immunohistochemically is coincident with elevated circulating MIF detected in the blood sera of irradiated mice. Given the robust tumor promoting activities of MIF, our results suggest that an innate host response to genotoxic stress may mitigate the beneficial effects of cancer therapy, and that MIF inhibition may improve therapeutic responses. PMID:26741693

  14. Cross-talk among myeloid-derived suppressor cells, macrophages, and tumor cells impacts the inflammatory milieu of solid tumors

    PubMed Central

    Beury, Daniel W.; Parker, Katherine H.; Nyandjo, Maeva; Sinha, Pratima; Carter, Kayla A.; Ostrand-Rosenberg, Suzanne

    2014-01-01

    MDSC and macrophages are present in most solid tumors and are important drivers of immune suppression and inflammation. It is established that cross-talk between MDSC and macrophages impacts anti-tumor immunity; however, interactions between tumor cells and MDSC or macrophages are less well studied. To examine potential interactions between these cells, we studied the impact of MDSC, macrophages, and four murine tumor cell lines on each other, both in vitro and in vivo. We focused on IL-6, IL-10, IL-12, TNF-α, and NO, as these molecules are produced by macrophages, MDSC, and many tumor cells; are present in most solid tumors; and regulate inflammation. In vitro studies demonstrated that MDSC-produced IL-10 decreased macrophage IL-6 and TNF-α and increased NO. IL-6 indirectly regulated MDSC IL-10. Tumor cells increased MDSC IL-6 and vice versa. Tumor cells also increased macrophage IL-6 and NO and decreased macrophage TNF-α. Tumor cell-driven macrophage IL-6 was reduced by MDSC, and tumor cells and MDSC enhanced macrophage NO. In vivo analysis of solid tumors identified IL-6 and IL-10 as the dominant cytokines and demonstrated that these molecules were produced predominantly by stromal cells. These results suggest that inflammation within solid tumors is regulated by the ratio of tumor cells to MDSC and macrophages and that interactions of these cells have the potential to alter significantly the inflammatory milieu within the tumor microenvironment. PMID:25170116

  15. High‐resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages

    PubMed Central

    Guo, Manman; Härtlova, Anetta; Dill, Brian D.; Prescott, Alan R.; Gierliński, Marek

    2015-01-01

    Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in‐depth proteomics characterization of phagosomes from RAW 264.7 and bone marrow derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicate that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec‐1. Moreover, bone marrow derived macrophages phagosomes mature considerably faster by fusion with endosomes and the lysosome which we validated using fluorogenic phagocytic assays. We provide a valuable resource for researcher in the field and recommend careful use of the RAW 264.7 cell line when studying phagosome functions. All MS data have been deposited in the ProteomeXchange with identifier PXD001293 (http://proteomecentral.proteomexchange.org/dataset/PXD001293). PMID:25504905

  16. Legionella pneumophila EnhC is required for efficient replication in tumor necrosis factor α-stimulated macrophages

    PubMed Central

    Liu, Mingyu; Conover, Gloria M.; Isberg, Ralph R.

    2008-01-01

    Legionella pneumophila enhC− mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighboring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the ΔenhC strain, while tumor necrosis factor α (TNF-α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila ΔenhC. The absence of EnhC also caused defective trafficking of the Legionella-containing vacuole in TNF-α treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the ΔenhC mutant was more susceptible to H2O2 treatment than the wild type strain. EnhC, therefore, is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication. PMID:18549456

  17. Effects of molecular liposomal hybrid compositions with oxidized dextrans and isonicotinic acid hydrazide on production of granulocytic macrophage colony-stimulating factor by macrophages.

    PubMed

    Shkurupy, V A; Arkhipov, S A; Troitsky, A V; Luzgina, N G; Zaikovskaja, M V; Ufimceva, E G; Iljine, D A; Akhramenko, E S; Gulyaeva, E P; Bistrova, T N

    2009-10-01

    The effects of molecular liposomal hybrid compositions consisting of liposomes (200-450 nm) containing oxidized dextrans (dextranals; 35-60 kDa) conjugated with isonicotinic acid hydrazide (dextrazides), their components, and native dextrans on the production of granulocytic macrophage CSF by peritoneal macrophages were studied in vitro. Dextranals proved to be more potent inductors of granulocytic macrophage CSF than native dextrans. Conjugation of nicotinic acid hydrazide with dextranals did not modify their capacity to stimulate the production of granulocytic macrophage CSF. Liposomes in the molecular liposomal hybrid compositions did not attenuate the dextrazide capacity to stimulate the production of granulocytic macrophage CSF. Molecular liposomal compositions containing 60 kDa dextrazide exhibited the most potent stimulatory effect on macrophage production of granulocytic macrophage CSF. PMID:20396775

  18. Human Leukocyte Antigen-Presented Macrophage Migration Inhibitory Factor Is a Surface Biomarker and Potential Therapeutic Target for Ovarian Cancer.

    PubMed

    Patterson, Andrea M; Kaabinejadian, Saghar; McMurtrey, Curtis P; Bardet, Wilfried; Jackson, Ken W; Zuna, Rosemary E; Husain, Sanam; Adams, Gregory P; MacDonald, Glen; Dillon, Rachelle L; Ames, Harold; Buchli, Rico; Hawkins, Oriana E; Weidanz, Jon A; Hildebrand, William H

    2016-02-01

    T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy. PMID:26719579

  19. Jellyfish skin polysaccharides: extraction and inhibitory activity on macrophage-derived foam cell formation.

    PubMed

    Zhang, Hai-Lin; Cui, Shao-Hua; Zha, Xue-Qiang; Bansal, Vibha; Xue, Lei; Li, Xiao-Long; Hao, Ran; Pan, Li-Hua; Luo, Jian-Ping

    2014-06-15

    In this work, response surface methodology was used to determine optimum conditions for extraction of polysaccharides from jellyfish skin (JSP). The optimum parameters were found to be raw material to water ratio 1:7.5 (w/v), extraction temperature 100°C and extraction time 4h. Under these conditions, the JSP yield reached 1.007 mg/g. Papain (15 U/mL) in combination with Sevag reagent was beneficial in removing proteins from JSP. After precipitation with ethanol at final concentration of 40%, 60% and 80% in turn, three polysaccharide fractions of JSP1, JSP2 and JSP3 were obtained from JSP, respectively. The three fractions exhibited different physicochemical properties with respect to molecular weight distribution, monosaccharide composition, infrared absorption spectra, and glycosyl bond composition. In addition, JSP3 showed strong inhibitory effects on oxidized low-density lipoprotein (oxLDL) induced conversion of macrophages into foam cells, which possibly attributed to the down-regulation of some atherogenesis-related gene expressions. PMID:24721094

  20. Pattern of cytokine and chemokine production by THP-1 derived macrophages in response to live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin Moreau strain

    PubMed Central

    Sousa-Vasconcelos, Periela da Silva; Seguins, Wellington da Silva; Luz, Eduardo de Souza; de Pinho, Rosa Teixeira

    2015-01-01

    Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1β] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria. PMID:26517663

  1. Modulation of Leishmania (L.) amazonensis Growth in Cultured Mouse Macrophages by Prostaglandins and Platelet Activating Factor

    PubMed Central

    Lonardoni, M. V. C.; Barbieri, C. L.; Russo, M.

    1994-01-01

    The role of endogenously synthesized PAF and prostaglandins on the infection of mouse macrophages by Letsbmanta (L.) amazonensis was investigated, as well as the possible correlation between the effects of these inflammatory mediators with nitric oxide production. It was found that pretreatment of macrophages with 10−5 M of the PAF antagonists, BN-52021 or WEB-2086, increased macrophage infection by 17 and 59%, respectively. The cyclooxygenase inhibitor, indomethacin (10 μg/ml), induced a significant inhibition which was reversed by addition of PGE (10-3 M) to the culture medium. These results suggested that the infection of macrophages by leisbmanla is inhibited by PAF and enhanced by prostaglandins and that these mediators are produced by macrophages during this infection. This was confirmed by addition of these mediators to the culture medium before infection; PAF (10−6, 10−9 and 10−12M) reduced significantly the infection whereas PGE2 (10−5 M) induced a marked enhancement. This effect of exogenous PAF on macrophage infection was reversed by the two PAF antagonists used in this study as well as by the inhibitor of nitric oxide synthesis, L-arginine methyl ester (100 mM). Taken together the data suggest that endogenous production of PAF and PGE2 exert opposing effects on Lesbmana–macrophage interaction and that nitric oxide may be involved in the augmented destruction of parasites induced by PAF. PMID:18472932

  2. Macrophage infection models for Mycobacterium tuberculosis.

    PubMed

    Johnson, Benjamin K; Abramovitch, Robert B

    2015-01-01

    Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging. PMID:25779326

  3. HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells.

    PubMed

    Jerebtsova, Marina; Kumari, Namita; Xu, Min; de Melo, Gustavo Brito Alvim; Niu, Xiaomei; Jeang, Kuan-Teh; Nekhai, Sergei

    2012-07-26

    A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs) through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages. PMID:22934150

  4. HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

    PubMed Central

    Jerebtsova, Marina; Kumari, Namita; Xu, Min; de Melo, Gustavo Brito Alvim; Niu, Xiaomei; Jeang, Kuan-Teh; Nekhai, Sergei

    2012-01-01

    A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs) through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages. PMID:22934150

  5. In vitro evaluation of inhibitory effect of nuclear factor-kappaB activity by small interfering RNA on pro-tumor characteristics of M2-like macrophages.

    PubMed

    Kono, Yusuke; Kawakami, Shigeru; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2014-01-01

    Tumor-associated macrophages (TAMs) have an alternatively activated macrophage phenotype (M2) and promote cancer cell proliferation, angiogenesis and metastasis. Nuclear factor-kappaB (NF-κB) is one of the master regulators of macrophage polarization. Here, we investigated the effect of inhibition of NF-κB activity by small interfering RNA (siRNA) on the pro-tumor response of macrophages located in the tumor microenvironment in vitro. We used mouse peritoneal macrophages cultured in conditioned medium from colon-26 cancer cells as an in vitro TAM model (M2-like macrophages). Transfection of NF-κB (p50) siRNA into M2-like macrophages resulted in a significant decrease in the secretion of interleukin (IL)-10 (a T helper 2 (Th2) cytokine) and a significant increase of T helper 1 (Th1) cytokine production (IL-12, tumor necrosis factor-α, and IL-6). Furthermore, vascular endothelial growth factor production and matrix metalloproteinase-9 mRNA expression in M2-like macrophages were suppressed by inhibition of NF-κB expression with NF-κB (p50) siRNA. In addition, there was a reduction of arginase mRNA expression and increase in nitric oxide production. The cytokine secretion profiles of macrophages cultured in conditioned medium from either B16BL6 or PAN-02 cancer cells were also converted from M2 to classically activated (M1) macrophages by transfection of NF-κB (p50) siRNA. These results suggest that inhibition of NF-κB activity in M2-like macrophages alters their phenotype toward M1. PMID:24141263

  6. Myeloid-Specific Krüppel-Like Factor 2 Inactivation Increases Macrophage and Neutrophil Adhesion and Promotes Atherosclerosis

    PubMed Central

    Lingrel, Jerry B; Pilcher-Roberts, Robyn; Basford, Joshua E.; Manoharan, Palanikumar; Neumann, Jon; Konaniah, Eddy S.; Srinivasan, Ramprasad; Bogdanov, Vladimir Y; Hui, David Y.

    2012-01-01

    Rationale and Objective Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. Methods and Results Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2-/-) mice were similar to myeKlf2+/+ macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2+/+ macrophages, myeKlf2-/- macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2-/- mice also adhered more robustly to endothelial cells, and less myeKlf2-/- neutrophils survived in culture over a 24 hr period in comparison with myeKlf2+/+ neutrophils. When myeKlf2-/- mice were mated to Ldlr-/- mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2-/-Ldlr-/- mice compared to myeKlf2+/+Ldlr-/- littermates. The increased atherosclerosis in myeKlf2-/-Ldlr-/- mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated- and nitrosylated-tyrosine epitopes in their lesion areas compared to myeKlf2+/+Ldlr-/- mice. Conclusions This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis. PMID:22474254

  7. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  8. Multipronged attenuation of macrophage-colony stimulating factor signaling by Epstein-Barr virus BARF1

    SciTech Connect

    Shim, Ann Hye-Ryong; Chang, Rhoda Ahn; Chen, Xiaoyan; Longnecker, Richard; He, Xiaolin

    2014-10-02

    The ubiquitous EBV causes infectious mononucleosis and is associated with several types of cancers. The EBV genome encodes an early gene product, BARF1, which contributes to pathogenesis, potentially through growth-altering and immune-modulating activities, but the mechanisms for such activities are poorly understood. We have determined the crystal structure of BARF1 in complex with human macrophage-colony stimulating factor (M-CSF), a hematopoietic cytokine with pleiotropic functions in development and immune response. BARF1 and M-CSF form a high-affinity, stable, ring-like complex in both solution and the crystal, with a BARF1 hexameric ring surrounded by three M-CSF dimers in triangular array. The binding of BARF1 to M-CSF dramatically reduces but does not completely abolish M-CSF binding and signaling through its cognate receptor FMS. A three-pronged down-regulation mechanism is proposed to explain the biological effect of BARF1 on M-CSF:FMS signaling. These prongs entail control of the circulating and effective local M-CSF concentration, perturbation of the receptor-binding surface of M-CSF, and imposition of an unfavorable global orientation of the M-CSF dimer. Each prong may reduce M-CSF:FMS signaling to a limited extent but in combination may alter M-CSF:FMS signaling dramatically. The downregulating mechanism of BARF1 underlines a viral modulation strategy, and provides a basis for understanding EBV pathogenesis.

  9. IDENTIFICATION AND EXPRESSION OF MACROPHAGE MIGRATION INHIBITORY FACTOR IN SARCOPTES SCABIEI

    PubMed Central

    COTE’, N.M.; JAWORSKI, D.C.; WASALA, N.B.; MORGAN, M.S.; ARLIAN, L. G.

    2013-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine produced by many mammalian tissues including skin. It is also found in many invertebrate parasites of mammals including ticks and may function to aid the parasite to evade the innate and adaptive immune responses in the host. In this study, the cDNA for a MIF gene was sequenced from Sarcoptes scabiei, the scabies mite, using RT-PCR and RACE molecular techniques. The resulting nucleotide sequence had a length of 405 base pairs and the putative amino acid sequences for the mite and tick (Dermacentor variabilis) proteins were identical. The initial steps for the project resulted in the production of expressed scabies mite cDNAs. A real time (qPCR) assay was performed with MIF from scabies mites and various tick species. Results show that mRNA encoding MIF homologues was three times more abundant in the mite samples when compared to RNA prepared from D. variabilis salivary glands and 1.3 times more abundant when compared with RNA prepared from D. variabilis midgut. PMID:23831036

  10. Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.

    PubMed

    Heinzelman, Pete; Carlson, Sharon J; Cox, George N

    2015-10-01

    Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a 'refacing effect' that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here. PMID:25855658

  11. [Emergency therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF)].

    PubMed

    Gratwohl, A; Dazzi, H; Tichelli, A; Stebler, C; Wernli, M; Thomssen, C; Kim, I; Dieterle, A; Obrist, R; Stern, A

    1991-03-23

    Granulocyte-macrophage colony stimulating factor (GM-CSF) has been tested for tolerability and efficacy on a compassionate need case basis in 17 patients (5 females, 12 males aged 4-72 years, median 35 years). GM-CSF was given at the rate of 3.5-32 micrograms/kg for 2-64 days as a continuous infusion for the following indications: impending rejection following bone marrow transplantation (5 patients), severe neutropenia secondary to chemotherapy in tumor patients (5), severe aplastic anemia (3), immune granulocytopenia (2) and accidental overdose with cytostatic agents (2 patients). Tolerance of GM-CSF was good in regard to doses of up to 16 micrograms/kg. Fever, myalgia and eosinophilia were the most frequent side effects. The patient treated with 32 micrograms/kg developed thrombosis of the vena cava. Efficacy is more difficult to assess in this heterogenous population, but 11 of 17 patients showed increased granulocyte counts and 3 patients clearly recovered from severe neutropenia. The role of GM-CSF in this recovery, however, cannot be proven. The results further indicate that GM-CSF cannot reverse ongoing rejection following allogenic BMT and cannot correct immune neutropenia. The value of GM-CSF therapy in patients with severe aplastic anemia and in the context of chemotherapy still needs to be defined. It is certainly indicated in patients with an accidental overdose of chemotherapeutic agents. PMID:2028244

  12. Macrophage Migration Inhibitory Factor Gene Polymorphisms and Plasma Levels in Children With Obstructive Sleep Apnea

    PubMed Central

    Khalyfa, Abdelnaby; Kheirandish-Gozal, Leila; Capdevila, Oscar Sans; Bhattacharjee, Rakesh; Gozal, David

    2012-01-01

    Introduction Obstructive sleep apnea (OSA) is associated with increased risk for cardiovascular and metabolic dysfunction in both adults and children. In adults with OSA, serum levels of macrophage migration inhibitory factor (MIF) are elevated. Therefore, we assessed plasma MIF levels and MIF allelic variant frequencies in children with and without OSA. Methods A total of 614 consecutive children ages 5–8 years were recruited. Children were divided into those with OSA and without OSA (NOSA) based on the apnea-hypopnea index (AHI). In addition to lipid profile, hsCRP, and fasting insulin and glucose levels, plasma MIF levels were assayed using ELISA, and 28 single-nucleotide polymorphisms (SNPs) covering the region were genotyped. Linkage disequilibrium and haplotype blocks were analyzed using Haploview version 4.2 software. Results Morning plasma MIF levels were increased in children with OSA. Of the 28 SNPs tested, the frequency of rs10433310 minor allele was significantly decreased in OSA. This SNP was also associated with reduced fasting insulin and hsCRP levels in OSA. The minor allele frequency of all other 27 SNPs was similar in OSA and NOSA groups. Conclusions Childhood OSA is associated with higher plasma MIF, hsCRP, and fasting insulin levels that promote cardiometabolic risk, and the MIF gene SNP rs10433310 may account for some the variance in such risk. PMID:22451332

  13. A small-molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease.

    PubMed

    Kithcart, Aaron P; Cox, Gina M; Sielecki, Thais; Short, Abigail; Pruitt, James; Papenfuss, Tracey; Shawler, Todd; Gienapp, Ingrid; Satoskar, Abhay R; Whitacre, Caroline C

    2010-11-01

    Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system (CNS) characterized by demyelination and axon loss. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be elevated in the cerebrospinal fluid of patients during relapses. The purpose of this study was to evaluate a new small-molecule inhibitor of MIF and its ability to reduce the severity of an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We utilized 2 structurally related isoxazolines, which show in vitro inhibition of MIF tautomerase activity. We found that administration of an inhibitor of MIF to mice with established EAE immediately reduced the severity of clinical signs and expanded a population of regulatory T lymphocytes. We also noted that the inhibitor reduced relapses of disease in a relapsing/remitting model of EAE. An analysis of leukocyte migration into the brain revealed that administration of inhibitor reduced entry of these cells. No effects on inflammatory cytokine production or T-cell activation in the periphery were noted. From these studies, we conclude that a small-molecule inhibitor of MIF reduces the severity of EAE and prevents access of immune cells into the CNS, which could be of therapeutic relevance to MS. PMID:20624927

  14. Molecular function of macrophage migration inhibitory factor and a novel therapy for inflammatory bowel disease.

    PubMed

    Nishihira, Jun

    2012-10-01

    Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD. PMID:23050964

  15. An alternative strategy to generate coding sequence of macrophage migration inhibitory factor-2 of Wuchereria bancrofti

    PubMed Central

    Chauhan, Nikhil; Hoti, S.L.

    2016-01-01

    Background & objectives: Different developmental stages of Wuchereria bancrofti, the major causal organism of lymphatic filariasis (LF), are difficult to obtain. Beside this limitation, to obtain complete coding sequence (CDS) of a gene one has to isolate mRNA and perform subsequent cDNA synthesis which is laborious and not successful at times. In this study, an alternative strategy employing polymerase chain reaction (PCR) was optimized and validated, to generate CDS of Macrophage migration Inhibitory Factor-2 (wbMIF-2), a gene expressed in the transition stage between L3 to L4. Methods: The genomic DNA of W. bancrofti microfilariae was extracted and used to amplify the full length wbMIF-2 gene (4.275 kb). This amplified product was used as a template for amplifying the exons separately, using the overlapping primers, which were then assembled through another round of PCR. Results: A simple strategy was developed based on PCR, which is used routinely in molecular biology laboratories. The amplified CDS of 363 bp of wbMIF-2 generated using genomic DNA splicing technique was devoid of any intronic sequence. Interpretation & conclusions: The cDNA of wbMIF-2 gene was successfully amplified from genomic DNA of microfilarial stage of W. bancrofti thus circumventing the use of inaccessible L3-L4 transitional stage of this parasite. This strategy is useful for generating CDS of genes from parasites that have restricted availability. PMID:27121522

  16. Macrophage Migration Inhibitory Factor Is a Novel Determinant of Cigarette Smoke–Induced Lung Damage

    PubMed Central

    Fallica, Jonathan; Boyer, Laurent; Kim, Bo; Serebreni, Leonid; Varela, Lidenys; Hamdan, Omar; Wang, Lan; Simms, Tiffany; Damarla, Mahendra; Kolb, Todd M.; Bucala, Richard; Mitzner, Wayne; Hassoun, Paul M.

    2014-01-01

    Cigarette smoke (CS) is the most common cause of chronic obstructive pulmonary diseases (COPD), including emphysema. CS exposure impacts all cell types within the airways and lung parenchyma, causing alveolar tissue destruction through four mechanisms: (1) oxidative stress; (2) inflammation; (3) protease-induced degradation of the extracellular matrix; and (4) enhanced alveolar epithelial and endothelial cell (EC) apoptosis. Studies in human pulmonary ECs demonstrate that macrophage migration inhibitory factor (MIF) antagonizes CS-induced apoptosis. Here, we used human microvascular ECs, an animal model of emphysema (mice challenged with chronic CS), and patient serum samples to address both the capacity of CS to alter MIF expression and the effects of MIF on disease severity. We demonstrate significantly reduced serum MIF levels in patients with COPD. In the murine model, chronic CS exposure resulted in decreased MIF mRNA and protein expression in the intact lung. MIF deficiency (Mif−/−) potentiated the toxicity of CS exposure in vivo via increased apoptosis of ECs, resulting in enhanced CS-induced tissue remodeling. This was linked to MIF’s capacity to protect against double-stranded DNA damage and suppress p53 expression. Taken together, MIF appears to antagonize CS-induced toxicity in the lung and resultant emphysematous tissue remodeling by suppressing EC DNA damage and controlling p53-mediated apoptosis, highlighting a critical role of MIF in EC homeostasis within the lung. PMID:24490973

  17. In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis

    SciTech Connect

    Aglietta, M.; Monzeglio, C.; Sanavio, F.; Apra, F.; Morelli, S.; Stacchini, A.; Piacibello, W.; Bussolino, F.; Bagnara, G.; Zauli, G. )

    1991-03-15

    The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patients with normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; Sandoz/Schering-Plough, 5.5 micrograms protein/kg/d, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit (CFU-Mk)) in S phase (evaluated by the suicide technique with high 3H-Tdr doses) increased from 31% +/- 16% to 88% +/- 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage I-III forms. Moreover, by autoradiography (after incubation of marrow cells with 125I-labeled GM-CSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitors was not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSF-induced proliferative stimulus into enhanced platelet production.

  18. Discovery of Novel Inhibitors of the Tautomerase Activity of Macrophage Migration Inhibitory Factor (MIF).

    PubMed

    Zapatero, Maria Cleofé; Pérez, Paloma; Vázquez, María Jesús; Colmenarejo, Gonzalo; de Los Frailes, Maite; Ramón, Fernando

    2016-06-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine associated with multiple diseases, including neurodegenerative disorders. With the ultimate goal of providing novel chemotypes as starting points for development of disease-modifying therapeutics for neurodegeneration, we endeavored to screen the GSK compound collection for MIF inhibitors using a miniaturized, activity-based kinetic assay. The assay monitors the increase in absorbance at 320 nm resulting from keto-to-enol tautomerization of 4-hydroxyphenylpyruvate, a reaction catalyzed by MIF. We ran a full-diversity screen evaluating the inhibitory activity of 1.6 million compounds. Primary hits were confirmed and retested in an orthogonal assay measuring tautomerization of l-dopachrome methyl ester by the decrease in absorbance at 475 nm in kinetic mode. Selected compounds were progressed to medium-throughput mode-of-inhibition studies, which included time dependence, enzyme concentration dependence, and reversibility of their inhibitory effect. With these results and after inspection of the physicochemical properties of compounds, 17 chemotypes were prioritized and progressed to further stages of validation and characterization to better assess their therapeutic potential. PMID:26933127

  19. Recovery from severe hematopoietic suppression using recombinant human granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Monroy, R.L.; Skelly, R.R.; Taylor, P.; Dubois, A.; Donahue, R.E.

    1988-01-01

    The ability of recombinant human granulocytemacrophage colony-stimulating factor (rhGM-CSF) to enhance recovery of a radiation-suppressed hematopoietic system was evaluated in a nonuniform radiation-exposure model using the rhesus monkey. Recombinant human GM-CSF treatment for 7 days after a lethal, nonuniform radiation exposure of 800 cGy was sufficient to enhance hematopoietic reconstitution, leading to an earlier recovery. Monkeys were treated with 72,000 U/kg/day of rhGm-CSF delivered continuously through an Alzet mini-osmotic pump implanted subcutaneously on day 3. Treated monkeys demonstrated effective granulocyte and platelet levels in the peripheral blood, 4 and 7 days earlier, respectively, than control monkeys. Granulocyte-macrophage colony-forming unit (CFU-GM) activity in the bone marrow was monitored to evaluate the effect of rhGM-CSF on marrow recovery. Treatment with rhGM-CSF led to an early recovery of CFU-GM activity suggesting that rhGM-CSF acted on an earlier stem cell population to generate CFU-GM. Thus, the effect of rhGM-CSF on hematopoietic regeneration, granulocyte recovery, and platelet recovery are evaluated.

  20. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  1. Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions.

    PubMed

    Kostevich, Valeria A; Sokolov, Alexey V; Grudinina, Natalia A; Zakharova, Elena T; Samygina, Valeria R; Vasilyev, Vadim B

    2015-10-01

    Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is a target for pharmacological treatment of sepsis and malignant tumors. Inhibition of tautomerase activity of MIF in reaction with p-hydroxyphenylpyruvate (HPP) was observed in the presence of ceruloplasmin (CP), a copper-containing plasma protein. Binding labile copper ions to CP (CP+Cu(II)) is a prerequisite for MIF inhibiting. CP+Cu(II) is shown to be an uncompetitive inhibitor of MIF (Ki ~ 37 nM), which suggests formation of a complex 'MIF-HPP-CP-Cu(II)'. Filtration of CP+Cu(II) on a column with Chelex-100, otherwise the presence of high concentrations of histidine, cysteine or methionine abrogated the inhibitory effect of CP. Adding salts of Co(II) and Ni(II) that replace copper ions in the labile sites prevented the inhibitory effect of CP+Cu(II). Limited proteolysis of CP by thrombin diminished its oxidase activity in reaction with p-phenylenediamine, but endowed it with the capacity of inhibiting MIF. Covalent modification of MIF by phenylmethylsulfonyl fluoride (PMSF) resulted in binding of MIF-PMSF to CP immobilized on CM5 chip, the dissociation constant being 4.2 μM. In D-galactosamine-sensitized mice CP+Cu(II) increased the LPS-induced lethality from 54 to 100%, while administration of antibodies against MIF prevented the lethal effect. The enhancement by CP+Cu(II) of the pro-inflammatory signal of MIF is discussed. PMID:26091949

  2. Nanocarrier-mediated inhibition of macrophage migration inhibitory factor attenuates secondary injury after spinal cord injury.

    PubMed

    Saxena, Tarun; Loomis, Kristin H; Pai, S Balakrishna; Karumbaiah, Lohitash; Gaupp, Eric; Patil, Ketki; Patkar, Radhika; Bellamkonda, Ravi V

    2015-02-24

    Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity

  3. Plasma Level of Placenta-Derived Macrophage-Stimulating Protein -Chain in Preeclampsia before 20 Weeks of Pregnancy

    PubMed Central

    Ma, Qingling; Chen, Ruihua

    2016-01-01

    Object This study aimed to investigate the diagnostic value of placenta-derived macrophage-stimulating protein α-chain (MSP-α) before the 20th week of gestation for the early diagnosis of preeclampsia (PE). Methods and Materials Two parts of this nested case-control study were simultaneously executed, and 1500 pregnant women were recruited. A total of 124 pregnant women were included in the plasma analysis part of this study. The MSP-α plasma level was measured before the 20th week of gestation, and the participants were followed until delivery. A case group of 62 women with PE and a control group of 62 women matched by gestational age, maternal age, and pre-pregnancy BMI (with normotensive pregnancies) were evaluated. In the placenta analysis part of this nested case-control study, the placentas of 34 pregnant women were randomly obtained. The placental levels of MSP were measured in 17 individuals with PE (case group) and in 17 women with a normotensive pregnancy matched by gestational age and maternal age (control group). Results The plasma level of MSP-α was higher in the PE group than in the control group before the 20th week of gestation (p < 0.001). In addition, compared to the women with severe features in the PE group, those without severe features had a significantly higher plasma MSP-α level before the 20th week of gestation (p < 0.001). The area under the receiver operating characteristic curve (AUC) of MSP-α before the 20th week of gestation was 0.905 (95% CI, 0.811–0.962) for the women with early-onset PE without severe features. With regard to the placenta, the PE group (accumulated optical density, IOD [SUM] = 8862.37 ± 2064.42) exhibited increased MSP staining (more intense MSP staining or more extensive staining) compared with the control group (normal pregnancies (IOD [SUM] = 447.92 ± 114.72, P < 0.001). Furthermore, increased MSP staining was detected among the women without severe features compared with those with severe features in

  4. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    SciTech Connect

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu . E-mail: nakanaka@kenroku.kanazawa-u.ac.jp

    2005-09-10

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.

  5. Garlic (Allium sativum) stimulates lipopolysaccharide-induced tumor necrosis factor-alpha production from J774A.1 murine macrophages.

    PubMed

    Sung, Jessica; Harfouche, Youssef; De La Cruz, Melissa; Zamora, Martha P; Liu, Yan; Rego, James A; Buckley, Nancy E

    2015-02-01

    Garlic (Allium sativum) is known to have many beneficial attributes such as antimicrobial, antiatherosclerotic, antitumorigenetic, and immunomodulatory properties. In the present study, we investigated the effects of an aqueous garlic extract on macrophage cytokine production by challenging the macrophage J774A.1 cell line with the garlic extract in the absence or presence of lipopolysaccharide (LPS) under different conditions. The effect of allicin, the major component of crushed garlic, was also investigated. Using enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction, it was found that garlic and synthetic allicin greatly stimulated tumor necrosis factor-alpha (TNF-α) production in macrophages treated with LPS. The TNF-α secretion levels peaked earlier and were sustained for a longer time in cells treated with garlic and LPS compared with cells treated with LPS alone. Garlic acted in a time-dependent manner. We suggest that garlic, at least partially via its allicin component, acts downstream from LPS to stimulate macrophage TNF-α secretion. PMID:25366263

  6. Kinetics of tumour necrosis factor and prostaglandin production by murine resident peritoneal macrophages as affected by dietary n-3 polyunsaturated fatty acids.

    PubMed Central

    Hardard'ottir, I; Whelan, J; Kinsella, J E

    1992-01-01

    Cell-associated and secreted tumour necrosis factor (TNF), prostaglandin (PG) E2, and 6-keto PGF1 alpha were monitored at various times following in vitro stimulation of resident peritoneal macrophages with lipopolysaccharide (LPS). Macrophages were obtained from mice maintained on diets containing 1.5 wt% n-3 polyunsaturated fatty acids (PUFA)+ 1.5 wt% n-6 fatty acids; 1.5 wt% n-6 fatty acids; or 3 wt% n-6 fatty acids, for 4 weeks. Cell-associated TNF increased transiently in the resident peritoneal macrophages from mice consuming all diets and decreased after TNF secretion had reached maximum and plateaued. Macrophages from mice consuming the n-3 PUFA contained more cell-associated TNF and secreted more TNF than macrophages from mice consuming diets containing n-6 fatty acids only, at all time-points studied. Macrophages from mice consuming the n-3 PUFA showed an earlier increase in cell-associated and secreted TNF compared with macrophages from mice consuming n-6 fatty acids only. Kinetics of maximum TNF production was not affected by the diets and dietary n-3 PUFA did not cause a prolonged increase in TNF secretion. Macrophages from mice consuming the n-3 PUFA produced less PG than macrophages from mice consuming the n-6 fatty acids only. PG secretion increased following appearance of cell-associated TNF but when PG had accumulated in the medium there was no further increase in TNF production. PMID:1398747

  7. Macrophage activation by OM-85 BV.

    PubMed

    Mauël, J

    1992-01-01

    Peritoneal or bone-marrow-derived murine macrophages were exposed for 24 h in vitro to dilutions of the bacterial extract OM-85 BV, in the presence or absence of other added compounds [macrophage-activating factor (MAF), recombinant murine interferon-gamma (IFN-gamma)]. Various metabolic responses and functional activities were then measured. Glucose oxidation through the hexose monophosphate shunt pathway was markedly stimulated in OM-85 BV-treated macrophages compared to control macrophages. Similarly, OM-85 BV primed macrophages for superoxide production upon triggering by phorbol myristate acetate. Both effects were further enhanced by simultaneous treatment of the cells with MAF with OM-85 BV. The bacterial extract also induced macrophages to release large amounts of nitrite (a marker of the activated state). As regards functional responses, coincubation with MAF and OM-85 BV activated macrophages to destroy target cells as well as intracellular microorganisms; in the latter case, similar results were obtained when MAF was replaced by IFN-gamma. In all these tests, the possibility that the observed effects were due to contamination of the bacterial extracts by endotoxin could be excluded. The above results indicate that OM-85 BV induces metabolic and functional properties in macrophages that are characteristic of the activated state and are important for host defence. PMID:1332156

  8. 1α,25-Dihydroxyvitamin D3 inhibits the differentiation and bone resorption by osteoclasts generated from Wistar rat bone marrow-derived macrophages

    PubMed Central

    WANG, DONG; GU, JIAN-HONG; CHEN, YANG; ZHAO, HONG-YAN; LIU, WEI; SONG, RUI-LONG; BIAN, JIAN-CHUN; LIU, XUE-ZHONG; YUAN, YAN; LIU, ZONG-PING

    2015-01-01

    The steroid hormone 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] plays an important role in maintaining a balance in calcium and bone metabolism. To study the effects of 1α,25-(OH)2D3 on osteoclast (OC) formation and bone resorption, OC differentiation was induced in bone marrow-derived mononuclear cells from Wistar rats with the addition of macrophage colony stimulating factor and receptor activator for nuclear factor-κB ligand in vitro. Cells were then treated with 1α,25-(OH)2D3 at 10−9, 10−8 or 10−7 mol/l. OCs were identified using tartrate-resistant acid phosphatase staining and activity was monitored in the absorption lacunae by scanning electron microscopy. Expression levels of functional proteins associated with bone absorption, namely carbonic anhydrase II, cathepsin K and matrix metalloproteinase-9 were evaluated by western blot analysis. The results showed that 1α,25-(OH)2D3 inhibited the formation and activation of OCs in a dose-dependent manner and downregulated the expression levels of bone absorption-associated proteins. PMID:26622436

  9. Macrophages derived from THP-1 promote the osteogenic differentiation of mesenchymal stem cells through the IL-23/IL-23R/β-catenin pathway.

    PubMed

    Tu, Bing; Liu, Shen; Liu, Guangwang; Yan, Wei; Wang, Yugang; Li, Zhiwei; Fan, Cunyi

    2015-11-15

    Abnormal bone formation is a clinically significant dilemma for many conditions in response to injury, inflammation or genetic disease. However, the effects of inflammation on the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. IL-23 secretion from macrophages might contribute to the development of bone formation. Here, we investigated the stimulatory effects of THP-1 macrophage conditioned medium (MΦ CM) on the osteogenic differentiation of human MSCs and the associated signaling pathways. The osteogenic differentiation of MSCs was induced after exposure to osteogenic differentiation medium (OM). MΦ CM significantly increased alkaline phosphate (ALP) activity and calcium mineralization in MSCs. Osteogenic marker genes, including RUNX2, ALP and osteocalcin (OCN), were also up-regulated in MSCs after exposure to MΦ CM. Moreover, western blotting revealed that MΦ CM treatment induced STAT3 and β-catenin activation in MSCs. Furthermore, blockade of IL-23 in MΦ CM not only impaired the osteogenic-promotion effects of macrophage but also decreased the expression of osteogenic maker genes. However, IL-23R silencing suppressed MΦ CM-induced calcium mineralization and osteogenic maker gene expression in MSCs. These data suggest that macrophages derived from THP-1 promote the osteoblastic differentiation of MSCs through the IL-23/IL-23R/β-catenin pathway and macrophages might contribute to the development of bone formation in inflammation. PMID:26477825

  10. Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells.

    PubMed

    Tynell, Janne; Westenius, Veera; Rönkkö, Esa; Munster, Vincent J; Melén, Krister; Österlund, Pamela; Julkunen, Ilkka

    2016-02-01

    In this study we assessed the ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to replicate and induce innate immunity in human monocyte-derived macrophages and dendritic cells (MDDCs), and compared it with severe acute respiratory syndrome coronavirus (SARS-CoV). Assessments of viral protein and RNA levels in infected cells showed that both viruses were impaired in their ability to replicate in these cells. Some induction of IFN-λ1, CXCL10 and MxA mRNAs in both macrophages and MDDCs was seen in response to MERS-CoV infection, but almost no such induction was observed in response to SARS-CoV infection. ELISA and Western blot assays showed clear production of CXCL10 and MxA in MERS-CoV-infected macrophages and MDDCs. Our data suggest that SARS-CoV and MERS-CoV replicate poorly in human macrophages and MDDCs, but MERS-CoV is nonetheless capable of inducing a readily detectable host innate immune response. Our results highlight a clear difference between the viruses in activating host innate immune responses in macrophages and MDDCs, which may contribute to the pathogenesis of infection. PMID:26602089

  11. Macrophage Migration Inhibitory Factor Contributes to Host Defense against Acute Trypanosoma cruzi Infection

    PubMed Central

    Reyes, José L.; Terrazas, Luis I.; Espinoza, Bertha; Cruz-Robles, David; Soto, Virgilia; Rivera-Montoya, Irma; Gómez-García, Lorena; Snider, Heidi; Satoskar, Abhay R.; Rodríguez-Sosa, Miriam

    2006-01-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is involved in the host defense against several pathogens. Here we used MIF−/− mice to determine the role of endogenous MIF in the regulation of the host immune response against Trypanosoma cruzi infection. MIF−/− mice displayed high levels of blood and tissue parasitemia, developed severe heart and skeletal muscle immunopathology, and succumbed to T. cruzi infection faster than MIF+/+ mice. The enhanced susceptibility of MIF−/− mice to T. cruzi was associated with reduced levels of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-12 (IL-12), IL-18, gamma interferon (IFN-γ), and IL-1β, in their sera and reduced production of IL-12, IFN-γ, and IL-4 by spleen cells during the early phase of infection. At all time points, antigen-stimulated splenocytes from MIF+/+ and MIF−/− mice produced comparable levels of IL-10. MIF−/− mice also produced significantly less Th1-associated antigen-specific immunoglobulin G2a (IgG2a) throughout the infection, but both groups produced comparable levels of Th2-associated IgG1. Lastly, inflamed hearts from T. cruzi-infected MIF−/− mice expressed increased transcripts for IFN-γ, but fewer for IL-12 p35, IL-12 p40, IL-23, and inducible nitric oxide synthase, compared to MIF+/+ mice. Taken together, our findings show that MIF plays a role in controlling acute T. cruzi infection. PMID:16714544

  12. Two macrophage migration inhibitory factors regulate starfish larval immune cell chemotaxis.

    PubMed

    Furukawa, Ryohei; Tamaki, Kana; Kaneko, Hiroyuki

    2016-04-01

    Immune cell recruitment is critical step in the inflammatory response and associated diseases. However, the underlying regulatory mechanisms are poorly understood in invertebrates. Mesenchyme cells of the starfish larvae, which allowed Metchnikoff to complete his landmark experiments, are important model for analysis of immune cell migration. The present study investigated the role of macrophage migration inhibitory factor (MIF)-an evolutionarily conserved cytokine that is functionally similar to chemokines-in the larvae of the starfish Patiria (Asterina) pectinifera, which were found to possess two orthologs, ApMIF1 and ApMIF2. ApMIF1 and ApMIF2 clustered with mammalian MIF and its homolog D-dopachrome tautomerase (DDT), respectively, in the phylogenetic analysis. In contrast to the functional similarity between mammalian MIF and DDT, ApMIF1 knockdown resulted in the excessive recruitment of mesenchyme cells in vivo, whereas ApMIF2 deficiency inhibited the recruitment of these cells to foreign bodies. Mesenchyme cells migrated along a gradient of recombinant ApMIF2 in vitro, whereas recombinant ApMIF1 completely blocked ApMIF2-induced directed migration. Moreover, the expression patterns of ApMIF1 and ApMIF2 messenger RNA in bacteria-challenged mesenchyme cells were consistent with in vivo observations of cell behaviors. These results indicate that ApMIF1 and ApMIF2 act as chemotactic inhibitory and stimulatory factors, respectively, and coordinately regulate mesenchyme cell recruitment during the immune response in starfish larvae. This is the first report describing opposing functions for MIF- and DDT-like molecules. Our findings provide novel insight into the mechanisms underlying immune regulation in invertebrates. PMID:26833025

  13. Modulatory effects of thymol and carvacrol on inflammatory transcription factors in lipopolysaccharide-treated macrophages.

    PubMed

    Gholijani, Nasser; Gharagozloo, Marjan; Farjadian, Shirin; Amirghofran, Zahra

    2016-01-01

    Inflammation is a crucial factor in the pathogenesis of numerous diseases. This study sought to evaluate the effects of thymol and carvacrol, the main components of Thymus vulgaris (thyme) essential oil, on transcription factors regulating inflammation. Lipopolysaccharide (LPS)-stimulated J774.1 mouse macrophages were examined by real time-PCR for interleukin (IL)-1β and tumor necrosis factor (TNF)-α gene expression in the presence of these compounds. Levels of inducible phospho-nuclear factor-κB (pNF-κB) p65, activator protein-1 [AP-1(c-Fos/c-Jun)], and nuclear factors of activated T-cells (NFATs) were also measured using Western blots. Levels of phosphorylation of stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinase (SAPK/JNK), signal transducer, and activator of transcription (STAT-3), p38, IκBα, and NF-κB p65, as well as total levels of IL-1β and TNFα were determined. The results indicated carvacrol significantly reduced both IL-1β and TNFα at the protein and mRNA levels; thymol also significantly reduced IL-1β expression. Western blot analyses of nuclear cell extracts revealed both agents caused significantly decreased expression of c-Fos, NFAT-1, and NFAT-2; decreased expression of c-Jun was only caused by carvacrol. Neither agent inhibited p-NF-κB p65 expression. At the protein level, carvacrol and thymol each caused decreases in inducible phospho-SAPK/JNK and phospho-STAT3 levels, whereas only carvacrol resulted in increased p-p38 levels in the total cell extract. Despite the reduction of phospho-IκBα caused by both agents, p-NF-κB p65 still increased in the presence of carvacrol. Based on these findings, it is concluded that carvacrol and thymol could contribute to reduction of inflammatory responses through modulation of the expression of JNK, STAT-3, AP-1, and NFATs. PMID:25812626

  14. The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS)-Stimulated Human THP-1 Macrophages.

    PubMed

    Robertson, Ruairi C; Guihéneuf, Freddy; Bahar, Bojlul; Schmid, Matthias; Stengel, Dagmar B; Fitzgerald, Gerald F; Ross, R Paul; Stanton, Catherine

    2015-08-01

    Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%-42% total fatty acids as n-3 PUFA and 5%-7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p < 0.05) and IL-8 (p < 0.05) while that of P. lutheri inhibited IL-6 (p < 0.01) production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1) by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases. PMID:26308008

  15. The Anti-Inflammatory Effect of Algae-Derived Lipid Extracts on Lipopolysaccharide (LPS)-Stimulated Human THP-1 Macrophages

    PubMed Central

    Robertson, Ruairi C.; Guihéneuf, Freddy; Bahar, Bojlul; Schmid, Matthias; Stengel, Dagmar B.; Fitzgerald, Gerald F.; Ross, R. Paul; Stanton, Catherine

    2015-01-01

    Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%–42% total fatty acids as n-3 PUFA and 5%–7% crude extract as pigments, including chlorophyll a, β-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p < 0.05) and IL-8 (p < 0.05) while that of P. lutheri inhibited IL-6 (p < 0.01) production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1) by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases. PMID:26308008

  16. Ontogeny of Tissue-Resident Macrophages

    PubMed Central

    Hoeffel, Guillaume; Ginhoux, Florent

    2015-01-01

    The origin of tissue-resident macrophages, crucial for homeostasis and immunity, has remained controversial until recently. Originally described as part of the mononuclear phagocyte system, macrophages were long thought to derive solely from adult blood circulating monocytes. However, accumulating evidence now shows that certain macrophage populations are in fact independent from monocyte and even from adult bone marrow hematopoiesis. These tissue-resident macrophages derive from sequential seeding of tissues by two precursors during embryonic development. Primitive macrophages generated in the yolk sac (YS) from early erythro-myeloid progenitors (EMPs), independently of the transcription factor c-Myb and bypassing monocytic intermediates, first give rise to microglia. Later, fetal monocytes, generated from c-Myb+ EMPs that initially seed the fetal liver (FL), then give rise to the majority of other adult macrophages. Thus, hematopoietic stem cell-independent embryonic precursors transiently present in the YS and the FL give rise to long-lasting self-renewing macrophage populations. PMID:26441990

  17. Lon Mutant of Brucella abortus Induces Tumor Necrosis Factor-Alpha in Murine J774.A1 Macrophage

    PubMed Central

    Park, Sungdo; Choi, Young-Sill; Park, Sang-Hee; Kim, Young-Rok; Chu, Hyuk; Hwang, Kyu-Jam; Park, Mi-Yeoun

    2013-01-01

    Objectives The objective of this study was to isolate a Brucella lon mutant and to analyze the cytokine response of B. lon mutant during macrophage infection. Methods A wild-type Brucella abortus strain was mutagenized by Tn5 transposition. From the mouse macrophage J774.A1 cells, total RNA was isolated at 0 hours, 6 hours, 12 hours, and 24 hours after infection with Brucella. Using mouse cytokine microarrays, we measured transcriptional levels of the cytokine response, and validated our results with a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to confirm the induction of cytokine messenger RNA (mRNA). Results In host J774.A1 macrophages, mRNA levels of T helper 1 (Th1)-type cytokines, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), and IL-3, were significantly higher in the lon mutant compared to wild-type Brucella and the negative control. TNF-α levels in cell culture media were induced as high as 2 μg/mL after infection with the lon mutant, a greater than sixfold change. Conclusion In order to understand the role of the lon protein in virulence, we identified and characterized a novel B. lon mutant. We compared the immune response it generates to the wild-type Brucella response in a mouse macrophage cell line. We demonstrated that the B. lon mutants induce TNF-α expression from the host J774.A1 macrophage. PMID:24524018

  18. Protective role of spleen-derived macrophages in lung inflammation, injury, and fibrosis induced by nitrogen mustard.

    PubMed

    Venosa, Alessandro; Malaviya, Rama; Gow, Andrew J; Hall, Leroy; Laskin, Jeffrey D; Laskin, Debra L

    2015-12-15

    Nitrogen mustard (NM) is a vesicant that causes lung injury and fibrosis, accompanied by a persistent macrophage inflammatory response. In these studies we analyzed the spleen as a source of these cells. Splenectomized (SPX) and sham control rats were treated intratracheally with NM (0.125 mg/kg) or PBS control. Macrophage responses were analyzed 1-7 days later. Splenectomy resulted in an increase in lung macrophages expressing CCR2, but a decrease in ATR-1α(+) cells, receptors important in bone marrow and spleen monocyte trafficking, respectively. Splenectomy was also associated with an increase in proinflammatory M1 (iNOS(+), CD11b(+)CD43(+)) macrophages in lungs of NM-treated rats, as well as greater upregulation of iNOS and COX-2 mRNA expression. Conversely, a decrease in CD11b(+)CD43(-) M2 macrophages was observed in SPX rats, with no changes in CD68(+), CD163(+), CD206(+), or YM-1(+) M2 macrophages, suggesting distinct origins of M2 subpopulations responding to NM. Macrophage expression of M2 genes including IL-10, ApoE, PTX-2, PTX-3, 5-HT2α, and 5-HT7 was also reduced in NM-treated SPX rats compared with shams, indicating impaired M2 activity. Changes in lung macrophages responding to NM as a consequence of splenectomy were correlated with exacerbated tissue injury and more rapid fibrogenesis. These data demonstrate that the spleen is a source of a subset of M2 macrophages with anti-inflammatory activity; moreover, in their absence, proinflammatory/cytotoxic M1 macrophages predominate in the lung, resulting in heightened pathology. Understanding the origin of macrophages and characterizing their phenotype after vesicant exposure may lead to more targeted therapeutics aimed at reducing toxicity and disease pathogenesis. PMID:26475734

  19. Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

    SciTech Connect

    Williams, D.E.; Bicknell, D.C.; Park, L.S.; Straneva, J.E.; Cooper, S.; Broxmeyer, H.E.

    1988-01-01

    Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with /sup 125/I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with /sup 125/I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor. Affinity crosslinking studies demonstrated that /sup 125/I-labeled GM-CSF bound specifically to two species of M/sub r/ 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The M/sub r/ 70,000 species is thought to be a proteolytic fragment of the intact M/sub r/ 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

  20. Identification and biochemical characterization of macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti.

    PubMed

    Chauhan, Nikhil; Sharma, Rohit; Hoti, S L

    2015-02-01

    Homologues of human macrophage migration inhibitory factor (hMIF) have been reported from vertebrates, invertebrates and prokaryotes, as well as plants. Filarial parasites produce two homologues of hMIF viz., MIF-1 and MIF-2, which play important role in the host immune modulation. Earlier, we have characterized MIF-1 (Wba-mif-1) from Wuchereria bancrofti, the major causal organism of human lymphatic filariasis. Here, we are reporting the molecular and biochemical characterization of MIF-2 from this parasite (Wba-mif-2). The complete Wba-mif-2 gene and its cDNA were amplified, cloned and sequenced. The size of Wba-mif-2 gene and cDNA were found to be 4.275 kb and 363 bp, respectively. The gene annotation revealed the presence of a large intron of 3.912 kb interspersed with two exons of 183 bp and 180 bp. The alignment of derived amino acid sequences of Wba-MIF-2 with Wba-MIF-1 showed 44% homology. The conserved CXXC oxido-reductase catalytic site present in Wba-mif-1 was found absent in Wba-mif-2 coding sequence. The amplified Wba-mif-2 cDNA was cloned into an expression vector pRSET-B and transformed into salt inducible Escherichia coli strain GJ1158. The expressed recombinant Wba-MIF-2 protein showed tautomerase activity against L-dopachrome methyl ester and the specific activity was determined to be 18.57±0.77 μmol/mg/min. Three known inhibitors of hMIF tautomerase activity significantly inhibited the tautomerase activity of recombinant Wba-MIF-2. Although the conserved CXXC oxido-reductase motif is absent in Wba-mif-2, the recombinant protein showed significant oxido-reductase activity in the insulin reduction assay, possibly because of the presence of vicinal cysteine residues. PMID:25446175

  1. Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression.

    PubMed

    Mittelbronn, Michel; Platten, Michael; Zeiner, Pia; Dombrowski, Yvonne; Frank, Brigitte; Zachskorn, Cornelia; Harter, Patrick N; Weller, Michael; Wischhusen, Jörg

    2011-09-01

    Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy. PMID:21773885

  2. Malvidin-3-O-β glucoside, major grape anthocyanin, inhibits human macrophage-derived inflammatory mediators and decreases clinical scores in arthritic rats.

    PubMed

    Decendit, Alain; Mamani-Matsuda, Maria; Aumont, Virginie; Waffo-Teguo, Pierre; Moynet, Daniel; Boniface, Katia; Richard, Emmanuel; Krisa, Stéphanie; Rambert, Jérôme; Mérillon, Jean-Michel; Mossalayi, M D

    2013-11-15

    Polyphenolic anthocyanins are major colorful compounds in red fruits, known to prevent cardiovascular and other diseases. Grape polyphenols are a mixture of various molecules and their exact contribution to above bioactivities remains to be clarified. In the present study, we first analyzed the effect of purified grape-derived compounds on human peripheral blood mononuclear cell (PBMC) survival, proliferation, as well as for their ability to inhibit the activation of human normal macrophages. Data indicated that malvidin-3-O-β glucoside (Malβg), the major grape anthocyanin, is bioactive with no toxicity on human PBMC. Malβg decreased the transcription of genes encoding inflammatory mediators, confirmed by the inhibition of TNFα, IL1, IL-6 and iNOS-derived nitric oxide (NO) secretion from activated macrophages. As Malβg also inhibited inflammatory response of rat macrophages, we investigated the anti-inflammatory potential of Malβg in chronic rat adjuvant-induced arthritis (AIA). Malβg significantly diminished inflammatory cachexia and arthritic paw scores in AIA rats at both therapeutic and preventive levels. In vivo effects of Malβg correlated with down-regulation of NO generation from AIA rats' peritoneal macrophages ex vivo. These data indicate that Malβg, major grape anthocyanin, is a potent anti-inflammatory agent in vitro and in vivo, without detectable toxic effect. PMID:23796750

  3. Macrophage activation for intracellular killing as induced by a Ca2+ ionophore. Dependence on L-arginine-derived nitrogen oxidation products.

    PubMed Central

    Buchmüller-Rouiller, Y; Corradin, S B; Mauël, J

    1992-01-01

    Mouse macrophages activated by interferon-gamma kill intracellular Leishmania by a process that depends on the generation of L-arginine-derived nitrogen oxidation products. Interferon-induced intracellular killing can be mimicked by exposure of macrophages to the Ca2+ ionophore A23187 in the presence of lipopolysaccharide. The mechanisms of this effect were therefore investigated. Destruction of the parasite was accompanied by accumulation of nitrite in the macrophage culture fluids. Leishmanicidal activity and nitrite production in cultures stimulated with ionophore A23187 and lipopolysaccharide were abrogated when cells were activated in medium containing arginase or the L-arginine analogues L-canavanine, guanidine or NG-monomethyl-L-arginine. L-Arginine was required during the lipopolysaccharide-induced triggering phase only. Indeed, macrophage priming with ionophore A23187 in L-arginine-depleted medium led to full microbicidal activity and nitrite generation provided that L-arginine was present during subsequent triggering by lipopolysaccharide. Addition of NG-monomethyl-L-arginine to ionophore-activated macrophages increased O2- production on phorbol myristate stimulation, while inhibiting glucose oxidation through the hexose monophosphate shunt pathway. Leishmanicidal activity and nitrite production were also inhibited when ionophore-treated cultures were incubated with excess iron, implying a role for iron as a defence mechanism against the toxicity of nitrogen derivatives. These results indicate that the ionophore-induced leishmanicidal activity occurs through a process similar to that evoked by interferon-gamma, i.e. the production of L-arginine-derived nitrogen oxidation products. PMID:1599422

  4. IFN-gamma reduces specific binding of tumor necrosis factor on murine macrophages

    SciTech Connect

    Drapier, J.C.; Wietzerbin, J. )

    1991-02-15

    Because IFN-gamma is the main cytokine activating macrophages and TNF cooperates in this activation, we assessed TNF binding capacity during the course of murine macrophage activation by IFN-gamma. TNF binding to elicited macrophages increased with time, was maximal by 8 h of culture, and required de novo protein synthesis. {sup 125}I-TNF bound to about 40,000 sites/cell with a Kd of 1 x 10(-9) M. Cross-linking experiments performed with a bifunctional cross-linking agent revealed a specific band with a m.w. of 94,000. Preincubation of macrophages with IFN-gamma prevented the binding of TNF to receptors. This effect was dose-dependent and maximal at 100 U/ml. IFN-gamma also reduced specific TNF binding to preexisting receptors (50% inhibition in 3 h), but IFN-gamma did not change the internalization rate of TNF. These studies showed that the number of TNF receptors increased on macrophages vs maturation in culture and was negatively controlled by IFN-gamma.

  5. Amino-Biphosphonate–Mediated MMP-9 Inhibition Breaks the Tumor-Bone Marrow Axis Responsible for Myeloid-Derived Suppressor Cell Expansion and Macrophage Infiltration in Tumor Stroma

    PubMed Central

    Melani, Cecilia; Sangaletti, Sabina; Barazzetta, Francesca M.; Werb, Zena; Colombo, Mario P.

    2009-01-01

    BALB-neuT mice expressing an activated rat c-erbB-2/neu transgene under the mouse mammary tumor virus long terminal repeat show enhanced hematopoiesis with hyperproduction of myeloid-derived suppressor cells (MDSC) because of vascular endothelial growth factor (VEGF) secreted by the tumor. Here, we show that both tumor and stromal cells express matrix metalloproteinase-9 (MMP-9), thereby increasing the levels of pro–MMP-9 in the sera of tumor-bearing mice. Treatment with amino-biphosphonates impaired tumor growth, significantly decreased MMP-9 expression and the number of macrophages in tumor stroma, and reduced MDSC expansion both in bone marrow and peripheral blood by dropping serum pro–MMP-9 and VEGF. We dissected the role of tumor-derived MMP-9 from that secreted by stromal leukocytes by transplanting bone marrow from MMP-9 knockout mice into BALB-neuT mice. Although bone marrow progenitor–derived MMP-9 had a major role in driving MDSC expansion, amino-biphosphonate treatment of bone marrow chimeras further reduced both myelopoiesis and the supportive tumor stroma, thus enhancing tumor necrosis. Moreover, by reducing MDSC, amino-biphosphonates overcome the tumor-induced immune suppression and improved the generation and maintenance of antitumor immune response induced by immunization against the p185/HER-2. Our data reveal that suppression of MMP-9 activity breaks the vicious loop linking tumor growth and myeloid cell expansion, thus reducing immunosuppression. Amino-biphosphonates disclose a specific MMP-9 inhibitory activity that may broaden their application above their current usage. PMID:18056472

  6. Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

    PubMed Central

    Tanaka, S; Takahashi, N; Udagawa, N; Tamura, T; Akatsu, T; Stanley, E R; Kurokawa, T; Suda, T

    1993-01-01

    The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts. Images PMID:8423223

  7. Safety of recombinant human granulocyte-macrophage colony-stimulating factor in healing pediatric severe burns.

    PubMed

    Chi, Y F; Chai, J K; Luo, H M; Zhang, Q X; Feng, R

    2015-01-01

    We explored the safety of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for healing burns in children. Subjects were randomly assigned to two groups: the experimental group received external rhGM-CSF gel, and the control group received rhGM-CSF gel matrix components, applied to the burn surface. Neither group was given any other drugs that promote wound healing. Each day we recorded the pulse, body temperature, and respiration status in the two groups. We detected the blood routine, urine routine, and hepatic and renal function before the patients received drug treatment and after 72 h. The wound scab and healing states in the two groups were recorded every 4 days to evaluate wound healing rate and time taken for complete healing. Adverse reactions and their rate of occurrence were also recorded. The median time of healing was 15 days in the experimental group and 19 days in the control group (log-rank χ(2) = 5.139, P < 0.05). After 10 days, the experimental group healing rate was consistently higher than that of the control group (significantly different using intuitive analysis), suggesting the experimental group method was more effective. There were no obvious adverse reactions. There was no significant difference between the blood routine, urine routine, and liver and kidney function in the two groups before the treatment and after 3 days (P > 0.05). Compared with saline treatment of severe burns, rhGM-CSF can effectively shorten the healing time without significant adverse reactions, and is an effective and safe treatment for burns in children. PMID:25867422

  8. High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates

    PubMed Central

    Schneider, Anina; Weier, Manuela; Sweep, Fred C. G. J.; Le Roy, Didier; Bernhagen, Jürgen; Calandra, Thierry; Giannoni, Eric

    2016-01-01

    The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis. PMID:26858459

  9. Filtration of Macrophage Migration Inhibitory Factor (MIF) in Patients with End Stage Renal Disease Undergoing Hemodialysis

    PubMed Central

    Pohl, Julia; Heisler, Martin; Totzeck, Matthias; Kleophas, Werner; Hetzel, Gerd R.; Kelm, Malte; Hendgen-Cotta, Ulrike; Rassaf, Tienush

    2015-01-01

    Background End stage renal disease (ESRD) patients are characterized by increased morbidity and mortality due to highest prevalence of cardiovascular disease. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that controls cellular signaling in human physiology, pathophysiology, and diseases. Increased MIF plasma levels promote vascular inflammation and development of atherosclerosis. We have shown that MIF is associated with vascular dysfunction in ESRD patients. Whether hemodialysis (HD) affects circulating MIF plasma levels is unknown. We here aimed to investigate whether HD influences the circulating MIF pool in ESRD patients. Methods and Results An observational single-center study was conducted. MIF plasma levels in ESRD patients were assessed before, during, and after a HD session (n = 29). Healthy age-matched volunteers served as controls to compare correlations of MIF plasma levels with inflammatory plasma components (n = 20). MIF removed from the circulating blood pool could be detected in the dialysate and allowed for calculation of totally removed MIF (MIF content in dialysate 219±4 μg/HD-session). MIF plasma levels were markedly decreased 2 hour after initiation of HD (MIF plasma level pre-HD 84.8±6 ng/ml to intra-HD 61.2±5 ng/ml p<0.001) and were replenished already 20 min after termination of HD to basal levels (intra-HD 61.2±5 ng/ml to post-HD 79.8±5 ng/ml, p<0.001). Conclusion MIF is a dialyzable plasma component that is effectively filtrated during HD from the patient blood pool in large amounts. After removal of remarkable amounts of MIF during a single HD session, MIF plasma pool is early reconstituted after termination of HD from unknown sources. PMID:26485680

  10. MicroRNA‑451 inhibits neuroblastoma proliferation, invasion and migration by targeting macrophage migration inhibitory factor.

    PubMed

    Liu, Geng; Xu, Zhengwei; Hao, Dingjun

    2016-03-01

    Neuroblastoma (NB) is the most prevalent type of extracranial solid tumour in young children. To improve current understanding of the mechanisms, which modulate cancer cell proliferation, invasion and migration, investigations have focused on microRNAs (miRs), a class of small non‑coding RNAs, which post‑transcriptionally regulate gene expression during various crucial cell processes. The present study aimed to investigate the role of miR‑451 in NB. Human NB tissue and adjacent normal tissue were surgically removed, and the expression of miR‑451, and development and pathological characteristics of NB were investigated. The expression of miR‑451 was reduced in the NB tissue, compared with that in the adjacent tissue, and correlations between the reduction in miR‑451 and unfavourable variables included tumour size (P=0.0081), differentiation (P=0.0217), lymph node metastasis (P=0.0489), tumour‑node‑metastasis stage (0.0220) and distant metastases (P=0.0201). Transfection of the SK‑N‑SH and GI‑LA‑N NB cell lines with miR‑451 inhibited cell growth, invasion and migration. Furthermore, the present study demonstrated that macrophage migration inhibitory factor (MIF) was regulated directly by miR‑451 and was a critical mediator of the biological effects of miR‑451 in NB. The re‑expression of MIF markedly reversed the carcinogenic inhibitory property of miR‑451. These data provide a more detailed understanding of the essential role of miR‑451 in NB, which relies on regulation of the expression of MIF. PMID:26783235

  11. Variation in macrophage migration inhibitory factor [MIF] immunoreactivity during bovine gestation.

    PubMed

    Paulesu, L; Pfarrer, C; Romagnoli, R; Ietta, F; Callesen, H; Hambruch, N; Dantzer, V

    2012-03-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in several aspects of the immune response. MIF appears to play important roles in materno-fetal immuno-tolerance during placental establishment, modulation and growth as studied in epitheliochorial porcine and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18-250), and endometrial tissues from two non-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining, the caruncular epithelium during pregnancy showed stronger staining for MIF. The intercaruncular epithelium in non-pregnant endometrium showed some reaction apically with increasing intensity at uterine gland openings; in contrast, at day 18 of gestation this staining was markedly increased. During gestation both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During reestablishment of vascularisation, the vasculature in the caruncular area showed MIF reactivity. While supporting involvement of MIF in different placental types, the spatio-temporal variation in the bovine placenta suggests a regulatory role for MIF mainly in the interhemal barrier and during vascular development. PMID:22200576

  12. Crystal structure of a macrophage migration inhibitory factor from Giardia lamblia

    SciTech Connect

    Buchko, Garry W.; Abendroth, Jan; Robinson, Howard; Zhang, Yanfeng; Hewitt, Stephen N.; Edwards, Tom E.; Van Voorhis, Wesley C.; Myler, Peter J.

    2013-06-15

    Macrophage migration inhibitory factor (MIF) is a eukaryotic cytokine that affects a broad spectrum of immune responses and its activation/inactivation is associated with numerous diseases. During protozoan infections MIF is not only expressed by the host, but, has also been observed to be expressed by some parasites and released into the host. To better understand the biological role of parasitic MIF proteins, the crystal structure of the MIF protein from Giardia lamblia (Gl-MIF), the etiological agent responsible for giardiasis, has been determined at 2.30 Å resolution. The 114-residue protein adopts an α/β fold consisting of a four-stranded β-sheet with two anti-parallel α-helices packed against a face of the β-sheet. An additional short β-strand aligns anti-parallel to β4 of the β-sheet in the adjacent protein unit to help stabilize a trimer, the biologically relevant unit observed in all solved MIF crystal structures to date, and form a discontinuous β-barrel. The structure of Gl-MIF is compared to the MIF structures from humans (Hs-MIF) and three Plasmodium species (falciparum, berghei, and yoelii). The structure of all five MIF proteins are generally similar with the exception of a channel that runs through the center of each trimer complex. Relative to Hs-MIF, there are differences in solvent accessibility and electrostatic potential distribution in the channel of Gl-MIF and the Plasmodium-MIFs due primarily to two “gate-keeper” residues in the parasitic MIFs. For the Plasmodium MIFs the gate-keeper residues are at positions 44 (Y==>R) and 100 (V==>D) and for Gl-MIF it is at position 100 (V==>R). If these gate-keeper residues have a biological function and contribute to the progression of parasitemia they may also form the basis for structure-based drug design targeting parasitic MIF proteins.

  13. Transcriptional regulation of mouse granulocyte-macrophage colony-stimulating factor/IL-3 locus

    SciTech Connect

    Osborne, C.S.; Vadas, M.A.; Cockerill, P.N.

    1995-07-01

    Granulocyte-macrophage (GM)-CSF and IL-3 are hemopoietic growth factors whose genes are closely linked in both humans and mice. In humans, the GM-CSF and IL-3 genes are regulated by a cyclosporin A-inhibitable enhancer located 3 kb upstream of the GM-CSF gene that is inducible by signals that mimic TCR activation. To search for a murine homologue of this enhancer we probed mouse genomic DNA and located a 400-bp element 2 kb upstream of the mouse GM-CSF gene that was 76% homologous with the human GM-CSF enhancer. Like the human GM-CSF enhancer, this element formed a cyclosporin A-inhibitable DNase I-hypersensitive site in the murine T cell line EL4 upon activation with phorbol ester and calcium ionophore. Transient transfection assays showed that this homologue of the human enhancer acted as an inducible enhancer of the thymidine kinase promoter, the mouse IL-3 promoter, and the human GM-CSF promoter. We observed, however, that the mouse GM-CSF promoter was significantly more active than the human GM-CSF promoter and found that it supported a level of activity equivalent to the combination of the human GM-CSF promoter and the human GM-CSF enhancer. Consequently, the activity of mouse GM-CSF promoter was not significantly elevated in the presence of the mouse GM-CSF enhancer. Because the mouse GM-CSF enhancer is considerably less active than its human homologue we suggest that the mouse GM-CSF gene has evolved with less dependence upon the upstream enhancer for its activation. 53 refs., 6 figs.

  14. A new class of isothiocyanate-based irreversible inhibitors of Macrophage Migration Inhibitory Factor (MIF)

    PubMed Central

    Ouertatani-Sakouhi, Hajer; El-Turk, Farah; Fauvet, Bruno; Roger, Thierry; Le Roy, Didier; Karpinar, Damla Pinar; Leng, Lin; Bucala, Richard; Zweckstetter, Markus; Calandra, Thierry; Lashuel, Hilal A.

    2013-01-01

    Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without affecting the oligomerization state of MIF. Different alkyl and arylalkyl ITCs-modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding and biological activity of MIF. Light scattering, analytical ultracentrifugation and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and loss of catalytic activity translated into reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo. PMID:19737008

  15. Dengue Virus Nonstructural Protein 1 Induces Vascular Leakage through Macrophage Migration Inhibitory Factor and Autophagy

    PubMed Central

    Chen, Hong-Ru; Chuang, Yung-Chun; Lin, Yee-Shin; Liu, Hsiao-Sheng; Liu, Ching-Chuan; Perng, Guey-Chuen

    2016-01-01

    Dengue virus (DENV) is the most common mosquito-borne flavivirus; it can either cause mild dengue fever or the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the characteristic features of DHF/DSS is vascular leakage; although DENV nonstructural protein 1 (NS1) has been proved to induce vascular leakage after binding to Toll-like receptor 4, the down-stream mechanism has not yet been fully understood. In the sera of DENV-infected patients, the concentrations of DENV NS1 and inflammatory cytokine macrophage migration inhibitory factor (MIF) are positively correlated with disease severity, but whether DENV NS1 induces vascular leakage through MIF secretion remains unknown. We demonstrated that recombinant NS1 induced vascular leakage and MIF secretion both in human endothelial cell line HMEC-1 and in mice. Furthermore, these phenomena were inhibited in the presence of anti-NS1 antibodies both in vitro and in vivo. DENV NS1 also induced LC3-I to LC3-II conversion and p62 degradation in endothelial cell line, which indicated the formation of autophagy. To clarify whether MIF or autophagy mediated DENV NS1-induced vascular leakage, various inhibitors were applied. The results showed that DENV NS1-induced vascular leakage and VE-cadherin disarray were blocked in the presence of MIF inhibitors, anti-MIF-antibodies or autophagy inhibitors. An Atg5 knockdown clone further confirmed that autophagy formation of endothelial cells was required in NS1-induced vascular leakage. Furthermore, DENV NS1-induced LC3 puncta were also decreased in the presence of MIF inhibitors, indicating that MIF mediated DENV NS1-induced autophagy. Taken together, the results suggest a potential mechanism of DENV-induced vascular leakage and provide possible therapeutic targets against DHF/DSS. PMID:27409803

  16. High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

    PubMed

    Roger, Thierry; Schneider, Anina; Weier, Manuela; Sweep, Fred C G J; Le Roy, Didier; Bernhagen, Jürgen; Calandra, Thierry; Giannoni, Eric

    2016-02-23

    The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis. PMID:26858459

  17. Macrophage migration inhibitory factor is an endogenous regulator of stress-induced extramedullary erythropoiesis.

    PubMed

    Vignjević Petrinović, Sanja; Budeč, Mirela; Marković, Dragana; Gotić, Mirjana; Mitrović Ajtić, Olivera; Mojsilović, Slavko; Stošić-Grujičić, Stanislava; Ivanov, Milan; Jovčić, Gordana; Čokić, Vladan

    2016-09-01

    Macrophage migration inhibitory factor is a well-known proinflammatory cytokine that is released during systemic stress response. Although MIF can affect erythrocyte production, the role of this cytokine in stress-induced erythropoiesis is completely unknown. To extend our previous findings showing that chronic psychological stress stimulates extramedullary erythropoiesis, here we examined whether MIF is involved in the control of stress-induced erythropoietic response. Adult male C57BL/6 wild-type (WT) and MIF-KO (knock-out) mice were subjected to 2-h daily restraint stress for either 7 or 14 consecutive days. The number of erythroid progenitors and CD71/Ter119 profile of erythroid precursors were analyzed in the bone marrow and spleen. Additionally, MIF protein expression was assessed in WT mice. Our results demonstrated that chronic restraint stress enhanced the number of both erythroid progenitors and precursors in the spleen. Stress-induced increase in the number of splenic late erythroid progenitors as well as in the percentage of CD71(+)Ter119(+)-double-positive precursors was significantly more pronounced in MIF-KO mice compared to WT animals. Furthermore, repeatedly stressed WT animals demonstrated an augmented MIF expression in the spleen. Unlike the spleen, the bone marrow of chronically stressed WT mice exhibited less prominent changes in erythropoietic stress response and no significant alteration in MIF expression. In addition, MIF deficiency did not influence the bone marrow erythropoiesis in stressed animals. These findings suggest that MIF regulates extramedullary erythropoiesis by inhibiting an overexpansion of splenic immature erythroid cells during chronic stress and indicate a novel role for this cytokine under chronic stress conditions. PMID:27129368

  18. Dengue Virus Nonstructural Protein 1 Induces Vascular Leakage through Macrophage Migration Inhibitory Factor and Autophagy.

    PubMed

    Chen, Hong-Ru; Chuang, Yung-Chun; Lin, Yee-Shin; Liu, Hsiao-Sheng; Liu, Ching-Chuan; Perng, Guey-Chuen; Yeh, Trai-Ming

    2016-07-01

    Dengue virus (DENV) is the most common mosquito-borne flavivirus; it can either cause mild dengue fever or the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). One of the characteristic features of DHF/DSS is vascular leakage; although DENV nonstructural protein 1 (NS1) has been proved to induce vascular leakage after binding to Toll-like receptor 4, the down-stream mechanism has not yet been fully understood. In the sera of DENV-infected patients, the concentrations of DENV NS1 and inflammatory cytokine macrophage migration inhibitory factor (MIF) are positively correlated with disease severity, but whether DENV NS1 induces vascular leakage through MIF secretion remains unknown. We demonstrated that recombinant NS1 induced vascular leakage and MIF secretion both in human endothelial cell line HMEC-1 and in mice. Furthermore, these phenomena were inhibited in the presence of anti-NS1 antibodies both in vitro and in vivo. DENV NS1 also induced LC3-I to LC3-II conversion and p62 degradation in endothelial cell line, which indicated the formation of autophagy. To clarify whether MIF or autophagy mediated DENV NS1-induced vascular leakage, various inhibitors were applied. The results showed that DENV NS1-induced vascular leakage and VE-cadherin disarray were blocked in the presence of MIF inhibitors, anti-MIF-antibodies or autophagy inhibitors. An Atg5 knockdown clone further confirmed that autophagy formation of endothelial cells was required in NS1-induced vascular leakage. Furthermore, DENV NS1-induced LC3 puncta were also decreased in the presence of MIF inhibitors, indicating that MIF mediated DENV NS1-induced autophagy. Taken together, the results suggest a potential mechanism of DENV-induced vascular leakage and provide possible therapeutic targets against DHF/DSS. PMID:27409803

  19. Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury.

    PubMed

    Mishra, Vivek; Sinha, Satyesh K; Rajavashisth, Tripathi B

    2016-01-01

    Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury. PMID:27135205

  20. Blocking macrophage migration inhibitory factor activity alleviates mouse acute otitis media in vivo.

    PubMed

    Zhang, Jin; Xu, Min; Zheng, Qingyin; Zhang, Yan; Ma, Weijun; Zhang, Zhaoqiang

    2014-11-01

    This study was to investigate the role of macrophage migration inhibitory factor (MIF) in mouse acute otitis media (AOM), we hypothesize that blocking MIF activity will relieve mouse AOM. A mouse AOM model was constructed by injecting lipopolysaccharide (LPS) into the middle ear of C57BL/6 mice through the tympanic membrane (TM). MIF levels were measured by real-time PCR (RT-PCR) and ELISA after LPS application. Normal or AOM mice were given PBS or ISO-1 (MIF antagonist) every day for 10 days and the hearing levels were determined by measuring auditory brainstem response (ABR) threshold. After the ABR test finished, H&E staining was conducted and the inflammation was also measured by detecting interleukin (IL)-1β, tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) levels with RT-PCR and ELISA. TLR-4 expression was determined by western blotting and NF-κB activation was determined by electrophoretic mobility shift assays. Compared with the normal control, MIF levels in the middle ear of LPS-induced AOM mice were significant increased. The ABR results showed that mean ABR thresholds in ISO-1 treated AOM mice were significantly reduced compared with PBS treated AOM mice since day 7, indicating that ISO-1 treatment potentially improved the hearing levels of AOM mice. H&E staining showed that ISO-1 treatment could reduce the mucosal thickness of AOM mice. In ISO-1 treated mice, TLR-4 expression and levels of IL-1β, TNF-α and VEGF were significantly lower compared with PBS treated AOM mice. ISO-1 treatment also significantly inhibited NF-κB activation in AOM mice compared with PBS treated AOM mice. These results suggested that blocking the activity of MIF by ISO-1 could reduce the inflammation in AOM mice in which process TLR-4 and NF-κB were involved. The reduction in MIF activity is conducive to alleviate mouse AOM, which may serve as a potential therapeutic target for the treatment of AOM. PMID:25108100

  1. Inhibition of proinflammatory biomarkers in THP1 macrophages by polyphenols derived from chamomile, meadowsweet and willow bark.

    PubMed

    Drummond, Elaine M; Harbourne, Niamh; Marete, Eunice; Martyn, Danika; Jacquier, Jc; O'Riordan, Dolores; Gibney, Eileen R

    2013-04-01

    Antiinflammatory compounds in the diet can alleviate excessive inflammation, a factor in the pathogenesis of common diseases such as rheumatoid arthritis, atherosclerosis and diabetes. This study examined three European herbs, chamomile (Matricaria chamomilla), meadowsweet (Filipendula ulmaria L.) and willow bark (Salix alba L.), which have been traditionally used to treat inflammation and their potential for use as antiinflammatory agents. Aqueous herbal extracts and isolated polyphenolic compounds (apigenin, quercetin and salicylic acid, 0-100 μM) were incubated with THP1 macrophages, and interleukin (IL)-1β, IL-6 and tumour necrosis factor-alpha (TNF-α) were measured. At concentrations of 10 μM, both apigenin and quercetin reduced IL-6 significantly ( p < 0.05). Apigenin at 10 μM and quercetin at 25 μM reduced TNF-α significantly ( p < 0.05). Amongst the herbal extracts, willow bark had the greatest antiinflammatory activity at reducing IL-6 and TNF-α production. This was followed by meadowsweet and then chamomile. The lowest effective antiinflammatory concentrations were noncytotoxic (MTT mitochondrial activity assay). The Comet assay, which was used to study the protective effect of the isolated phenols against oxidative damage, showed positive results for all three polyphenols. These are the first findings that demonstrate the antiinflammatory capacity of these herbal extracts. PMID:22711544

  2. Nerve Growth Factor Regulation by TNF-α and IL-1β in Synovial Macrophages and Fibroblasts in Osteoarthritic Mice

    PubMed Central

    Takano, Shotaro; Inoue, Gen; Aikawa, Jun; Iwase, Dai; Minatani, Atsushi; Iwabuchi, Kazuya; Takaso, Masashi

    2016-01-01

    To investigate the role of macrophages as a regulator and producer of nerve growth factor (NGF) in the synovial tissue (ST) of osteoarthritis (OA) joints, the gene expression profiles of several inflammatory cytokines in the ST, including synovial macrophages and fibroblasts, of OA mice (STR/Ort) were characterized. Specifically, real-time polymerase chain reaction analysis was used to evaluate the expression of tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, IL-6, and NGF in CD11b+ and CD11b– cells isolated from the ST of a murine OA model. The effects of TNF-α, IL-1β, and IL-6 on the expression of NGF in cultured synovial cells were also examined. The expression of TNF-α, IL-1β, IL-6, and NGF in the ST of STR/Ort was higher than that in C57/BL6J mice. Compared to the CD11b– cell fraction, higher expression levels of TNF-α, IL-1β, and IL-6 were detected in the CD11b+ cell fraction, whereas no differences in the expression of NGF were detected between the two cell fractions. Notably, TNF-α upregulated NGF expression in synovial fibroblasts and macrophages and IL-1β upregulated NGF expression in synovial fibroblasts. IL-1β and TNF-α may regulate NGF signaling in OA joints and be suitable therapeutic targets for treating OA pain.

  3. C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.

    PubMed

    Hoeffel, Guillaume; Chen, Jinmiao; Lavin, Yonit; Low, Donovan; Almeida, Francisca F; See, Peter; Beaudin, Anna E; Lum, Josephine; Low, Ivy; Forsberg, E Camilla; Poidinger, Michael; Zolezzi, Francesca; Larbi, Anis; Ng, Lai Guan; Chan, Jerry K Y; Greter, Melanie; Becher, Burkhard; Samokhvalov, Igor M; Merad, Miriam; Ginhoux, Florent

    2015-04-21

    Although classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs. PMID:25902481

  4. Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages

    SciTech Connect

    Renard, C.; Vanderhaeghe, H.J.; Claes, P.J.; Zenebergh, A.; Tulkens, P.M.

    1987-03-01

    beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, /sup 14/C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of /sup 14/C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al., in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.

  5. Dendritic cell-derived tumor necrosis factor α modifies airway epithelial cell responses.

    PubMed

    Lutfi, R; Ledford, J R; Zhou, P; Lewkowich, I P; Page, K

    2012-01-01

    Mucosal dendritic cells (DC) are intimately associated with the airway epithelium and thus are ideally situated to be first responders to pathogens. We hypothesize that DC drive innate immune responses through early release of tumor necrosis factor (TNF) α, which drives airway epithelial cell responses. In a mouse model, TNFα release was significantly increased following a single exposure to German cockroach (GC) frass, an event independent of neutrophil recruitment into the airways. While lung epithelial cells and alveolar macrophages failed to release TNFα following GC frass exposure, bone marrow-derived DC (BMDC) produced substantial amounts of TNFα suggesting their importance as early responding cells. This was confirmed by flow cytometry of pulmonary myeloid DC. Addition of GC frass-pulsed BMDC or conditioned media from GC frass-pulsed BMDC to primary mouse tracheal epithelial cells (MTEC) or MLE-15 cells induced chemokine (C-C) motif ligand (CCL) 20 and granulocyte macrophage (GM) colony-stimulating factor (CSF), both of which are important for DC recruitment, survival and differentiation. Importantly, DC do not produce CCL20 or GM-CSF following allergen exposure. Blocking TNFα receptor 1 (TNFR1) completely abolished chemokine production, suggesting that BMDC-derived TNFα induced airway epithelial cell activation and enhancement of the innate immune response. Lastly, blocking TNFR1 in vivo resulted in significantly decreased CCL20 and GM-CSF production in the lungs of mice. Together, our data strongly suggest that DC-derived TNFα plays a crucial role in the initiation of innate immune responses through the modification of airway epithelial cell responses. PMID:22517116

  6. Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor-beta.

    PubMed Central

    Evanko, S. P.; Raines, E. W.; Ross, R.; Gold, L. I.; Wight, T. N.

    1998-01-01

    The accumulation of proteoglycans (PGs) in atherosclerosis contributes to disease progression and stenosis and may partly depend on local regulation by growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta. In this study, the distribution of the major extracellular PGs is compared with that of PDGF and TGF-beta isoforms in developing lesions of atherosclerosis from hypercholesterolemic nonhuman primates. Strong immunostaining for decorin, biglycan, versican, and hyaluronan is observed in both intermediate and advanced lesions. Perlecan staining is weak in intermediate lesions but strong in advanced lesions in areas bordering the plaque core. Immunostaining for PDGF-B and TGF-beta1 is particularly prominent in macrophages in intermediate and advanced lesions. In contrast, TGF-beta2 and TGF-beta3 and PDGF-A are present in both macrophages and smooth muscle cells. Overall, PG deposits parallel areas of intense growth factor immunostaining, with trends in relative localization that suggest interrelationships among certain PGs and growth factors. Notably, decorin and TGF-beta1 are distributed similarly, predominantly in the macrophage-rich core, whereas biglycan is prominent in the smooth muscle cell matrix adjoining TGF-beta1-positive macrophages. Versican and hyaluronan are enriched in the extracellular matrix adjacent to both PDGF- and TGF-beta1-positive cells. These data demonstrate that PG accumulation varies with lesion severity, structural characteristics, and the proximity of PDGF and TGF-beta. Images Figure 1 p537-a Figure 2 p539-a Figure 3 Figure 4 Figure 5 Figure 6 PMID:9466580

  7. Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

    PubMed Central

    Séguin, R; Mann, B J; Keller, K; Chadee, K

    1995-01-01

    The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6 PMID:8618866

  8. HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages.

    PubMed

    Girona, Josefa; LaVille, Agnes E; Solà, Rosa; Motta, Claude; Masana, Lluís

    2003-09-22

    Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL. PMID:14499733

  9. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities

    PubMed Central

    Heinz, Sven; Benner, Christopher; Spann, Nathanael; Bertolino, Eric; Lin, Yin C.; Laslo, Peter; Cheng, Jason X.; Murre, Cornelis; Singh, Harinder; Glass, Christopher K.

    2010-01-01

    Summary Genome-scale studies have revealed extensive, cell type-specific co-localization of transcription factors, but the mechanisms underlying this phenomenon remain poorly understood. Here we demonstrate in macrophages and B cells that collaborative interactions of the common factor PU.1 with small sets of macrophage- or B celllineage-determining transcription factors establish cell-specific binding sites that are associated with the majority of promoter-distal H3K4me1-marked genomic regions. PU.1 binding initiates nucleosome remodeling followed by H3K4 monomethylation at large numbers of genomic regions associated with both broadly and specifically expressed genes. These locations serve as beacons for additional factors, exemplified by liver X receptors, which drive both cell-specific gene expression and signal-dependent responses. Together with analyses of transcription factor binding and H3K4me1 patterns in other cell types, these studies suggest that simple combinations of lineage-determining transcription factors can specify the genomic sites ultimately responsible for both cell identity and cell type-specific responses to diverse signaling inputs. PMID:20513432

  10. Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms.

    PubMed

    Pierce, G F; Mustoe, T A; Lingelbach, J; Masakowski, V R; Griffin, G L; Senior, R M; Deuel, T F

    1989-07-01

    Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In

  11. Macrophage heterogeneity in the context of rheumatoid arthritis.

    PubMed

    Udalova, Irina A; Mantovani, Alberto; Feldmann, Marc

    2016-08-01

    Macrophages are very important in the pathogenesis of rheumatoid arthritis (RA). The increase in the number of sublining macrophages in the synovium is an early hallmark of active rheumatic disease, and high numbers of macrophages are a prominent feature of inflammatory lesions. The degree of synovial macrophage infiltration correlates with the degree of joint erosion, and depletion of these macrophages from inflamed tissue has a profound therapeutic benefit. Research has now uncovered an unexpectedly high level of heterogeneity in macrophage origin and function, and has emphasized the role of environmental factors in their functional specialization. Although the heterogeneous populations of macrophages in RA have not been fully characterized, preliminary results in mouse models of arthritis have contributed to our understanding of the phenotype and ontogeny of synovial macrophages, and to deciphering the properties of monocyte-derived infiltrating and tissue-resident macrophages. Elucidating the molecular mechanisms that drive polarization of macrophages towards proinflammatory or anti-inflammatory phenotypes could lead to identification of signalling pathways that inform future therapeutic strategies. PMID:27383913

  12. Macrophages regulate corpus luteum development during embryo implantation in mice

    PubMed Central

    Care, Alison S.; Diener, Kerrilyn R.; Jasper, Melinda J.; Brown, Hannah M.; Ingman, Wendy V.; Robertson, Sarah A.

    2013-01-01

    Macrophages are prominent in the uterus and ovary at conception. Here we utilize the Cd11b-Dtr mouse model of acute macrophage depletion to define the essential role of macrophages in early pregnancy. Macrophage depletion after conception caused embryo implantation arrest associated with diminished plasma progesterone and poor uterine receptivity. Implantation failure was alleviated by administration of bone marrow–derived CD11b+F4/80+ monocytes/macrophages. In the ovaries of macrophage-depleted mice, corpora lutea were profoundly abnormal, with elevated Ptgs2, Hif1a, and other inflammation and apoptosis genes and with diminished expression of steroidogenesis genes Star, Cyp11a1, and Hsd3b1. Infertility was rescued by exogenous progesterone, which confirmed that uterine refractoriness was fully attributable to the underlying luteal defect. In normally developing corpora lutea, macrophages were intimately juxtaposed with endothelial cells and expressed the proangiogenic marker TIE2. After macrophage depletion, substantial disruption of the luteal microvascular network occurred and was associated with altered ovarian expression of genes that encode vascular endothelial growth factors. These data indicate a critical role for macrophages in supporting the extensive vascular network required for corpus luteum integrity and production of progesterone essential for establishing pregnancy. Our findings raise the prospect that disruption of macrophage-endothelial cell interactions underpinning corpus luteum development contributes to infertility in women in whom luteal insufficiency is implicated. PMID:23867505

  13. Macrophages regulate corpus luteum development during embryo implantation in mice.

    PubMed

    Care, Alison S; Diener, Kerrilyn R; Jasper, Melinda J; Brown, Hannah M; Ingman, Wendy V; Robertson, Sarah A

    2013-08-01

    Macrophages are prominent in the uterus and ovary at conception. Here we utilize the Cd11b-Dtr mouse model of acute macrophage depletion to define the essential role of macrophages in early pregnancy. Ma