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Sample records for macrophage derived factor

  1. Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.

    PubMed

    Akagawa, Kiyoko S

    2002-07-01

    Macrophages have various functions and play a critical role in host defense and the maintenance of homeostasis. However, macrophages are heterogeneous and exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression, and function. When blood monocytes are cultured in medium alone in vitro, monocytes die, and colony-stimulating factors (CSFs) such as macrophage (M)-CSF or granulocyte-macrophage (GM)-CSF are necessary for their survival and differentiation into macrophages. However, M-CSF-induced monocyte-derived macrophages (M-Mphi) and GM-CSF-induced monocyte-derived macrophages (GM-Mphi) are distinct in their morphology, cell surface antigen expression, and functions, including Fcgamma receptor mediated-phagocytosis, H2O2 production, H2O2 sensitivity, catalase activity, susceptibility to human immunodeficiency virus type 1 and Mycobacterium tuberculosis, and suppressor activity. The characteristics of GM-Mphi resemble those of human alveolar macrophages.

  2. Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.

    PubMed

    Akagawa, Kiyoko S; Komuro, Iwao; Kanazawa, Hiroko; Yamazaki, Toshio; Mochida, Keiko; Kishi, Fumio

    2006-01-01

    Macrophages (Mphis) have various functions and play a critical role in host defense and the maintenance of homeostasis. Mphis exist in every tissue in the body, but Mphis from different tissues exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression and function, and are called by different names. However, the precise mechanism of the generation of macrophage heterogeneity is not known. In the present study, the authors examined the functional heterogeneity of Mphis generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF). CD14 positive human monocytes (Mos) were incubated with M-CSF and GM-CSF for 6-7 days to stimulate the generation of M-CSF-induced monocyte-derived Mphis (M-Mphis) and GM-CSF-induced monocyte-derived Mphis (GM-Mphis), respectively. The expression of cell surface antigens and several functions such as antigen presenting cell activity, susceptibility to oxidant stress, and the susceptibility to HIV-1 and mycobacterium tuberculosis infection were examined. GM-Mphis and M-Mphis are distinct in their morphology, cell surface antigen expression, and functions examined. The phenotype of GM-Mphis closely resembles that of human Alveolar-Mphis (A-Mphis), indicating that CSF-induced human monocyte-derived Mphis are useful to clarify the molecular mechanism of heterogeneity of human Mphis, and GM-Mphis will become a model of human A-Mphis.

  3. Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

    PubMed

    Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

    2003-01-01

    Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

  4. Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1

    PubMed Central

    Wang, Huayang; Shao, Qianqian; Sun, Jintang; Ma, Chao; Gao, Wenjuan; Wang, Qingjie; Zhao, Lei; Qu, Xun

    2016-01-01

    ABSTRACT Tumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Colony stimulating factor 1 (CSF1) is a primary chemoattractant and functional regulator for macrophages, and therefore would be a feasible intervention for the macrophage-targeting therapeutics. However, the expression of CSF1 in colon cancer microenvironment and its roles in cancer development is largely unknown. In the present study, we found that CSF1 was over-expressed exclusively in colon cancer cells and was correlated with macrophages infiltration. The high CSF1 expression and macrophages infiltration were related to the tumor–node-metastasis (TNM) stage of colon cancer, and suggested to be positively associated with survival of colon cancer patients. In the in vitro studies based on an indirect Transwell system, we found that co-culture with macrophage promoted CSF1 production in colon cancer cells. Further investigation on regulatory mechanisms suggested that CSF1 production in colon cancer cells was dependent on PKC pathway, which was activated by IL-8, mainly produced by macrophages. Moreover, colon cancer cell-derived CSF1 drove the recruitment of macrophages and re-educated their secretion profile, including the augment of IL-8 production. The mice tumor xenografts study also found that over-expression of CSF1 in colon cancer cells promoted intratumoral infiltration of macrophages, and partially suppressed tumor growth. In all, our results demonstrated that CSF1 was an important factor in the colon cancer microenvironment, involving in the interactions between colon cancer cells and tumor-infiltrated macrophages. PMID:27141406

  5. Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1.

    PubMed

    Wang, Huayang; Shao, Qianqian; Sun, Jintang; Ma, Chao; Gao, Wenjuan; Wang, Qingjie; Zhao, Lei; Qu, Xun

    2016-04-01

    Tumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Colony stimulating factor 1 (CSF1) is a primary chemoattractant and functional regulator for macrophages, and therefore would be a feasible intervention for the macrophage-targeting therapeutics. However, the expression of CSF1 in colon cancer microenvironment and its roles in cancer development is largely unknown. In the present study, we found that CSF1 was over-expressed exclusively in colon cancer cells and was correlated with macrophages infiltration. The high CSF1 expression and macrophages infiltration were related to the tumor-node-metastasis (TNM) stage of colon cancer, and suggested to be positively associated with survival of colon cancer patients. In the in vitro studies based on an indirect Transwell system, we found that co-culture with macrophage promoted CSF1 production in colon cancer cells. Further investigation on regulatory mechanisms suggested that CSF1 production in colon cancer cells was dependent on PKC pathway, which was activated by IL-8, mainly produced by macrophages. Moreover, colon cancer cell-derived CSF1 drove the recruitment of macrophages and re-educated their secretion profile, including the augment of IL-8 production. The mice tumor xenografts study also found that over-expression of CSF1 in colon cancer cells promoted intratumoral infiltration of macrophages, and partially suppressed tumor growth. In all, our results demonstrated that CSF1 was an important factor in the colon cancer microenvironment, involving in the interactions between colon cancer cells and tumor-infiltrated macrophages.

  6. Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.

    PubMed

    Eisenman, S T; Gibbons, S J; Verhulst, P-J; Cipriani, G; Saur, D; Farrugia, G

    2017-04-01

    Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow-derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high-powered field. Bone marrow-derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT-PCR. Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2-CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1-CM, including tumor necrosis factor alpha (TNF-α). ELISA detected 0.697±0.03 ng mL(-1) TNF-α in M1-CM. Recombinant mouse TNF-α reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50 = 0.817 ng mL(-1) ). Blocking M1-CM TNF-α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z-VAD.fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z-VAD.fmk, n=6, P<.05). This work demonstrates that TNF-α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase-dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis. © 2016 John Wiley & Sons Ltd.

  7. Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors.

    PubMed

    Lorber, Barbara; Berry, Martin; Logan, Ann

    2005-04-01

    In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.

  8. Monocyte-derived macrophage microparticles impart tissue factor activity to biomaterial surfaces.

    PubMed

    Patchipulusu, Sirisha; Turturro, Michael; Hall, Connie L

    2010-02-01

    The initiation of coagulation on biomaterials is attributed to the contact pathway of coagulation. However, recent discoveries of blood-borne tissue factor (TF) activity suggest that the TF pathway of coagulation may contribute to thrombosis on biomaterials. To evaluate the role of TF bearing microparticles to biomaterial thrombogenicity, the adhesion of monocyte-derived macrophage microparticles (MMPs) to bare, bovine serum albumin (BSA) blocked, and plasma-coated materials was examined. MMP suspensions consisted of 20-37% TF positive particles that exhibited TF activity. Data from static experiments with polyethylene (PE), polydimethylsiloxane (PDMS), polystyrene (PS), and glass knitted and woven Dacron(R) grafts showed that MMPs adhered to uncoated, and plasma coated surfaces supported TF activity, whereas surfaces blocked with BSA supported less activity. Flow studies were performed on plasma-coated glass and tissue culture-treated polystyrene (TCPS) as a model system to demonstrate deposition and firm adhesion of microparticles under physiologic flow conditions. MMPs deposited and imparted TF activity to plasma-coated glass at wall shear rates of 100, 400, and 1200 sec(-1). Deposition on TCPS was comparable to glass at 100 sec(-1), but virtually nonexistent at the two higher shear rates after a 1 h perfusion, implying material and shear dependent adhesion. The localization of procoagulant MMPs to biomaterial surfaces could lead to an increased risk of thrombosis on cardiovascular implants beyond that anticipated by the contact pathway alone. (c) 2009 Wiley Periodicals, Inc.

  9. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. 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.

  11. Effect of the Gc-derived macrophage-activating factor precursor (preGcMAF) on phagocytic activation of mouse peritoneal macrophages.

    PubMed

    Uto, Yoshihiro; Yamamoto, Syota; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Nakata, Eiji; Hori, Hitoshi

    2011-07-01

    The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of β-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages. We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF. PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.

  12. Wound healing: the effect of macrophage and tumour derived angiogenesis factors on skin graft vascularization.

    PubMed Central

    Arnold, F.; West, D.; Kumar, S.

    1987-01-01

    Angiogenic factors prepared from rat Walker 256 mammary carcinoma, (TAF) and activated mouse peritoneal macrophages (MAF), were tested for their ability to stimulate vascularization during healing. They were applied to one of a pair of bilaterally symmetrical, autologous, isotopic, full thickness skin grafts in mice. Blood flow to treated and untreated graft pairs was compared by their uptake of injected 86Rb Cl, at 3 and 7 days after grafting. No difference was detected after treatment with either agent. We conclude that while angiogenic factors are important in vascularization during healing, this normally occurs at a near maximal rate and cannot be further enhanced. PMID:2443156

  13. Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

    PubMed

    Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

    2002-09-04

    The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

  14. 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.

  15. 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

  16. Pigment epithelium-derived factor (PEDF) promotes tumor cell death by inducing macrophage membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

    PubMed

    Ho, Tsung-Chuan; Chen, Show-Li; Shih, Shou-Chuan; Chang, Shing-Jyh; Yang, Su-Lin; Hsieh, Jui-Wen; Cheng, Huey-Chuan; Chen, Lee-Jen; Tsao, Yeou-Ping

    2011-10-14

    Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.

  17. Monocytes-derived macrophages mediated stable expression of human brain-derived neurotrophic factor, a novel therapeutic strategy for neuroAIDS.

    PubMed

    Tong, Jing; Buch, Shilpa; Yao, Honghong; Wu, Chengxiang; Tong, Hsin-I; Wang, Youwei; Lu, Yuanan

    2014-01-01

    HIV-1 associated dementia remains a significant public health burden. Clinical and experimental research has shown that reduced levels of brain-derived neurotrophic factor (BDNF) may be a risk factor for neurological complications associated with HIV-1 infection. We are actively testing genetically modified macrophages for their possible use as the cell-based gene delivery vehicle for the central nervous system (CNS). It can be an advantage to use the natural homing/migratory properties of monocyte-derived macrophages to deliver potentially neuroprotective BDNF into the CNS, as a non-invasive manner. Lentiviral-mediated gene transfer of human (h)BDNF plasmid was constructed and characterized. Defective lentiviral stocks were generated by transient transfection of 293T cells with lentiviral transfer plasmid together with packaging and envelope plasmids. High titer lentiviral vector stocks were harvested and used to transduce human neuronal cell lines, primary cultures of human peripheral mononocyte-derived macrophages (hMDM) and murine myeloid monocyte-derived macrophages (mMDM). These transduced cells were tested for hBDNF expression, stability, and neuroprotective activity. The GenomeLab GeXP Genetic Analysis System was used to evaluate transduced cells for any adverse effects by assessing gene profiles of 24 reference genes. High titer vectors were prepared for efficient transduction of neuronal cell lines, hMDM, and mMDM. Stable secretion of high levels of hBDNF was detected in supernatants of transduced cells using western blot and ELISA. The conditioned media containing hBDNF were shown to be protective to neuronal and monocytic cell lines from TNF-α and HIV-1 Tat mediated cytotoxicity. Lentiviral vector-mediated gene transduction of hMDM and mMDM resulted in high-level, stable expression of the neuroprotective factorBDNF in vitro. These findings form the basis for future research on the potential use of BDNF as a novel therapy for neuroAIDS.

  18. Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

    PubMed

    Mohamad, Saharuddin Bin; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

    2002-01-01

    Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

  19. 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

  20. 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.

  1. Receptor binding and cellular uptake studies of macrophage migration inhibitory factor (MIF): use of biologically active labeled MIF derivatives.

    PubMed

    Kleemann, Robert; Grell, Matthias; Mischke, Ralf; Zimmermann, Gudrun; Bernhagen, Jürgen

    2002-03-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target

  2. Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells.

    PubMed

    Na, Yi Rang; Hong, Ji Hye; Lee, Min Yong; Jung, Jae Hun; Jung, Daun; Kim, Young Won; Son, Dain; Choi, Murim; Kim, Kwang Pyo; Seok, Seung Hyeok

    2015-10-01

    Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells*

    PubMed Central

    Na, Yi Rang; Hong, Ji Hye; Lee, Min Yong; Jung, Jae Hun; Jung, Daun; Kim, Young Won; Son, Dain; Choi, Murim; Kim, Kwang Pyo; Seok, Seung Hyeok

    2015-01-01

    Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs. PMID:26229149

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells.

    PubMed

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

    2012-01-01

    Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic membrane (CAM) assay. DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential.

  10. 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.

  11. Monocyte-derived factors including PLA2G7 induced by macrophage-nasopharyngeal carcinoma cell interaction promote tumor cell invasiveness

    PubMed Central

    Low, Heng Boon; Png, Chin Wen; Li, Chunwei; Wang, De Yun; Wong, Soon Boon Justin; Zhang, Yongliang

    2016-01-01

    The non-keratinizing undifferentiated subtype of nasopharyngeal carcinoma (NPC) is a malignancy characterized by an intimate relationship between neoplastic cells and a non-neoplastic lymphoid component. Tumor-associated macrophages (TAMs) foster tumor progression through production of soluble mediators that support proliferation, angiogenesis, survival and invasion of malignant cells. However, the role of macrophages in the progression of NPC remains poorly understood. This study aims to investigate the functional and phenotypic changes that occur to macrophages in macrophage-NPC cell co-culture systems, and how these changes influence tumor cells. We found that monocytes, including THP-1 cells and primary human monocytes, co-cultured with C666-1 NPC cells upregulate expression of pro-inflammatory cytokines at the early stages, followed by the induction of metastasis-related genes and interferon-stimulated genes at the later stage of coculture, indicating that TAMs are “educated” by NPC cells for cancer progression. Importantly, the induction of these factors from the TAMs was also found to enhance the migratory capabilities of the NPC cells. We have also identified one of these macrophage-derived factor, phospholipase A2 Group 7 (PLA2G7), to be important in regulating tumor cell migration and a novel tumor-promoting factor in NPC. Further studies to characterize the role of PLA2G7 in tumor metastasis may help determine its potential as a therapeutic target in NPC. PMID:27487154

  12. 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.

  13. Ascorbic acid stimulates the resorption of canine articular cartilage induced by a factor derived from activated rabbit macrophages.

    PubMed

    Dean, D D; Sellers, A; Howell, D S; Kerwar, S S; Woessner, J F

    1985-01-01

    Articular cartilage explants from the knees of mongrel dogs release 5-10% of their proteoglycan content spontaneously when cultured for 4 days in serum-free modified Bigger's medium. A factor synthesized and secreted by lipopolysaccharide-stimulated rabbit macrophages can stimulate this release of proteoglycan by 2 to 3-fold. The release of proteoglycan in response to macrophage factor is maximal in the presence of 1.5-50 micrograms/ml L-ascorbic acid. In the absence of ascorbate, or with high levels of ascorbate (150 micrograms/ml), the effect of the factor is diminished by 50%. D-isoascorbate, reduced glutathione, or dithiothreitol cannot substitute for L-ascorbate in producing this effect, while dehydroascorbate can.

  14. 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.

  15. 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.

  16. 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.

  17. Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection

    PubMed Central

    de Melo, Gabrielly L.; Anidi, Chioma; Hamburger, Rebecca; Pham, Jennifer

    2016-01-01

    Dynamic regulation of leukocyte population size and activation state is crucial for an effective immune response. In malaria, Plasmodium parasites elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. Here we show that myeloid expansion during P. chabaudi infection is dependent upon both CD4+ T cells and the cytokine Macrophage Colony Stimulating Factor (MCSF). Single-cell RNA-Seq analysis on antigen-experienced T cells revealed robust expression of Csf1, the gene encoding MCSF, in a sub-population of CD4+ T cells with distinct transcriptional and surface phenotypes. Selective deletion of Csf1 in CD4+ cells during P. chabaudi infection diminished proliferation and activation of certain myeloid subsets, most notably lymph node-resident CD169+ macrophages, and resulted in increased parasite burden and impaired recovery of infected mice. Depletion of CD169+ macrophages during infection also led to increased parasitemia and significant host mortality, confirming a previously unappreciated role for these cells in control of P. chabaudi. This work establishes the CD4+ T cell as a physiologically relevant source of MCSF in vivo; probes the complexity of the CD4+ T cell response during type 1 infection; and delineates a novel mechanism by which T helper cells regulate myeloid cells to limit growth of a blood-borne intracellular pathogen. PMID:27923070

  18. 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.

  19. 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.

  20. 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

  1. Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages

    PubMed Central

    Wooden, Jessica; Ciborowski, Pawel

    2014-01-01

    The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a “one size fits all” rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM)a, we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10 minutes. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge. PMID:25220915

  2. 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

  3. Thymus-derived lymphocytes and their interactions with macrophages are required for the production of osteoclast-activating factor in the mouse.

    PubMed Central

    Horowitz, M; Vignery, A; Gershon, R K; Baron, R

    1984-01-01

    A bone-resorbing factor, comparable to the osteoclast-activating factor (OAF) produced from peripheral blood leukocytes, is shown to be produced by murine spleen cells activated with the T-cell mitogen Con A. Murine OAF is demonstrated here as being a product of the interaction between thymus-derived T lymphocytes and macrophages. Activation of T cells in the presence of macrophages with Con A yields culture supernatants with OAF activity. This OAF activity is not dialyzable and is not extracted by lipid solvents. Purified B cells in the presence or absence of macrophages and cocultured with Con A or activated with the B-cell-specific mitogen lipopolysaccharide yield culture supernatants with no detectable OAF activity. Similarly, macrophages cocultured with Con A or activated with lipopolysaccharide fail to yield culture supernatants with bone resorbing activity. These types of immune cell interactions are similar to that required for the production of lymphokines. These data support the hypothesis that one aspect of regulation of bone remodeling is through cells of the immune system. PMID:6609360

  4. 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

  5. Tumor-Derived Granulocyte-Macrophage Colony-Stimulating Factor and Granulocyte Colony-Stimulating Factor Prolong the Survival of Neutrophils Infiltrating Bronchoalveolar Subtype Pulmonary Adenocarcinoma

    PubMed Central

    Wislez, Marie; Fleury-Feith, Jocelyne; Rabbe, Nathalie; Moreau, Joelle; Cesari, Danielle; Milleron, Bernard; Mayaud, Charles; Antoine, Martine; Soler, Paul; Cadranel, Jacques

    2001-01-01

    We evaluated the role of the tumor environment in the regulation of apoptosis of tumor-infiltrating neutrophils, the number of which correlates negatively with outcome, in patients with adenocarcinoma of the bronchioloalveolar (BAC) subtype. We examined three different parameters of apoptosis, namely morphological aspect, annexin-V expression, and DNA fragmentation. Bronchoalveolar lavage fluid (BALF) supernatants from patients with BAC significantly inhibited the 24-hour spontaneous apoptosis of normal peripheral blood neutrophils in vitro compared to BALF supernatants from control patients (64 ± 4% versus 90 ± 2% measured by annexin-V flow cytometry, P = 0.04). The alveolar neutrophil count correlated positively with the granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations in the patient’s BALF. Furthermore, neutralizing antibodies (Abs) against GM-CSF and G-CSF significantly inhibited BALF anti-apoptotic activity (15 to 40% and 34 to 63% inhibition, respectively), whereas neutralizing Abs against interleukin (IL)-8, IL-6, IL-1β and tumor necrosis factor-α had no significant effect. In an attempt to identify the cell origin of anti-apoptotic cytokines, we tested in vitro the effect of BAC cells (A549 cell line and primary culture derived from a patient’s BAC tumor) on the apoptosis of peripheral blood neutrophils. Cell-free supernatants from tumor cells did not inhibit neutrophil apoptosis. In contrast, cell-free supernatants from tumor cells previously exposed to conditioned media from peripheral blood mononuclear cells and alveolar macrophages significantly inhibited spontaneous neutrophil apoptosis. This inhibition was partially lifted when conditioned media from mononuclear cells were previously treated with Abs against IL-1β and tumor necrosis factor-α. As in vivo, neutralizing Abs against GM-CSF significantly inhibited the anti-apoptotic activity of cell culture supernatants

  6. Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages.

    PubMed

    Shioi, Atsushi; Katagi, Miwako; Okuno, Yasuhisa; Mori, Katsuhito; Jono, Shuichi; Koyama, Hidenori; Nishizawa, Yoshiki

    2002-07-12

    Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-gamma (IFN-gamma) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-gamma and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-gamma and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of beta-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-alpha (TNF-alpha) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-alpha and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-gamma and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-alpha and OSM.

  7. 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

  8. 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

    2013-07-08

    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.

  9. Mechanism of macrophage migration inhibitory factor-induced decrease of T-type Ca(2+) channel current in atrium-derived cells.

    PubMed

    Rao, Fang; Deng, Chun-Yu; Wu, Shu-Lin; Xiao, Ding-Zhang; Huang, Wei; Deng, Hai; Kuang, Su-Juan; Lin, Qiu-Xiong; Shan, Zhi-Xin; Liu, Xiao-Ying; Zhu, Jie-Ning; Yu, Xi-Yong

    2013-01-01

    The T-type Ca(2+) current (I(Ca,T)) plays an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate the role of macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, in the regulation of T-type Ca(2+) channels (TCCs) in atrial myocytes. We used the whole-cell voltage-clamp technique and biochemical assays to study the regulation and expression of I(Ca,T) in atrial myocytes. Gene levels of the α1G and α1H subunit of TCCs were decreased in human atrial tissue of patients with AF. In cultured atrium-derived myocytes (HL-1 cells), mouse recombinant MIF (20 or 40 nm, 24 h) suppressed peak I(Ca,T) in a concentration-dependent manner, impaired the voltage-dependent activation of I(Ca,T) and downregulated TCC α1G and α1H mRNA. The Src inhibitors genistein and PP1 significantly enhanced I(Ca,T). The reduction of I(Ca,T) and TCC subunit mRNA induced by recombinant MIF could be reversed by genistein and PP1. The TCC α1G associated with Src in HL-1 cells and mouse cardiomycytes. Macrophage migration inhibitory factor is involved in the pathogenesis of AF, probably by decreasing the T-type calcium current in atrium-derived myocytes through impairment of channel function and activation of c-Src kinases, representing a potential pathogenic mechanism in atrial fibrillation.

  10. Glioma-derived macrophage migration inhibitory factor (MIF) promotes mast cell recruitment in a STAT5-dependent manner.

    PubMed

    Põlajeva, Jelena; Bergström, Tobias; Edqvist, Per-Henrik; Lundequist, Anders; Sjösten, Anna; Nilsson, Gunnar; Smits, Anja; Bergqvist, Michael; Pontén, Fredrik; Westermark, Bengt; Pejler, Gunnar; Forsberg Nilsson, Karin; Tchougounova, Elena

    2014-02-01

    Recently, glioma research has increased its focus on the diverse types of cells present in brain tumors. We observed previously that gliomas are associated with a profound accumulation of mast cells (MCs) and here we investigate the underlying mechanism. Gliomas express a plethora of chemoattractants. First, we demonstrated pronounced migration of human MCs toward conditioned medium from cultures of glioma cell lines. Subsequent cytokine array analyses of media from cells, cultured in either serum-containing or -free conditions, revealed a number of candidates which were secreted in high amounts in both cell lines. Among these, we then focused on macrophage migration inhibitory factor (MIF), which has been reported to be pro-inflammatory and -tumorigenic. Infiltration of MCs was attenuated by antibodies that neutralized MIF. Moreover, a positive correlation between the number of MCs and the level of MIF in a large cohort of human glioma tissue samples was observed. Further, both glioma-conditioned media and purified MIF promoted differential phosphorylation of a number of signaling molecules, including signal transducer and activator of transcription 5 (STAT5), in MCs. Inhibition of pSTAT5 signaling significantly attenuated the migration of MCs toward glioma cell-conditioned medium shown to contain MIF. In addition, analysis of tissue microarrays (TMAs) of high-grade gliomas revealed a direct correlation between the level of pSTAT5 in MCs and the level of MIF in the medium. In conclusion, these findings indicate the important influence of signaling cascades involving MIF and STAT5 on the recruitment of MCs to gliomas.

  11. CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

    PubMed Central

    Moriuchi, Hiroyuki; Moriuchi, Masako; Combadiere, Christophe; Murphy, Philip M.; Fauci, Anthony S.

    1996-01-01

    It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors. PMID:8986813

  12. Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes.

    PubMed

    Baron, C; Raposo, G; Scholl, S M; Bausinger, H; Tenza, D; Bohbot, A; Pouillart, P; Goud, B; Hanau, D; Salamero, J

    2001-03-01

    The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

  13. Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

    PubMed

    Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

    2012-05-05

    Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

    PubMed Central

    Dohlman, Thomas H.; Ding, Julia; Dana, Reza; Chauhan, Sunil K.

    2017-01-01

    Purpose Growing evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) contributes to T helper 17 (Th17) cell–associated immunoinflammatory diseases. The purpose of this study was to evaluate the effect of T cell–derived GM-CSF on CD11b+ myeloid cell function in dry eye disease (DED). Methods In a murine model of DED, quantitative real-time PCR and ELISA were used to measure GM-CSF expression at the ocular surface, and flow cytometry was used to enumerate GM-CSF producing Th17 cells. A granulocyte-macrophage colony-stimulating factor neutralizing antibody was used topically in vivo and in an in vitro culture system to evaluate the role of GM-CSF in recruiting and maturing CD11b+ cells. Clinical disease severity was evaluated after topical administration of GM-CSF neutralizing antibody. Results In dry eye disease, GM-CSF is significantly upregulated at the ocular surface and the frequency of GM-CSF producing Th17 cells is significantly increased in the draining lymph nodes. In vitro neutralization of GM-CSF from CD4+ T cells derived from DED mice suppresses major histocompatibility complex II expression by CD11b+ cells and CD11b+ cell migration. Topical neutralization of GM-CSF in a murine model of DED suppresses CD11b+ maturation and migration, as well as Th17 cell induction, yielding a reduction in clinical signs of disease. Conclusions T helper 17 cell–derived GM-CSF contributes to DED pathogenesis by promoting CD11b+ cell activation and migration to the ocular surface. PMID:28241321

  15. T Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration.

    PubMed

    Dohlman, Thomas H; Ding, Julia; Dana, Reza; Chauhan, Sunil K

    2017-02-01

    Growing evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) contributes to T helper 17 (Th17) cell-associated immunoinflammatory diseases. The purpose of this study was to evaluate the effect of T cell-derived GM-CSF on CD11b+ myeloid cell function in dry eye disease (DED). In a murine model of DED, quantitative real-time PCR and ELISA were used to measure GM-CSF expression at the ocular surface, and flow cytometry was used to enumerate GM-CSF producing Th17 cells. A granulocyte-macrophage colony-stimulating factor neutralizing antibody was used topically in vivo and in an in vitro culture system to evaluate the role of GM-CSF in recruiting and maturing CD11b+ cells. Clinical disease severity was evaluated after topical administration of GM-CSF neutralizing antibody. In dry eye disease, GM-CSF is significantly upregulated at the ocular surface and the frequency of GM-CSF producing Th17 cells is significantly increased in the draining lymph nodes. In vitro neutralization of GM-CSF from CD4+ T cells derived from DED mice suppresses major histocompatibility complex II expression by CD11b+ cells and CD11b+ cell migration. Topical neutralization of GM-CSF in a murine model of DED suppresses CD11b+ maturation and migration, as well as Th17 cell induction, yielding a reduction in clinical signs of disease. T helper 17 cell-derived GM-CSF contributes to DED pathogenesis by promoting CD11b+ cell activation and migration to the ocular surface.

  16. Platelet-derived growth factor-C (PDGF-C) induces anti-apoptotic effects on macrophages through Akt and Bad phosphorylation.

    PubMed

    Son, Dain; Na, Yi Rang; Hwang, Eung-Soo; Seok, Seung Hyeok

    2014-02-28

    PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

  17. 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.

  18. 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.

  19. 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.

  20. Proximal tubule-derived Colony Stimulating Factor-1 mediates polarization of renal macrophages and dendritic cells, and recovery in acute kidney injury

    PubMed Central

    Wang, Yinqiu; Chang, Jian; Yao, Bing; Niu, Aolei; Kelly, Emily; Breeggemann, Matthew C.; Abboud Werner, Sherry L.; Harris, Raymond C.; Zhang, Ming-Zhi

    2015-01-01

    Infiltrating cells play an important role in both the development of and recovery from acute kidney injury (AKI). Macrophages and renal dendritic cells are of particular interest because they can exhibit distinctly different functional phenotypes, broadly characterized as proinflammatory (M1) or tissue reparative (M2). Resident renal macrophages and dendritic cells participate in recovery from AKI in response to either ischemia/reperfusion or a model of selective proximal tubule injury induced by diphtheria toxin-induced apoptosis in transgenic mice expressing the human diphtheria toxin receptor on proximal tubule cells. Colony Stimulating Factor-1 (CSF-1) is an important factor mediating the recovery from AKI, and CSF-1 can stimulate macrophage and dendritic cell proliferation and polarization during the recovery phase of AKI. The kidney, and specifically the proximal tubule, is a major source of intrarenal CSF-1 production in response to AKI. We induced selective deletion of proximal tubule CSF-1 to determine its role in expansion and proliferation of renal macrophages and dendritic cells and in recovery from AKI. In both models of AKI, there was decreased M2 polarization, delayed functional and structural recovery and increased tubulointerstitial fibrosis. Thus, intrarenal CSF-1 is an important mediator of macrophage/dendritic cell polarization and recovery from AKI. PMID:26422503

  1. Up-regulation of alveolar macrophage platelet-derived growth factor-B (PDGF-B) mRNA by interferon-gamma from Mycobacterium tuberculosis antigen (PPD)-stimulated lymphocytes.

    PubMed Central

    Wangoo, A; Taylor, I K; Haynes, A R; Shaw, R J

    1993-01-01

    Macrophage production of PDGF-B is believed to be important in the pathogenesis of diseases where chronic lung inflammation develops into fibrosis. Since tuberculosis is characterized by chronic inflammation and tissue fibrosis, we asked if lymphokines from lymphocytes stimulated by the Mycobacterium tuberculosis antigen PPD, contained factors capable of increasing human alveolar macrophage PDGF-B mRNA. Supernatants from both phytohaemagglutinin (PHA)- and purified protein derivative (PPD)-stimulated lymphocytes, when added to macrophages, induced an increase in the mRNA of PDGF-B, but not transforming growth factor-beta (TGF-beta). When lymphocytes from contacts of patients with tuberculosis, patients with tuberculosis, and normal subjects were compared following PPD stimulation, the lymphocytes from the contacts had the greatest proliferation response, the greatest production of interferon-gamma (IFN-gamma), and their lymphokines induced the greatest increase in PDGF-B mRNA in macrophages. Recombinant human IFN-gamma reproduced this ability of lymphokines to increase macrophage PDGF-B mRNA. Finally, the increase in macrophage PDGF-B mRNA following incubation with supernatants from PPD-stimulated lymphocytes was shown to be due to IFN-gamma, when the increase in macrophage PDGF-B mRNA was prevented by addition of anti-human IFN-gamma antibody to the lymphocyte supernatant. This study indicated that antigen-stimulated lymphocytes released IFN-gamma, which in turn resulted in an increase in PDGF-B mRNA in alveolar macrophages. Such a mechanism provides a link between the DTH response and the first stages of a fibrotic reaction, and may offer an explanation for the progression of chronic inflammation to fibrosis, as occurs in the lungs of patients with untreated pulmonary tuberculosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8403516

  2. Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells.

    PubMed

    Li, Gong-Bo; Lu, Guang-Xiu

    2010-01-01

    A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as "non-dendritic cells" or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these "junk cells" are actually qualified dendritic cells.

  3. Data for proteomic analysis of Human monocyte-derived macrophages.

    PubMed

    Eligini, S; Brioschi, M; Fiorelli, S; Tremoli, E; Colli, S; Banfi, C

    2015-09-01

    This data article is referred to the research article entitled Human monocyte-derived macrophages are heterogeneous: proteomic profile of different phenotypes by Eligini et al. Eligini S., Brioschi M., Fiorelli S., Tremoli E., Banfi C., Colli S. Human monocyte-derived macrophages are heterogeneous: proteomic profile of different phenotypes. J. Proteomics 124, 2015, 112-123. Macrophages obtained in vitro from blood monocytes are largely used as surrogate model of tissue macrophages that are heterogeneous and not easy to obtain and handle. Under spontaneous differentiation in vitro, monocyte-derived macrophages (MDMs) display two dominant subsets (round and spindle) that show different transcriptional, antigenic, and functional profiles mimicking, at least in part, the heterogeneity of tissue macrophages. This article reports the nano-LC-MS(E) analysis of the proteome of round and spindle MDMs allowing a deeper comprehension of macrophage heterogeneity.

  4. Tertiary-butylhydroquinone upregulates expression of ATP-binding cassette transporter A1 via nuclear factor E2-related factor 2/heme oxygenase-1 signaling in THP-1 macrophage-derived foam cells.

    PubMed

    Lu, Qian; Tang, Shi-Lin; Liu, Xiao-Yan; Zhao, Guo-Jun; Ouyang, Xin-Ping; Lv, Yun-Cheng; He, Ping-Ping; Yao, Feng; Chen, Wu-Jun; Tang, Yan-Yan; Zhang, Min; Zhang, Da-Wei; Yin, Kai; Tang, Chao-Ke

    2013-01-01

    Tert-butylhydroquinone (tBHQ), a synthetic phenolic antioxidant, is commonly used as a food preservative because of its potent antilipid peroxidation activity. Several lines of evidence have demonstrated that dietary supplementation with antioxidants has an antiatherogenic function through reducing cholesterol uptake or promoting reverse cholesterol transport. In this study, we investigated whether tBHQ affects expression of ATP-binding cassette transporter A1 (ABCA1) and the potential subsequent effect on cellular cholesterol homeostasis. tBHQ increased ABCA1 protein levels and markedly enhanced cholesterol efflux from THP-1 macrophage-derived foam cells. Furthermore, tBHQ reduced calpain-mediated ABCA1 proteolysis via activation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Inhibition of HO-1 with a pharmacological inhibitor or siRNA and knockdown of Nrf2 suppressed the stimulatory effects of tBHQ on ABCA1 expression and calpain activity. Nrf2/HO-1 signaling is required for the regulation by tBHQ of ABCA1 expression and cholesterol efflux in macrophage-derived foam cells and an antiatherogenic role of tBHQ is suggested.

  5. Synthesis of granulocyte–macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material

    PubMed Central

    Zhang, Qiang; Johnston, Eric V.; Shieh, Jae-Hung; Moore, Malcolm A. S.; Danishefsky, Samuel J.

    2014-01-01

    Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synthetic congeners. PMID:24516138

  6. 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

    Jones, Anna C; Antillon, Kresta S; Jenkins, Shannon M; Janos, Sara N; Overton, Heidi N; 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

  7. 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

  8. Migration Inhibitory Factor and Macrophage Bactericidal Function

    PubMed Central

    Simon, Harvey B.; Sheagren, John N.

    1972-01-01

    A homogeneous population of immunologically active lymphocytes was obtained from peritoneal exudates of guinea pigs with delayed hypersensitivity to bovine gamma globulin (BGG). The lymphocytes were cultured with and without BGG for 24 hr, and cell-free supernatant fluids were then assayed simultaneously for their ability to influence two in vitro parameters of macrophage function: migration from capillary tubes and bactericidal capacity. In four consecutive experiments, supernatants from antigenically stimulated lymphocytes exhibited substantial migration-inhibitory-factor activity without enhancing the ability of macrophages to kill Listeria monocytogenes. Lymphocyte lysates were inactive in both assays. Possible mechanisms of lymphocyte-macrophage interactions are discussed. PMID:4120244

  9. Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro.

    PubMed

    Hashimoto, Atsushi; Tanaka, Takahiro; Itoh, Yuko; Yamagata, Akira; Kitamura, Nobutaka; Tazawa, Ryushi; Nakagaki, Kazuhide; Nakata, Koh

    2014-08-01

    To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli- or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60pM). Intermediate-term stimulation (24h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168h) was higher at low concentrations of crhGM-CSF (15-30pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Tumor-associated macrophages induce capillary morphogenesis of lymphatic endothelial cells derived from human gastric cancer.

    PubMed

    Tauchi, Yukie; Tanaka, Hiroaki; Kumamoto, Kanako; Tokumoto, Mao; Sakimura, Chie; Sakurai, Katsunobu; Kimura, Kenjiro; Toyokawa, Takahiro; Amano, Ryosuke; Kubo, Naoshi; Muguruma, Kazuya; Yashiro, Masakazu; Maeda, Kiyoshi; Ohira, Masaichi; Hirakawa, Kosei

    2016-08-01

    Tumor lymphangiogenesis is a major prognostic indicator of gastric cancer. Tumor-induced inflammation has been shown to attract tumor-associated macrophages that affect lymphangiogenesis. However, detailed mechanisms of macrophage-induced lymphangiogenesis have not been elucidated. Here, we evaluated the interaction between tumor-associated macrophages and lymphatic endothelial cells (LECs) derived from lymph nodes (LNs) of human gastric cancer. Lymphatic endothelial cells were directly or indirectly cocultured with macrophages from healthy human blood, with or without the supernatant of the gastric cancer cell line, OCUM-12. We analyzed the effect of cancer pretreated macrophages and of macrophages from metastatic LNs of gastric cancer on LECs. We observed morphological changes of LECs in coculture and assessed the gene expression of possible lymphangiogenic molecules of macrophages and LECs after contact coculture, and of cancer pretreated macrophages, by quantitative RT-PCR. Specimens of metastatic LN of gastric cancer were immunofluorescently stained. We found that tubulogenesis of LECs was observed only in the contact coculture model. OCUM-12 cells promoted macrophage-induced tubulogenesis of LECs. Relative gene expression of MMP and adhesion molecules was significantly upregulated in both capillary-forming LECs and cocultured macrophages. Cancer pretreated macrophages upregulated lymphangiogenic factors including inflammatory cytokines, MMPs, adhesion molecules, and vascular endothelial growth factor-C. Blocking of intercellular adhesion molecule-1 and macrophage activation suppressed tubulogenesis of LECs. Immunohistochemistry showed macrophages localized around lymphatic vessels. Our results suggested that interaction between LECs and macrophages may be an important initial step of tumor lymphangiogenesis developing LN metastasis. Understanding of its mechanisms could be useful for future therapeutics of gastric cancer.

  11. Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis.

    PubMed

    Linehan, Eimear; Dombrowski, Yvonne; Snoddy, Rachel; Fallon, Padraic G; Kissenpfennig, Adrien; Fitzgerald, Denise C

    2014-08-01

    Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age-related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow-derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow-derived macrophages and bone marrow monocytes did not exhibit age-related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age-related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell-derived IL-10 was increased in resting and LPS-activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue-resident peritoneal macrophages, but not by bone marrow-derived macrophages/monocytes, and suggest that age-related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.

  12. 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.

  13. Granulocyte-macrophage colony-stimulating factor-cultured bone marrow-derived macrophages reveal accessory cell function and synthesis of MHC class II determinants in the absence of external stimuli.

    PubMed

    Fischer, H G; Opel, B; Reske, K; Reske-Kunz, A B

    1988-08-01

    The antigen-mediated activation of a number of T cell clones by bone marrow (BM) cells cultivated in the presence of various colony-stimulating factor (CSF) preparations was investigated. BM macrophages (BMM phi) grown in L929 cell supernatant as a crude source of macrophage colony-stimulating factor (M-CSF) as well as BM cells propagated in the presence of recombinant M-CSF exhibited transient antigen presentation potential to some T cell clones, being maximal on day 7 and having declined to a low level by day 19 of in vitro culture. Treatment of these long-term-cultivated BMM phi populations with recombinant interferon-gamma (IFN-gamma) resulted in predominant antigen presentation capacity. In contrast, BM cells differentiated in the presence of recombinant granulocyte (G)M-CSF developed highly efficient accessory cell function to all T cell clones examined. This function became apparent earlier, was retained during the time period tested (up to day 19 of continuous culture) and did not require prior stimulation by IFN-gamma. The functionally competent cells were shown to belong to the monocyte/macrophage lineage. These findings are consistent with the demonstration of substantial levels of major histocompatibility complex (MHC) class II molecules synthesized by GM-CSF-cultured BM cells in the absence of exogenous IFN-gamma. In contrast, M-CSF grown BM cells synthesized only minute amounts of Ia antigens unless they were stimulated by IFN-gamma. Because GM-CSF-cultivated BM cells proved clearly superior to M-CSF-grown and IFN-gamma-activated BM cells with respect to antigen-presenting capacity but exhibited lower levels of MHC class II molecules, other properties acting in addition to surface Ia antigens might be responsible for their pronounced T cell accessory function.

  14. Effects of mushroom-derived beta-glucan-rich polysaccharide extracts on nitric oxide production by bone marrow-derived macrophages and nuclear factor-kappaB transactivation in Caco-2 reporter cells: can effects be explained by structure?

    PubMed

    Volman, Julia J; Helsper, Johannes P F G; Wei, Song; Baars, Johan J P; van Griensven, Leo J L D; Sonnenberg, Anton S M; Mensink, Ronald P; Plat, Jogchum

    2010-02-01

    Mushrooms are known for their immune-modulating and anti-tumour properties. The polysaccharide fraction, mainly beta-glucans, is responsible for the immune-modulating effects. Fungal beta-glucans have been shown to activate leukocytes, which depend on structural characteristics of beta-glucans. As edible mushrooms come in contact with the intestinal immune system, effects on enterocytes are also interesting. Our aim was to evaluate the effect of mushroom polysaccharide extracts varying in beta-glucan structure on nitric oxide production by bone marrow-derived macrophages (BMMs) from mice and on nuclear factor-kappaB transactivation in human intestinal Caco-2 cells. We demonstrated that extracts from Agaricus bisporus stimulated nitric oxide production by BMM, whereas extracts from Coprinus comatus and spores of Ganoderma lucidum had only minor effects. Furthermore, extracts of A. blazei Murill and Phellinus linteus had no effect at all. Almost all mushroom extracts lowered nuclear factor-kappaB transactivation in Caco-2 cells. Structural analysis of A. bisporus compared with A. blazei Murill suggests that branching of the beta-glucan chain is essential for immune-stimulating activity. In conclusion, extracts from A. bisporus activate BMM, without activating enterocytes. These characteristics make A. bisporus an attractive candidate as a nutritional compound to stimulate the immune response in depressed states of immunity.

  15. 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.

  16. Macrophage migration inhibitory factor promotes clearance of pneumococcal colonization.

    PubMed

    Das, Rituparna; LaRose, Meredith I; Hergott, Christopher B; Leng, Lin; Bucala, Richard; Weiser, Jeffrey N

    2014-07-15

    Human genetic polymorphisms associated with decreased expression of macrophage migration inhibitory factor (MIF) have been linked to the risk of community-acquired pneumonia. Because Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and nasal carriage is a precursor to invasive disease, we explored the role of MIF in the clearance of pneumococcal colonization in a mouse model. MIF-deficient mice (Mif(-/-)) showed prolonged colonization with both avirulent (23F) and virulent (6A) pneumococcal serotypes compared with wild-type animals. Pneumococcal carriage led to both local upregulation of MIF expression and systemic increase of the cytokine. Delayed clearance in the Mif(-/-) mice was correlated with reduced numbers of macrophages in upper respiratory tract lavages as well as impaired upregulation of MCP-1/CCL2. We found that primary human monocyte-derived macrophages as well as THP-1 macrophages produced MIF upon pneumococcal infection in a pneumolysin-dependent manner. Pneumolysin-induced MIF production required its pore-forming activity and phosphorylation of p38-MAPK in macrophages, with sustained p38-MAPK phosphorylation abrogated in the setting of MIF deficiency. Challenge with pneumolysin-deficient bacteria demonstrated reduced MIF upregulation, decreased numbers of macrophages in the nasopharynx, and less effective clearance. Mif(-/-) mice also showed reduced Ab response to pneumococcal colonization and impaired ability to clear secondary carriage. Finally, local administration of MIF was able to restore bacterial clearance and macrophage accumulation in Mif(-/-) mice. Our work suggests that MIF is important for innate and adaptive immunity to pneumococcal colonization and could be a contributing factor in genetic differences in pneumococcal disease susceptibility.

  17. 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

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

    PubMed

    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

    2015-03-10

    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.

  19. 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

  20. Macrophage-derived microvesicles promote proliferation and migration of Schwann cell on peripheral nerve repair

    SciTech Connect

    Zhan, Chuan Ma, Cheng-bin; Yuan, Hong-mou; Cao, Bao-yuan; Zhu, Jia-jun

    2015-12-04

    Background: Macrophages have been implicated in peripheral nerve regeneration. However, whether macrophages-derived microvesicles (MVs) are involved in this process remains unknown. In the present study, the effects of macrophages-derived MVs on proliferation and migration of Schwann cells (SCs) were evaluated in both in vitro and in vivo. Methods: Human monocytic leukaemia cell line (THP-1) was successfully driven to M1 and M2 phenotypes by delivery of either IFN-γ or IL-4, respectively. SCs incubated with M1 or M2 macrophages-derived MVs, the cell migration and proliferation were assessed, and expression levels of nerve growth factor (NGF) and Laminin were measured. A rat model of sciatic nerve was established and the effects of macrophages-derived MVs on nerve regeneration were investigated. Results: M2-derived MVs elevated migration, proliferation, NFG and Laminin protein levels of SCs compared with M1-or M0-derived MVs. The relative expression levels of miR-223 were also increased in M2 macrophages and M2-derived MVs. Transfected M2 macrophages with miR-223 inhibitor then co-incubated with SCs, an inhibition of cell migration and proliferation and a down-regulated levels of NFG and Laminin protein expression were observed. In vivo, M2-derived MVs significantly increased the infiltration and axon number of SCs. Conclusion: M2-derived MVs promoted proliferation and migration of SCs in vitro and in vivo, which provided a therapeutic strategy for nerve regeneration. - Highlights: • M2 macrophages-derived MVs elevated migration and proliferation of SCs. • M2 macrophages-derived MVs up-regulated NFG and Laminin expression of SCs. • MiR-223 expression was increased in M2 macrophages-derived MVs. • MiR-223 inhibitor reduced migration and proliferation of SCs co-incubated with MVs. • MiR-223 inhibitor down-regulated NFG and Laminin levels of SCs co-incubated with MVs.

  1. Sialoadhesin expression by bone marrow macrophages derived from Ehrlich-tumor-bearing mice.

    PubMed

    Kusmartsev, S; Ruiz de Morales, J M; Rullas, J; Danilets, M G; Subiza, J L

    1999-12-01

    Sialoadhesin (sheep erythrocyte receptor, SER) is a macrophage-restricted adhesion molecule that binds certain sialylated ligands. It is borne by bone marrow stromal macrophages, promoting the interaction with developing myeloid cells, and by a subset of tissue macrophages involved in antigen presentation and activation of tumor-reactive T cells. The expression of sialoadhesin on SER+ macrophages is not constitutive but requires the continuous supply of a sialoadhesin-inducing serum factor. Tumor growth is often associated with marked alterations of myelopoiesis and impairment of T cell activation; yet the expression of sialoadhesin in macrophages derived from tumor bearers has not been addressed. The aim of this study was to assess whether Ehrlich tumor (ET) - a murine mammary carcinoma - growth may modify the sialoadhesin expression by bone marrow macrophages and/or sialoadhesin-inducing activity in ET-bearing sera. Moreover, putative functional sialoadhesin inhibitors produced by ET cells were tested. The results indicate that bone marrow cells from ET bearers show a seven- to eight-fold decrease in SER+ cells as detected by flow cytometry. This is accompanied by an overall decrease in sheep erythrocyte binding to tumor-bearer-derived bone marrow cells, but also by lower numbers of plastic-adherent cells. Functional sialoadhesin expression is preserved at the single-cell level and no inhibitors are found in ET-bearing sera or ET cell culture supernatants. Tumor progression does not impair the sialoadhesin-inducing activity of ET-bearing sera, or the ability of SER- macrophages (e.g. peritoneal macrophages) to respond to such an induction. In conclusion, while SER+ macrophages are greatly decreased in bone marrow from ET bearers, this is not due to a down-regulation of sialoadhesin expression, nor to an impairment of sialoadhesin-inducing factor or to the presence of sialoadhesin-binding moieties of tumor origin, but, more likely, to a decrease of fully mature

  2. 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

  3. 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.

  4. Exosomes derived from gastric cancer cells activate NF-κB pathway in macrophages to promote cancer progression.

    PubMed

    Wu, Lijun; Zhang, Xu; Zhang, Bin; Shi, Hui; Yuan, Xiao; Sun, Yaoxiang; Pan, Zhaoji; Qian, Hui; Xu, Wenrong

    2016-09-01

    Exosomes are nano-sized membrane vesicles secreted by both normal and cancer cells. Emerging evidence indicates that cancer cells derived exosomes contribute to cancer progression through the modulation of tumor microenvironment. However, the effects of exosomes derived from gastric cancer cells on macrophages are not well understood. In this study, we investigated the biological role of gastric cancer cells derived exosomes in the activation of macrophages. We demonstrated that gastric cancer cells derived exosomes activated macrophages to express increased levels of proinflammatory factors, which in turn promoted tumor cell proliferation and migration. In addition, gastric cancer cells derived exosomes remarkably upregulated the phosphorylation of NF-κB in macrophages. Inhibiting the activation of NF-κB reversed the upregulation of proinflammatory factors in macrophages and blocked their promoting effects on gastric cancer cells. Moreover, we found that gastric cancer cells derived exosomes could also activate macrophages from human peripheral blood monocytes through the activation of NF-κB. In conclusion, our results suggest that gastric cancer cells derived exosomes stimulate the activation of NF-κB pathway in macrophages to promote cancer progression, which provides a potential therapeutic approach for gastric cancer by interfering with the interaction between exosomes and macrophages in tumor microenvironment.

  5. 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

  6. Prevotella intermedia lipopolysaccharide stimulates release of tumor necrosis factor-alpha through mitogen-activated protein kinase signaling pathways in monocyte-derived macrophages.

    PubMed

    Kim, Sung-Jo; Choi, Eun-Young; Kim, Eun Gyung; Shin, Su-Hwa; Lee, Ju-Youn; Choi, Jeom-Il; Choi, In-Soon

    2007-11-01

    The purpose of this study was to investigate the effects of lipopolysaccharide from Prevotella intermedia, a major cause of inflammatory periodontal disease, on the production of tumor necrosis factor (TNF)-alpha and the expression of TNF-alpha mRNA in differentiated THP-1 cells, a human monocytic cell line. The potential involvement of the three main mitogen-activated protein kinase (MAPK) signaling pathways in the induction of TNF-alpha production was also investigated. Lipopolysaccharide from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. It was found that P. intermedia lipopolysaccharide can induce TNF-alpha mRNA expression and stimulate the release of TNF-alpha in differentiated THP-1 cells without additional stimuli. Treatment of the cells with P. intermedia lipopolysaccharide resulted in a simultaneous activation of three MAPKs [extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38]. Pretreatment of the cells with MAPK inhibitors effectively suppressed P. intermedia lipopolysaccharide-induced TNF-alpha production without affecting the expression of TNF-alpha mRNA. These data thus provided good evidence that the MAPK signaling pathways are required for the regulation of P. intermedia lipopolysaccharide-induced TNF-alpha synthesis at the level of translation more than at the transcriptional level.

  7. Soybean-derived Bowman-Birk Inhibitor (BBI) Inhibits HIV Replication in Macrophages

    PubMed Central

    Ma, Tong-Cui; Zhou, Run-Hong; Wang, Xu; Li, Jie-Liang; Sang, Ming; Zhou, Li; Zhuang, Ke; Hou, Wei; Guo, De-Yin; Ho, Wen-Zhe

    2016-01-01

    The Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, is known to have anti-inflammatory effect in both in vitro and in vivo systems. Macrophages play a key role in inflammation and immune activation, which is implicated in HIV disease progression. Here, we investigated the effect of BBI on HIV infection of peripheral blood monocyte-derived macrophages. We demonstrated that BBI could potently inhibit HIV replication in macrophages without cytotoxicity. Investigation of the mechanism(s) of BBI action on HIV showed that BBI induced the expression of IFN-β and multiple IFN stimulated genes (ISGs), including Myxovirus resistance protein 2 (Mx2), 2′,5′-oligoadenylate synthetase (OAS-1), Virus inhibitory protein (viperin), ISG15 and ISG56. BBI treatment of macrophages also increased the expression of several known HIV restriction factors, including APOBEC3F, APOBEC3G and tetherin. Furthermore, BBI enhanced the phosphorylation of IRF3, a key regulator of IFN-β. The inhibition of IFN-β pathway by the neutralization antibody to type I IFN receptor (Anti-IFNAR) abolished BBI-mediated induction of the anti-HIV factors and inhibition of HIV in macrophages. These findings that BBI could activate IFN-β-mediated signaling pathway, initialize the intracellular innate immunity in macrophages and potently inhibit HIV at multiple steps of viral replication cycle indicate the necessity to further investigate BBI as an alternative and cost-effective anti-HIV natural product. PMID:27734899

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

    PubMed Central

    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

    2016-01-01

    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

  9. Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages

    PubMed Central

    Cockrell, Diane C.; Long, Carrie M.; Robertson, Shelly J.; Shannon, Jeffrey G.; Miller, Heather E.; Myers, Lara; Larson, Charles L.; Starr, Tregei; Beare, Paul A.

    2017-01-01

    Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1

  10. Increased Susceptibility of  Thymocytes to Apoptosis in Mice Lacking AIM, a Novel Murine Macrophage-derived Soluble Factor Belonging to the Scavenger Receptor Cysteine-rich Domain Superfamily

    PubMed Central

    Miyazaki, Toru; Hirokami, Yumiko; Matsuhashi, Nobuyuki; Takatsuka, Hisakazu; Naito, Makoto

    1999-01-01

    Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection. PMID:9892623

  11. Bovine monocyte-derived macrophage function in induced copper deficiency.

    PubMed

    Cerone, S; Sansinanea, A; Streitenberger, S; García, C; Auza, N

    2000-03-01

    The effect of molybdenum-induced copper deficiency on monocyte-derived macrophage function was examined. Five female calves were given molybdenum (30 ppm) and sulphate (225 ppm) to induce experimental secondary copper deficiency. Oxidant production by bovine macrophages was measured after stimulation with phorbol myristate acetate (PMA) and opsonized zymosan (OpZ). Lipoperoxidative effects inside of macrophage, superoxide dismutase activity, superoxide anion and hydrogen peroxide formation were determined. Copper deficiency was confirmed from decreased serum copper levels, and animals with values less than 5.9 micromol/l were considered deficient. The content of intracellular copper decreased about 40% in deficient cells compared with the controls. The respiratory burst activity determined by nitroblue tetrazolium reduction was significantly impaired with both stimulants used. Superoxide anion formation was less affected than hydrogen peroxide generation. In addition, increased lipid peroxidation was observed. It could be concluded that the effect of these changes may impair the monocyte-derived macrophage function in the immune system.

  12. CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages.

    PubMed

    Neu, Caroline; Sedlag, Anne; Bayer, Carina; Förster, Sabine; Crauwels, Peter; Niess, Jan-Hendrik; van Zandbergen, Ger; Frascaroli, Giada; Riedel, Christian U

    2013-01-01

    Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model

  13. 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.

  14. 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.

  15. Macrophage colony-stimulating factor-induced macrophage differentiation promotes regrowth in atrophied skeletal muscles and C2C12 myotubes.

    PubMed

    Dumont, Nicolas A; Frenette, Jérôme

    2013-02-01

    Skeletal muscle injury and regeneration are closely associated with an inflammatory reaction that is usually characterized by sequential recruitment of neutrophils and monocytes or macrophages. Selective macrophage depletion models have shown that macrophages are essential for complete regeneration of muscle fibers after freeze injuries, toxin injuries, ischemia-reperfusion, and hindlimb unloading and reloading. Although there is growing evidence that macrophages possess major myogenic capacities, it is not known whether the positive effects of macrophages can be optimized to stimulate muscle regrowth. We used in vivo and in vitro mouse models of atrophy to investigate the effects of stimulating macrophages with macrophage colony-stimulating factor (M-CSF) on muscle regrowth. When atrophied soleus muscles were injected intramuscularly with M-CSF, we observed a 1.6-fold increase in macrophage density and a faster recovery in muscle force (20%), combined with an increase in muscle fiber diameter (10%), after 7 days of reloading, compared with PBS-injected soleus muscles. Furthermore, coculture of atrophied myotubes with or without bone marrow-derived macrophages (BMDM) and/or M-CSF revealed that the combination of BMDMs and M-CSF was required to promote myotube growth (15%). More specifically, M-CSF promoted the anti-inflammatory macrophage phenotype, which in turn decreased protein degradation and MuRF-1 expression by 25% in growing myotubes. These results indicate that specific macrophage subsets can be stimulated to promote muscle cell regrowth after atrophy.

  16. Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

    PubMed

    Taya, Kahoru; Nakayama, Emi E; Shioda, Tatsuo

    2014-01-01

    Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

  17. Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

    PubMed Central

    Dalal, Samay; Horstmann, Sarah A.; Richens, Tiffany R.; Tanaka, Takeshi; Doe, Jenna M.; Boe, Darren M.; Voelkel, Norbert F.; Taraseviciene-Stewart, Laimute; Janssen, William J.; Lee, Chun G.; Elias, Jack A.; Bratton, Donna; Tuder, Rubin M.; Henson, Peter M.; Vandivier, R. William

    2012-01-01

    Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance. PMID:22307908

  18. Biological functions of macrophage-derived Wnt5a, and its roles in human diseases

    PubMed Central

    Shao, Yue; Zheng, Qianqian; Wang, Wei; Xin, Na; Song, Xiaowen; Zhao, Chenghai

    2016-01-01

    Wnt5a is implicated in development and tissue homeostasis by activating β-catenin-independent pathway. Excessive production of Wnt5a is related to some human diseases. Macrophage recruitment is a character of inflammation and cancer, therefore macrophage-derived Wnt5a is supposed to be a player in these conditions. Actually, macrophage-derived Wnt5a maintains macrophage immune function, stimulates pro-inflammatory cytokine release, and induces angiogenesis and lymphangiogenesis. Furthermore, macrophage-derived Wnt5a is involved in insulin resistance, atherosclerosis and cancer. These findings indicate that macrophage-derived Wnt5a may be a target in the treatment of these diseases. Notably, unlike macrophages, the exact role of macrophage-derived Wnt5a in bacterial infection remains largely unknown. PMID:27608847

  19. Global Metabolomics Reveals the Metabolic Dysfunction in Ox-LDL Induced Macrophage-Derived Foam Cells

    PubMed Central

    Xu, Wenjuan; Wei, Ziyi; Dong, Jiaojiao; Duan, Feipeng; Chen, Kuikui; Chen, Chang; Liu, Jie; Yang, Xiaowei; Chen, Lianming; Xiao, Hongbin; Liu, An

    2017-01-01

    Atherosclerosis (AS) is a chronic disorder of large arteries that is a major risk factors of high morbidity and mortality. Oxidative modification LDL is one of the important contributors to atherogenesis. Macrophages take up ox-LDL and convert into foam cells, which is the hallmark of AS. To advance the understanding of the metabolic perturbation involved in ox-LDL induced macrophage-derived foam cells and discover the potential biomarkers of early AS, a global metabolomics approach was applied based on UHPLC-QTOF/MS. Multivariate statistical analyses identified five metabolites (25-azacholesterol, anandamide, glycocholate, oleoyl ethanolamide, and 3-oxo-4, 6-choladienoate) for distinguishing foamy macrophages from controls. Among the six main metabolic pathways, the unsaturated fatty acid, especially arachidonic acid metabolism, contributed importantly to early AS. A new biomarker, anandamide (AEA), whose synthesis and metabolism in macrophages are disturbed by overloaded ox-LDL, results in metabolic obstruction. This study is the first to investigate the metabolic disturbance in macrophage-derived foam cells induced by ox-LDL and screen potential biomarkers and metabolic pathways associated with early AS. Our findings provide a new insight in the underlying pathophysiological mechanisms and also help to identify novel targets for the intervention of AS. PMID:28912717

  20. In vivo effects of adding singular or combined anti-oxidative vitamins and/or minerals to diets on the immune system of tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages.

    PubMed

    Hung, Shao-Wen; Tu, Ching-Yu; Wang, Way-Shyan

    2007-01-15

    Macrophage function is an important factor for resistance to infection and anti-oxidative vitamins and minerals can affect how macrophages function in fish. We report the in vivo effect of adding singular or combined vitamins (A, C, and E) and/or minerals (Se, Zn, Cu, Mn, and Fe) in diets on the immune system of tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages. An optimal dose of vitamins and/or minerals in diets increased macrophage proliferation and protective activity, maintained macrophage viability, increased body weight and length, and increased lysozyme activity, however, at improper doses and combinations of vitamins or minerals a decrease was observed. Furthermore, vitamins and/or minerals at any doses and combinations in diets decreased superoxide and nitric oxide production. Therefore, appropriate doses and combinations of vitamins and/or minerals in diets may increase tilapia macrophages immunity.

  1. Tumor necrosis factor alpha acts as an autocrine second signal with gamma interferon to induce nitric oxide in group B streptococcus-treated macrophages.

    PubMed Central

    Goodrum, K J; Dierksheide, J; Yoder, B J

    1995-01-01

    Nitric oxide production by mouse macrophages treated with group B streptococci and gamma interferon was inhibited by cytochalasin B or by antibody neutralization of macrophage-derived tumor necrosis factor alpha. Phagocytosis-induced tumor necrosis factor alpha is responsible for group B streptococcus-induced nitric oxide production in interferon-treated macrophages. PMID:7642312

  2. Growth of Mycobacterium lepraemurium in nonstimulated and stimulated mouse peritoneal-derived and bone marrrow-derived macrophages in vitro.

    PubMed Central

    Alexander, J; Smith, C C

    1978-01-01

    Mycobacterium lepraemurium cells were found to multiply in normal mouse peritoneal-derived and bone marrow-derived macrophages in vitro. Whereas activated peritoneal-derived macrophages demonstrated marked bacteriostasis for M. lepraemurium, significant bactericidal activity was exhibited by activated bone marrow-derived macrophages. However, only a small proportion of the bacterial were killed by activated bone marrow-derived macrophages with subsequent and enhanced bacteria growth. It is suggested that a rapid turnover of monocytes in active lesions is required to control mycobacterial infections in vivo. These results would suggest that careful consideration be given to the choice of the host cell in studies involving obligate intracellular parasites. PMID:365762

  3. Functional characterization of the turkey macrophage migration inhibitory factor

    USDA-ARS?s Scientific Manuscript database

    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...

  4. 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.

  5. Human monocyte-derived macrophages spontaneously differentiated in vitro show distinct phenotypes.

    PubMed

    Eligini, Sonia; Crisci, Mauro; Bono, Elisa; Songia, Paola; Tremoli, Elena; Colombo, Gualtiero I; Colli, Susanna

    2013-07-01

    Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co-exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round- and spindle-shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte-derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)-10, and transforming growth factor (TGF) β2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C-C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non-inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro-inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions. Copyright © 2012 Wiley Periodicals, Inc.

  6. 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.

  7. Neurons promote macrophage proliferation by producing transforming growth factor-beta2.

    PubMed

    Dobbertin, A; Schmid, P; Gelman, M; Glowinski, J; Mallat, M

    1997-07-15

    The infiltration of bone marrow-derived macrophages into the CNS contributes to growth and reactions of microglia during development or after brain injury. The proliferation of microglial cells is stimulated by colony-stimulating factor 1 (CSF-1), an astrocyte-produced growth factor that acts on mononuclear phagocytes. In the present study, we have shown, using an in vitro model system, that rodent neurons obtained from the developing cerebral cortex produce a soluble factor that strongly enhances the proliferation of macrophages cultured in the presence of CSF-1. Both macrophages isolated from the developing brain and those from the adult bone marrow were stimulated. Kinetic analyses of [3H]thymidine incorporation into macrophages indicated that their response to the neuron-derived factor involved a shortening of the cycle of proliferating cells. The effect of neurons on macrophages was blocked in the presence of antibodies neutralizing transforming growth factor-beta2 (TGF-beta2), whereas recombinant TGF-beta2 stimulated macrophage proliferation in the presence of CSF-1. Neuronal secretion of TGF-beta2 was confirmed by reverse transcription-PCR detection of TGF-beta2 transcripts and immunodetection of the protein within neurons and in their culture medium. In situ hybridization and immunohistochemical experiments showed neuronal expression of TGF-beta2 in sections of cerebral cortex obtained from 6-d-old rats, an age at which extensive developmental recruitment of macrophages occurs in this cerebral region. Altogether, our results provide direct evidence that neurons have the capacity to promote brain macrophage proliferation and demonstrate the role of TGF-beta2 in this neuronal function.

  8. Oral Colostrum Macrophage-activating Factor for Serious Infection and Chronic Fatigue Syndrome: Three Case Reports.

    PubMed

    Inui, Toshio; Kubo, Kentaro; Kuchiike, Daisuke; Uto, Yoshihiro; Nishikata, Takahito; Sakamoto, Norihiro; Mette, Martin

    2015-08-01

    Gc protein-derived macrophage-activating factor (GcMAF) immunotherapy has been steadily advancing over the last two decades. Oral colostrum macrophage-activating factor (MAF) produced from bovine colostrum has shown high macrophage phagocytic activity. GcMAF-based immunotherapy has a wide application for use in treating many diseases via macrophage activation or for use as supportive therapy. Three case studies demonstrate that oral colostrum MAF can be used for serious infection and chronic fatigue syndrome (CFS) without adverse effects. We demonstrate that colostrum MAF shows promising clinical results in patients with infectious diseases and for symptoms of fatigue, which is common in many chronic diseases. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    PubMed Central

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

    2015-01-01

    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+CX3CR1hi 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

  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. Copyright © 2015 the American Physiological Society.

  11. Vitamin C suppresses lipopolysaccharide-induced procoagulant response of human monocyte-derived macrophages.

    PubMed

    Parahuleva, M S; Jung, J; Burgazli, M; Erdogan, A; Parviz, B; Hölschermann, H

    2016-05-01

    Although vitamin C is a strong antioxidant, the epidemiologic evidence to support its role in lowering risk of cardiovascular disease is inconsistent. In order to define the role of vitamin C in vascular pathophysiology, we have investigated the effect of vitamin C on the tissue factor (TF) and Factor VII Activating Protease (FSAP) expression induced by lipopolysaccharide (LPS) in human monocyte-derived macrophages. Vitamin C at clinically relevant doses was tested to its ability to influence the LPS- and reactive oxygen species (ROS) - generating system of xanthine/xanthine oxidase (X/XO) NF-kB activity in human monocyte-derived macrophages. Vitamin C-treatment prevents LPS- and ROS-induced DNA-binding activity of NF-kB in a concentration-dependent fashion. Vitamin C also inhibited the phosphorylation and proteolytic degradation of the inhibitor protein IkBa. In parallel to regulate NF-kB activity, vitamin C reduced the expression of TF and FSAP, genes known to be induced by bacterial LPS and triggered the extrinsic coagulation cascade and linked thrombosis with inflammation. Vitamin C alters pro-inflammatory and pro-coagulatory processes via inhibition of NF-kB activation and exerts beneficial antiatherogenic effects on human monocyte-derived macrophages in addition to its anti-oxidant properties.

  12. Elevation of macrophage-derived chemokine in eosinophilic pneumonia: a role of alveolar macrophages.

    PubMed

    Manabe, Kazuyoshi; Nishioka, Yasuhiko; Kishi, Jun; Inayama, Mami; Aono, Yoshinori; Nakamura, Yoichi; Ogushi, Fumitaka; Bando, Hiroyasu; Tani, Kenji; Sone, Saburo

    2005-02-01

    Macrophage-derived chemokine (MDC/CCL22) and thymus-and activation-regulated chemokine (TARC/CCL17) are ligands for CC chemokine receptor 4. Recently, TARC has been reported to play a role in the pathogenesis of idiopathic eosinophilic pneumonia (IEP). The purpose of this study was to evaluate the role of MDC in IEP and other interstitial lung diseases (ILDs). MDC and TARC in the bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay in patients with ILDs and healthy volunteers (HV). We also examined the expression of MDC mRNA in alveolar macrophages (AM) by real-time quantitative reverse transcriptase-polymerase chain reaction. Both MDC and TARC were detected only in BALF obtained from IEP patients. The concentration of MDC was higher than that of TARC in all cases. The level of MDC in IEP correlated with that of TARC. AM from IEP patients expressed a significantly higher amount of MDC than that from HV at the levels of protein and mRNA. MDC in BALF from IEP dramatically decreased when patients achieved remission. These findings suggest that MDC, in addition to TARC, might be involved in the pathogenesis of IEP, and AM play a role in the elevation of MDC in IEP.

  13. Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration.

    PubMed

    Varga, Tamas; Mounier, Rémi; Patsalos, Andreas; Gogolák, Péter; Peloquin, Matthew; Horvath, Attila; Pap, Attila; Daniel, Bence; Nagy, Gergely; Pintye, Eva; Póliska, Szilárd; Cuvellier, Sylvain; Larbi, Sabrina Ben; Sansbury, Brian E; Spite, Matthew; Brown, Chester W; Chazaud, Bénédicte; Nagy, Laszlo

    2016-11-15

    Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Effects of Two Fullerene Derivatives on Monocytes and Macrophages.

    PubMed

    Pacor, Sabrina; Grillo, Alberto; Đorđević, Luka; Zorzet, Sonia; Lucafò, Marianna; Da Ros, Tatiana; Prato, Maurizio; Sava, Gianni

    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.

  15. 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

  16. Lipid profiling of polarized human monocyte-derived macrophages.

    PubMed

    Montenegro-Burke, J Rafael; Sutton, Jessica A; Rogers, Lisa M; Milne, Ginger L; McLean, John A; Aronoff, David M

    2016-12-01

    The highly orchestrated transcriptional and metabolic reprogramming during activation drastically transforms the main functions and physiology of human macrophages across the polarization spectrum. Lipids, for example, can modify protein function by acting remotely as signaling molecules but also locally by altering the physical properties of cellular membranes. These changes play key roles in the functions of highly plastic immune cells due to their involvement in inflammation, immune responses, phagocytosis and wound healing processes. We report an analysis of major membrane lipids of distinct phenotypes of resting (M0), classically activated (M1), alternatively activated (M2a) and deactivated (M2c) human monocyte derived macrophages from different donors. Samples were subjected to supercritical fluid chromatography-ion mobility-mass spectrometry analysis, which allowed separations based on lipid class, facilitating the profiling of their fatty acid composition. Different levels of arachidonic acid mobilization as well as other fatty acid changes were observed for different lipid classes in the distinct polarization phenotypes, suggesting the activation of highly orchestrated and specific enzymatic processes in the biosynthesis of lipid signaling molecules and cell membrane remodeling. Thromboxane A2 production appeared to be a specific marker of M1 polarization. These alterations to the global composition of lipid bi-layer membranes in the cell provide a potential methodology for the definition and determination of cellular and tissue activation states.

  17. T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology.

    PubMed

    Baliram, R; Latif, R; Morshed, S A; Zaidi, M; Davies, T F

    2016-09-01

    TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

  18. Primed Activation of Macrophages by Oral Administration of Lipopolysaccharide Derived from Pantoea agglomerans.

    PubMed

    Inagawa, Hiroyuki; Kobayashi, Yutaro; Kohchi, Chie; Zhang, Ran; Shibasaki, Yasuhiro; Soma, Gen-Ichiro

    2016-01-01

    Bacterial lipopolysaccharide (LPS) is involved in the activation of the innate immune responses on monocytes/macrophages in vitro, and by intravenous injection. Although small quantities of LPS are usually found in traditional Chinese medicines, vegetables and fruits, the mode of action of orally administered LPS is still unclear. LPS derived from Pantoea agglomerans (LPSp) was orally administered to C3H/HeN or C3H/HeJ mice ad libitum. The LPSp treatment enhanced phagocytosis by resident peritoneal macrophages of C3H/HeN mice but not of C3H/HeJ mice. This activation can be defined as primed activation because no augmentation of inflammatory cytokines production was detected. LPSp in peritoneal fluid was detected and successfully quantified. Moreover, the LPSp reduced the expression of avian reticuloendotheliosis viral oncogene-related B (RelB) in the macrophages without degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα). Orally administered LPSp can reach the peritoneum, and enhance phagocytosis via Toll-like receptor 4 signaling pathway in resident peritoneal macrophages. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. Macrophage fusion factor elicited from BGG-sensitized lymphocytes.

    PubMed Central

    Warfel, A. H.; Hadden, J. W.

    1978-01-01

    Lymphocytes obtained from rabbit lymph nodes sensitized to bovine gamma globulin produce in vitro the lymphokine macrophage fusion factor (MFF) which mediates the fusion of approximately 100% of normal alveolar and oil-induced peritoneal macrophages. Giant cells (GC) of Langhans and foreign body type form large syncytia containing as many as several hundred nuclei per cell. Nuclei of GC appear more spherical and larger than those of the normal mononucleated macrophages, and they possess several prominent nucleoli. Giant cells of peritoneal macrophage origin show enhanced intracytoplasmic vacuolization. Normal macrophages cultured as a monolayer in MFF-rich supernatants form cell clusters which progressively fuse during the 24-hour incubation period. A signoid dose-response curve was obtained for cell fusion with MFF-rich supernatants possessing high titers, ie, the latter supernatants undiluted partially inhibited macrophage fusion. MIF-like activity was detected in MFF-rich supernatants as well as a factor(s) which inhibited 3H-thymidine uptake by giant cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:362945

  20. Shear stress is required for the endocytic uptake of the factor VIII-von Willebrand factor complex by macrophages.

    PubMed

    Castro-Núñez, L; Dienava-Verdoold, I; Herczenik, E; Mertens, K; Meijer, A B

    2012-09-01

    Low-density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)-von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte-derived macrophages. A confocal microscopy study was employed to study the uptake by monocyte-derived macrophages of a functional green fluorescent FVIII-GFP derivative in the presence and absence of VWF. The results revealed that FVIII-GFP is internalized by macrophages. We found that FVIII-GFP co-localizes with LDL receptor-related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII-GFP. However, FVIII-GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII-VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII-GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII-GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII-GFP and VWF was inhibited. The results show that shear stress is required for macrophages to internalize both constituents of the FVIII-VWF complex. © 2012 International Society on Thrombosis and Haemostasis.

  1. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. CXCL4 induces a unique transcriptome in monocyte-derived macrophages

    PubMed Central

    Gleissner, Christian A.; Shaked, Iftach; Little, Kristina M.; Ley, Klaus

    2012-01-01

    In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors like macrophage colony-stimulation factor (MCSF) and chemokines like platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with MCSF-induced macrophages or macrophages polarized with IFN-γ/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for six days. mRNA expression was measured by Affymetrix gene chips and differences were analyzed by Local Pooled Error test, Profile of Complex Functionality and Gene Set Enrichment Analysis. 375 genes were differentially expressed between MCSF- and CXCL4-induced macrophages, 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, antigen processing/presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level, however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently up- or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters higher expression in CXCL4- than MCSF-induced macrophages, resulting in lower LDL content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4. PMID:20335529

  3. 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.

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

    PubMed Central

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

    2015-01-01

    Summary 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

  5. 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

    2015-07-08

    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.

  6. Macrophage colony-stimulating factor induces indirect angiogenesis in vivo.

    PubMed

    Phillips, G D; Aukerman, S L; Whitehead, R A; Knighton, D R

    1993-01-01

    The cytokine macrophage colony-stimulating factor was implanted in the rabbit cornea over a wide dose range (1 ng to 100 microg) to assay its angiogenic activity in vivo. Neovascularization occurred in a dose-dependent manner, and maximum angiogenesis occurred only with 100 microg. Histologic analysis revealed that the corneas were free of inflammation at the lower doses, but had slight inflammation at 50 and 100 microg. Nonspecific esterase staining of frozen sections and transmission electron microscopy revealed that the inflammatory cells were predominantly macrophages, with very few neutrophils present. This association of capillary formation with inflammation suggests an indirect mechanism of angiogenesis. The lack of neutrophils within the inflammatory cell infiltrate demonstrates that indirect angiogenesis can proceed without the local presence of neutrophils. This distinguishes macrophage colony-stimulating factor from other indirect-acting angiogenesis factors that have been identified to date.

  7. Effects of periodontal therapy on serum C-reactive protein, sE-selectin, and tumor necrosis factor-alpha secretion by peripheral blood-derived macrophages in diabetes. A pilot study.

    PubMed

    Lalla, E; Kaplan, S; Yang, J; Roth, G A; Papapanou, P N; Greenberg, S

    2007-06-01

    Diabetes is associated with an increased risk for vascular disease and periodontitis. The aim of this study was to assess the effects of periodontal treatment in diabetes with respect to alterations in the pro-inflammatory potential of peripheral blood mononuclear cells. Ten patients with diabetes and moderate to severe periodontitis received full-mouth subgingival debridement. Blood samples for serum/plasma and mononuclear cell isolation were collected prior to and 4 wk after therapy. Mononuclear cells were analyzed by flow cytometry and stimulated with lipopolysaccharide or ionomycin/phorbol ester to determine the pro-inflammatory capacity of macrophages and lymphocytes, respectively. Following periodontal treatment, all patients demonstrated a significant improvement in clinical periodontal status (p < 0.05), despite only modest reduction in subgingival bacterial load or homologous serum immunoglobulin G titers. CD14(+) blood monocytes decreased by 47% (p < 0.05), and the percentage of macrophages spontaneously releasing tumor necrosis factor-alpha decreased by 78% (p < 0.05). There were no significant changes in the capacity of lymphocytes to secrete interferon-gamma. Among a number of serum inflammatory markers tested, high-sensitivity-C-reactive protein significantly decreased by 37% (p < 0.01) and soluble E-selectin decreased by 16.6% (p < 0.05). These data suggest a reduced tendency for monocyte/macrophage-driven inflammation with periodontal therapy and a potential impact on atherosclerosis-related complications in diabetic individuals.

  8. TNF-alpha, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O:3 derivatives.

    PubMed

    Carlos, Iracilda Zeppone; Silva Monnazzi, Luis Gustavo; Falcão, Deise Pasetto; Machado de Medeiros, Beatriz Maria

    2004-02-01

    In this study, the effect of Yersinia derivatives on nitric oxide (NO), hydrogen peroxide (H2O2) and tumor necrosis factor-alpha (TNF-alpha) production by murine peritoneal macrophages was investigated. Addition of lipopolysaccharide (LPS) to the macrophage culture resulted in NO production that was dose dependent. On the other hand, bacterial cellular extract (CE) and Yersinia outer proteins (Yops) had no effect on NO production. The possible inhibitory effect of Yops on macrophage cultures stimulated with LPS was investigated. Yops partially inhibited NO production (67.4%) when compared with aminoguanidine. The effects of Yersinia derivatives on H2O2 production by macrophages were similar to those on NO production. LPS was the only derivative that stimulated H2O2 release in a dose-dependent manner. All Yersinia derivatives provoked the production of TNF-alpha, but LPS had the strongest effect, as observed for NO production. CE and Yops stimulated TNF-alpha production to a lesser extent than LPS. The results indicate the possibility that in vivo Yops may aid the evasion of the bacteria from the host defense mechanism by impairing the secretion of NO by macrophages.

  9. Chemically-modified polysaccharide extract derived from Leucaena leucocephala alters Raw 264.7 murine macrophage functions.

    PubMed

    Gamal-Eldeen, Amira M; Amer, Hassan; Helmy, Wafaa A; Talaat, Roba M; Ragab, Halla

    2007-06-01

    In this study, a chemical modification of the polysaccharides extract (E) derived from Leucaena leucocephala seeds was performed to prepare C-glycosidic 2-propanol derivative (PE), and its sulphated derivative (SPE). This study aimed to characterize immunomodulatory activities of the original extract and its derivatives by exploring their effects on Raw macrophage 264.7 functions and their antioxidant activity. Our results indicated that PE was an effective radical scavenger to hydroxyl, peroxyl, and superoxide anion radicals, and SPE was a peroxyl radical scavenger. PE and SPE were found to influence the macrophage functions. Both of PE and SPE enhanced the macrophage proliferation and phagocytosis of FITC-zymosan; PE inhibited nitric oxide (NO) generation and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide (LPS)-stimulated Raw macrophage 264.7. In contrast, SPE over-induced NO generation and TNF-alpha secretion. Moreover, PE strongly inhibited the binding affinity of FITC-LPS to Raw 264.7, as indicated by flow cytometry analysis. These findings revealed that PE may act as a potent anti-inflammatory agent; however SPE may act as an inducer of macrophage functions against pathogens.

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

    PubMed

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

    2011-05-12

    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-1(op/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.

  11. Tissue-resident versus monocyte-derived macrophages in the tumor microenvironment.

    PubMed

    Lahmar, Qods; Keirsse, Jiri; Laoui, Damya; Movahedi, Kiavash; Van Overmeire, Eva; Van Ginderachter, Jo A

    2016-01-01

    The tumor-promoting role of macrophages has been firmly established in most cancer types. However, macrophage identity has been a matter of debate, since several levels of complexity result in considerable macrophage heterogeneity. Ontogenically, tissue-resident macrophages derive from yolk sac progenitors which either directly or via a fetal liver monocyte intermediate differentiate into distinct macrophage types during embryogenesis and are maintained throughout life, while a disruption of the steady state mobilizes monocytes and instructs the formation of monocyte-derived macrophages. Histologically, the macrophage phenotype is heavily influenced by the tissue microenvironment resulting in molecularly and functionally distinct macrophages in distinct organs. Finally, a change in the tissue microenvironment as a result of infectious or sterile inflammation instructs different modes of macrophage activation. These considerations are relevant in the context of tumors, which can be considered as sites of chronic sterile inflammation encompassing subregions with distinct environmental conditions (for example, hypoxic versus normoxic). Here, we discuss existing evidence on the role of macrophage subpopulations in steady state tissue and primary tumors of the breast, lung, pancreas, brain and liver.

  12. Myeloid cell-derived inducible nitric oxide synthase suppresses M1 macrophage polarization.

    PubMed

    Lu, Geming; Zhang, Ruihua; Geng, Shuo; Peng, Liang; Jayaraman, Padmini; Chen, Chun; Xu, Feifong; Yang, Jianjun; Li, Qin; Zheng, Hao; Shen, Kimberly; Wang, Juan; Liu, Xiyu; Wang, Weidong; Zheng, Zihan; Qi, Chen-Feng; Si, Chuanping; He, John Cijiang; Liu, Kebin; Lira, Sergio A; Sikora, Andrew G; Li, Liwu; Xiong, Huabao

    2015-03-27

    Here we show that iNOS-deficient mice display enhanced classically activated M1 macrophage polarization without major effects on alternatively activated M2 macrophages. eNOS and nNOS mutant mice show comparable M1 macrophage polarization compared with wild-type control mice. Addition of N6-(1-iminoethyl)-L-lysine dihydrochloride, an iNOS inhibitor, significantly enhances M1 macrophage polarization while S-nitroso-N-acetylpenicillamine, a NO donor, suppresses M1 macrophage polarization. NO derived from iNOS mediates nitration of tyrosine residues in IRF5 protein, leading to the suppression of IRF5-targeted M1 macrophage signature gene activation. Computational analyses corroborate a circuit that fine-tunes the expression of IL-12 by iNOS in macrophages, potentially enabling versatile responses based on changing microenvironments. Finally, studies of an experimental model of endotoxin shock show that iNOS deficiency results in more severe inflammation with an enhanced M1 macrophage activation phenotype. These results suggest that NO derived from iNOS in activated macrophages suppresses M1 macrophage polarization.

  13. Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages.

    PubMed

    Singh, Mahavir; Kapoor, Aniruddh; McCracken, James; Hill, Bradford; Bhatnagar, Aruni

    2017-03-01

    Macrophages are critical drivers of the immune response during infection and inflammation. The pathogenesis of several inflammatory conditions, such as diabetes, cancer and sepsis has been linked with aldose reductase (AR), a member of the aldo-keto reductase (AKR) superfamily. However, the role of AR in the early stages of innate immunity such as phagocytosis remains unclear. In this study, we examined the role of AR in regulating the growth and the phagocytic activity of bone marrow-derived mouse macrophages (BMMs) from AR-null and wild-type (WT) mice. We found that macrophages derived from AR-null mice were larger in size and had a slower growth rate than those derived from WT mice. The AR-null macrophages also displayed higher basal, and lipopolysaccharide (LPS) stimulated phagocytic activity than WT macrophages. Moreover, absence of AR led to a marked increase in cellular levels of both ATP and NADPH. These data suggest that metabolic pathways involving AR suppress macrophage energy production, and that inhibition of AR could induce a favorable metabolic state that promotes macrophage phagocytosis. Hence, modulation of macrophage metabolism by inhibition of AR might represent a novel strategy to modulate host defense responses and to modify metabolism to promote macrophage hypertrophy and phagocytosis under inflammatory conditions.

  14. Macrophage migration inhibitory factor induces phagocytosis of foreign particles by macrophages in autocrine and paracrine fashion.

    PubMed Central

    Onodera, S; Suzuki, K; Matsuno, T; Kaneda, K; Takagi, M; Nishihira, J

    1997-01-01

    Exposure to foreign particles sometimes causes inflammatory reactions through production of cytokines and chemoattractants by phagocytic cells. In this study, we focused on macrophage migration inhibitory factor (MIF) to evaluate its pathophysiological role in the phagocytic process. Immunohistochemical analysis of human pseudosynovial tissues retrieved at revision of total hip arthroplasty showed that infiltrating mononuclear and multinuclear cells were positively stained by both an anti-CD68 antibody and anti-human MIF antibody. For in vitro study, MIF was released from murine macrophage-like cells (RAW 264.7) in response to phagocytosis of fluorescent-latex beads in a particle dose-dependent manner. Northern blot analysis showed marked elevation of the MIF mRNA level in the phagocytic macrophage-like cells. Moreover, pretreatment of RAW 264.7 cells with rat recombinant MIF increased the extent of phagocytosis by 1.6-fold compared with the control. Taken together, these results suggest that MIF plays an important role by activating macrophages in autocrine and paracrine fashion to phagocytose foreign particles. Images Figure 1 Figure 3 Figure 5 PMID:9370935

  15. Characterization of Neospora caninum macrophage migration inhibitory factor

    USDA-ARS?s Scientific Manuscript database

    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...

  16. 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

  17. Alcohol Enhances HIV Infection of Cord Blood Monocyte-Derived Macrophages

    PubMed Central

    Mastrogiannis, Dimitrios S.; Wang, Xu; Dai, Min; Li, Jieliang; Wang, Yizhong; Zhou, Yu; Sakarcan, Selin; Peña, Juliet Crystal; Ho, Wenzhe

    2014-01-01

    Alcohol consumption or alcohol abuse is common among pregnant HIV+ women and has been identified as a potential behavioral risk factor for the transmission of HIV. In this study, we examined the impact of alcohol on HIV infection of cord blood monocyte-derived macrophages (CBMDM). We demonstrated that alcohol treatment of CBMDM significantly enhanced HIV infection of CBMDM. Investigation of the mechanisms of alcohol action on HIV demonstrated that alcohol inhibited the expression of several HIV restriction factors, including anti-HIV microRNAs, APOBEC3G and APOBEC3H. Additionally, alcohol also suppressed the expression of IFN regulatory factor 7 (IRF-7) and retinoic acid-inducible gene I (RIG-I), an intracellular sensor of viral infection. The suppression of these IFN regulatory factors was associated with reduced expression of type I IFN. These experimental findings suggest that maternal alcohol consumption may facilitate HIV infection, promoting vertical transmission of HIV. PMID:25053361

  18. 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.

  19. 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.

  20. Macrophage colony stimulating factor: not just for macrophages anymore! A gateway into complex biologies.

    PubMed

    Douglass, Thomas G; Driggers, Lara; Zhang, Jian Gang; Hoa, Neil; Delgado, Christina; Williams, Christopher C; Dan, Qinhong; Sanchez, Ramon; Jeffes, Edward W B; Wepsic, H Terry; Myers, Michael P; Koths, Kirston; Jadus, Martin R

    2008-10-01

    Macrophage colony stimulating factor (M-CSF, also called colony stimulating factor-1) has traditionally been viewed as a growth/differentiation factor for monocytes, macrophages, and some female-specific tumors. As a result of alternative mRNA splicing and post-translational processing, several forms of M-CSF protein are produced: a secreted glycoprotein, a longer secreted form containing proteoglycan, and a short membrane-bound isoform. These different forms of M-CSF all initiate cell signaling in cells bearing the M-CSF receptor, called c-fms. Here we review the biology of M-CSF, which has important roles in bone physiology, the intestinal tract, cancer metastases to the bone, macrophage-mediated tumor cell killing and tumor immunity. Although this review concentrates mostly on the membrane form of human M-CSF (mM-CSF), the biology of the soluble forms and the M-CSF receptor will also be discussed for comparative purposes. The mechanisms of the biological effects of the membrane-bound M-CSF reveal that this cytokine is unexpectedly involved in many complex molecular events. Recent experiments suggest that a tumor vaccine based on membrane-bound M-CSF-transduced tumor cells, combined with anti-angiogenic therapy, should be evaluated further for use in clinical trials.

  1. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid.

    PubMed

    Colegio, Oscar R; Chu, Ngoc-Quynh; Szabo, Alison L; Chu, Thach; Rhebergen, Anne Marie; Jairam, Vikram; Cyrus, Nika; Brokowski, Carolyn E; Eisenbarth, Stephanie C; Phillips, Gillian M; Cline, Gary W; Phillips, Andrew J; Medzhitov, Ruslan

    2014-09-25

    Macrophages have an important role in the maintenance of tissue homeostasis. To perform this function, macrophages must have the capacity to monitor the functional states of their 'client cells': namely, the parenchymal cells in the various tissues in which macrophages reside. Tumours exhibit many features of abnormally developed organs, including tissue architecture and cellular composition. Similarly to macrophages in normal tissues and organs, macrophages in tumours (tumour-associated macrophages) perform some key homeostatic functions that allow tumour maintenance and growth. However, the signals involved in communication between tumours and macrophages are poorly defined. Here we show that lactic acid produced by tumour cells, as a by-product of aerobic or anaerobic glycolysis, has a critical function in signalling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumour-associated macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumour growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumour cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumours to promote their growth.

  2. Promoting macrophage survival delays progression of pre-existing atherosclerotic lesions through macrophage-derived apoE.

    PubMed

    Bouchareychas, Laura; Pirault, John; Saint-Charles, Flora; Deswaerte, Virginie; Le Roy, Tiphaine; Jessup, Wendy; Giral, Philippe; Le Goff, Wilfried; Huby, Thierry; Gautier, Emmanuel L; Lesnik, Philippe

    2015-10-01

    Macrophage apoptosis is a prominent feature of atherosclerosis, yet whether cell death-protected macrophages would favour the resolution of already established atherosclerotic lesions, and thus hold therapeutic potential, remains unknown. We irradiated then transplanted into Apoe(-/-) or LDLr(-/-) recipient mice harbouring established atherosclerotic lesions, bone marrow cells from mice displaying enhanced macrophage survival through overexpression of the antiapoptotic gene hBcl-2 (Mø-hBcl2 Apoe(-/-) or Mø-hBcl2 Apoe(+/+) LDLr(-/-)). Both recipient mice exhibited decreased lesional apoptotic cell content and reduced necrotic areas when repopulated with Mø-hBcl2 mouse-derived bone marrow cells. In contrast, only LDLr(-/-) recipients showed a reduction in plasma cholesterol levels and in atherosclerotic lesions. The absence of significant reduction of plasma cholesterol levels in the context of apoE deficiency highlighted macrophage-derived apoE as key in both the regulation of plasma and tissue cholesterol levels and the progression of pre-existing lesion. Accordingly, hBcl2 expression in macrophages was associated with larger pools of Kupffer cells and Ly-6C(low) monocytes, both high producers of apoE. Additionally, increased Kupffer cells population was associated with improved clearance of apoptotic cells and modified lipoproteins. Collectively, these data show that promoting macrophage survival provides a supplemental source of apoE, which hinders pre-existing plaque progression. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  3. Differential gene expression in human, murine, and cell line-derived macrophages upon polarization.

    PubMed

    Spiller, Kara L; Wrona, Emily A; Romero-Torres, Saly; Pallotta, Isabella; Graney, Pamela L; Witherel, Claire E; Panicker, Leelamma M; Feldman, Ricardo A; Urbanska, Aleksandra M; Santambrogio, Laura; Vunjak-Novakovic, Gordana; Freytes, Donald O

    2016-09-10

    The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP-1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages

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

    PubMed Central

    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; Unanue, Emil R.; Colonna, Marco; Randolph, Gwendalyn J.; Mann, Douglas L.

    2014-01-01

    Summary Cardiac macrophages are crucial for tissue repair after cardiac injury but have not been 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, Ly6chi 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. PMID:24439267

  5. 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

  6. The macrophage migration inhibitory factor homolog of Entamoeba histolytica binds to and immunomodulates host macrophages.

    PubMed

    Moonah, Shannon N; Abhyankar, Mayuresh M; Haque, Rashidul; Petri, William A

    2014-09-01

    The host inflammatory response contributes to the tissue damage that occurs during amebic colitis, with tumor necrosis factor alpha (TNF-α) being a key mediator of the gut inflammation observed. Mammalian macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the exacerbation of a wide range of inflammatory diseases, including colitis. We identified a MIF gene homolog in the Entamoeba histolytica genome, raising the question of whether E. histolytica MIF (EhMIF) has proinflammatory activity similar to that of mammalian MIF. In this report, we describe the first functional characterization of EhMIF. Antibodies were prepared against recombinantly expressed EhMIF and used to demonstrate that EhMIF is expressed as a 12-kDa protein localized to the cytoplasm of trophozoites. In a manner similar to that of mammalian MIF, EhMIF interacted with the MIF receptor CD74 and bound to macrophages. EhMIF induced interleukin-6 (IL-6) production. In addition, EhMIF enhanced TNF-α secretion by amplifying TNF-α production by lipopolysaccharide (LPS)-stimulated macrophages and by inhibiting the glucocorticoid-mediated suppression of TNF-α secretion. EhMIF was expressed during human infection, as evidenced by the presence of anti-EhMIF antibodies in the sera of children living in an area where E. histolytica infection is endemic. Anti-EhMIF antibodies did not cross-react with human MIF. The ability of EhMIF to modulate host macrophage function may promote an exaggerated proinflammatory immune response and contribute to the tissue damage seen in amebic colitis.

  7. 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

  8. Human monocyte-derived macrophages are heterogenous: Proteomic profile of different phenotypes.

    PubMed

    Eligini, S; Brioschi, M; Fiorelli, S; Tremoli, E; Banfi, C; Colli, S

    2015-06-21

    Tissue macrophages play a key role in many aspects of human physiology and pathology. These cells are heterogeneous both in term of morphology and function. As an example, heterogeneity has been reported within the atherosclerotic lesions where distinct populations exert opposite functions driving plaque progression or stability. Tissue macrophages are not easily obtained and differentiated blood-derived monocytes are largely used as surrogate model. We previously reported that human macrophages spontaneously differentiated from adherent monocytes show two dominant subsets, distinct for morphology (spindle and round) and functions. The aim of this study was to evaluate the intracellular proteome of these two macrophage subsets by means of a microproteomic workflow properly set up to simultaneously identify and quantify proteins from a minimal number of morphotypically heterogeneous cells in culture. We report two distinct proteomic profiles that distinguish round from spindle macrophages. In particular, differential abundances were observed for proteins involved in membrane traffic regulation, lipid handling, efferocytosis, and protection against stress conditions. Results reinforce and extend previous data on the functional and antigenic profile of these macrophage phenotypes strengthening the suitability of our model to focus on macrophage heterogeneity. Tissue macrophages patrol homeostatic functions, immune surveillance, and resolution of inflammation. The spectrum of macrophage activation states is, therefore, wide and gives ground for the heterogeneity of these cells, documented in health and disease. This study provides knowledge of the distinct proteome that characterises the two dominant morphotypes (round and spindle) of human macrophages that, in our culture condition, are generated by spontaneous differentiation from blood-derived monocytes. Results extend previous data about the different antigenic, transcriptional, and functional profiles of these

  9. Embryonic stem cell-derived M2-like macrophages delay cutaneous wound healing.

    PubMed

    Dreymueller, Daniela; Denecke, Bernd; Ludwig, Andreas; Jahnen-Dechent, Willi

    2013-01-01

    In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar-free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2-like macrophages. Embryonic/fetal macrophages are M2-like, and this may promote scar-free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell-derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow-derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2-like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll-like receptors, and reduced bacterial phagocytosis. Despite this anti-inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell-free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds. © 2012 by the Wound Healing Society.

  10. 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.

  11. 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

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

    PubMed

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

    2014-08-01

    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 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 lipopolysaccharide-stimulated cells were sufficient to cause nuclear factor-κB activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage-derived exosomes. Macrophage-derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. 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.

  13. Adsorbed fibrinogen enhances production of bone- and angiogenic-related factors by monocytes/macrophages.

    PubMed

    Maciel, Joana; Oliveira, Marta I; Colton, Erica; McNally, Amy K; Oliveira, Carla; Anderson, James M; Barbosa, Mário A

    2014-01-01

    Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since protein adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for 10 days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory protein-1delta (MIP-1δ), platelet-derived growth factor-BB, bone morphogenetic protein (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in bone homeostasis and wound healing, such as growth hormone, transforming growth factor-β3, and insulin-like growth factor

  14. Adsorbed Fibrinogen Enhances Production of Bone- and Angiogenic-Related Factors by Monocytes/Macrophages

    PubMed Central

    Maciel, Joana; Oliveira, Marta I.; Colton, Erica; McNally, Amy K.; Oliveira, Carla; Anderson, James M.

    2014-01-01

    Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since protein adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for 10 days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory protein-1delta (MIP-1δ), platelet-derived growth factor-BB, bone morphogenetic protein (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in bone homeostasis and wound healing, such as growth hormone, transforming growth factor-β3, and insulin-like growth factor

  15. Tumor-Infiltrating Macrophages in Post-Transplant, Relapsed Classical Hodgkin Lymphoma Are Donor-Derived.

    PubMed

    Crane, Genevieve M; Samols, Mark A; Morsberger, Laura A; Yonescu, Raluca; Thiess, Michele L; Batista, Denise A S; Ning, Yi; Burns, Kathleen H; Vuica-Ross, Milena; Borowitz, Michael J; Gocke, Christopher D; Ambinder, Richard F; Duffield, Amy S

    Tumor-associated inflammatory cells in classical Hodgkin lymphoma (CHL) typically outnumber the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. The composition of the inflammatory infiltrate, particularly the fraction of macrophages, has been associated with clinical behavior. Emerging work from animal models demonstrates that most tissue macrophages are maintained by a process of self-renewal under physiologic circumstances and certain inflammatory states, but the contribution from circulating monocytes may be increased in some disease states. This raises the question of the source of macrophages involved in human disease, particularly that of CHL. Patients with relapsed CHL following allogeneic bone marrow transplant (BMT) provide a unique opportunity to begin to address this issue. We identified 4 such patients in our archives. Through molecular chimerism and/or XY FISH studies, we demonstrated the DNA content in the post-BMT recurrent CHL was predominantly donor-derived, while the H/RS cells were derived from the patient. Where possible to evaluate, the cellular composition of the inflammatory infiltrate, including the percentage of macrophages, was similar to that of the original tumor. Our findings suggest that the H/RS cells themselves define the inflammatory environment. In addition, our results demonstrate that tumor-associated macrophages in CHL are predominantly derived from circulating monocytes rather than resident tissue macrophages. Given the association between tumor microenvironment and disease progression, a better understanding of macrophage recruitment to CHL may open new strategies for therapeutic intervention.

  16. Tumor-Infiltrating Macrophages in Post-Transplant, Relapsed Classical Hodgkin Lymphoma Are Donor-Derived

    PubMed Central

    Morsberger, Laura A.; Yonescu, Raluca; Thiess, Michele L.; Batista, Denise A. S.; Ning, Yi; Burns, Kathleen H.; Vuica-Ross, Milena; Borowitz, Michael J.; Gocke, Christopher D.; Ambinder, Richard F.; Duffield, Amy S.

    2016-01-01

    Tumor-associated inflammatory cells in classical Hodgkin lymphoma (CHL) typically outnumber the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. The composition of the inflammatory infiltrate, particularly the fraction of macrophages, has been associated with clinical behavior. Emerging work from animal models demonstrates that most tissue macrophages are maintained by a process of self-renewal under physiologic circumstances and certain inflammatory states, but the contribution from circulating monocytes may be increased in some disease states. This raises the question of the source of macrophages involved in human disease, particularly that of CHL. Patients with relapsed CHL following allogeneic bone marrow transplant (BMT) provide a unique opportunity to begin to address this issue. We identified 4 such patients in our archives. Through molecular chimerism and/or XY FISH studies, we demonstrated the DNA content in the post-BMT recurrent CHL was predominantly donor-derived, while the H/RS cells were derived from the patient. Where possible to evaluate, the cellular composition of the inflammatory infiltrate, including the percentage of macrophages, was similar to that of the original tumor. Our findings suggest that the H/RS cells themselves define the inflammatory environment. In addition, our results demonstrate that tumor-associated macrophages in CHL are predominantly derived from circulating monocytes rather than resident tissue macrophages. Given the association between tumor microenvironment and disease progression, a better understanding of macrophage recruitment to CHL may open new strategies for therapeutic intervention. PMID:27685855

  17. 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.

  18. 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

  19. Schistosomal-derived lysophosphatidylcholine triggers M2 polarization of macrophages through PPARγ dependent mechanisms.

    PubMed

    Assunção, Leonardo Santos; Magalhães, Kelly G; Carneiro, Alan Brito; Molinaro, Raphael; Almeida, Patrícia E; Atella, Georgia C; Castro-Faria-Neto, Hugo C; Bozza, Patrícia T

    2017-02-01

    Mansonic schistosomiasis is a disease caused by the trematode Schistosoma mansoni, endemic to tropical countries. S. mansoni infection induces the formation of granulomas and potent polarization of Th2-type immune response. There is great interest in understanding the mechanisms used by this parasite that causes a modulation of the immune system. Recent studies from our group demonstrated that lipids of S. mansoni, including lysophosphatidylcholine (LPC) have immunomodulatory activity. In the present study, our aim was to investigate the role of lipids derived from S. mansoni in the activation and polarization of macrophages and to characterize the mechanisms involved in this process. Peritoneal macrophages obtained from wild type C57BL/6mice or bone marrow derived macrophages were stimulated in vitro with lipids extracted from adult worms of S. mansoni. We demonstrated that total schistosomal-derived lipids as well as purified LPC induced alternatively activated macrophages/M2 profile observed by increased expression of arginase-1, mannose receptor, Chi3l3, TGFβ and production of IL-10 and PGE2 24h after stimulation. The involvement of the nuclear receptor PPARγ in macrophage response against LPC was investigated. Through Western blot and immunofluorescence confocal microscopy we demonstrated that schistosomal-derived LPC induces increased expression of PPARγ in macrophages. The LPC-induced increased expression of arginase-1 were significantly inhibited by the PPAR-γ antagonist GW9662. Together, these results demonstrate an immunomodulatory role of schistosomal-derived LPC in activating macrophages to a profile of the type M2 through PPARγ-dependent mechanisms, indicating a novel pathway for macrophage polarization triggered by parasite-derived LPC with potential implications to disease pathogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Development and Functional Differentiation of Tissue-Resident Versus Monocyte-Derived Macrophages in Inflammatory Reactions.

    PubMed

    Italiani, Paola; Boraschi, Diana

    2017-01-01

    Mononuclear phagocytes are key cells in tissue integrity and defense. Tissue-resident macrophages are abundantly present in all tissues of the body and have a complex role in ensuring tissue functions and homeostatic balance. Circulating blood monocytes can enter tissue both in steady-state conditions, for helping in replenishing the tissue-resident macrophage pool and, in particular, for acting as potent effector cells during inflammatory events such as infections, traumas, and diseases. The heterogeneity of monocytes and macrophages depends on their ontogeny, their tissue location, and their functional programming, with both monocytes and macrophages able to exert distinct or similar functions depending on the tissue-specific and stimulus-specific microenvironment. In this short review, we will review the current hypotheses on tissue-resident macrophage ontogeny and functions, as compared to blood-derived monocytes, with a particular focus on inflammatory conditions.

  1. GEC-derived SFRP5 inhibits Wnt5a-induced macrophage chemotaxis and activation.

    PubMed

    Zhao, Chenghai; Bu, Xianmin; Wang, Wei; Ma, Tingxian; Ma, Haiying

    2014-01-01

    Aberrant macrophage infiltration and activation has been implicated in gastric inflammation and carcinogenesis. Overexpression of Wnt5a and downregulation of SFRP5, a Wnt5a antagonist, were both observed in gastric cancers recently. This study attempted to explore whether Wnt5a/SFRP5 axis was involved in macrophage chemotaxis and activation. It was found that both Wnt5a transfection and recombinant Wnt5a (rWnt5a) treatment upregulated CCL2 expression in macrophages, involving JNK and NFκB signals. Conditioned medium from Wnt5a-treated macrophages promoted macrophage chemotaxis mainly dependent on CCL2. SFRP5 from gastric epithelial cells (GECs) inhibited Wnt5a-induced CCL2 expression and macrophage chemotaxis. In addition, Wnt5a treatment stimulated macrophages to produce inflammatory cytokines and COX-2/PGE2, which was also suppressed by SFRP5 from GECs. These results demonstrate that Wnt5a induces macrophage chemotaxis and activation, which can be blocked by GEC-derived SFRP5, suggesting that Wnt5a overproduction and SFRP5 deficiency in gastric mucosa may together play an important role in gastric inflammation and carcinogenesis.

  2. A novel assay system for macrophage-activating factor activity using a human U937 cell line.

    PubMed

    Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

    2014-08-01

    Macrophages play important roles in antitumor immunity, and immunotherapy with the group-specific component protein-derived macrophage-activating factor (GcMAF) has been reported to be effective in patients with various types of cancers. However, in macrophage research, it is important to properly evaluate macrophage activity. U937 macrophages were induced by 12-O-tetradecanoyl-13-phorbolacetate (TPA). The phagocytic activity of macrophages was evaluated as the internalized beads ratio. The MAF activity was assessed at 30 min after MAF addition as the activation ratio. We established a novel assay for phagocytic activities using differentiated U937 macrophages. The novel protocol was simple and rapid and was sensitive for GcMAF. This protocol should be useful not only for basic studies, such as those on molecular mechanisms underlying macrophage activation, but also for clinical studies, such as assessment of GcMAF activity prior to clinical use. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types

    PubMed Central

    2011-01-01

    Background Macrophages play an important role in neuroinflammatory diseases such as multiple sclerosis (MS) and spinal cord injury (SCI), being involved in both damage and repair. The divergent effects of macrophages might be explained by their different activation status: classically activated (CA/M1), pro-inflammatory, macrophages and alternatively activated (AA/M2), growth promoting, macrophages. Little is known about the effect of macrophages with these phenotypes in the central nervous system (CNS) and how they influence pathogenesis. The aim of this study was therefore to determine the characteristics of these phenotypically different macrophages in the context of the CNS in an in vitro setting. Results Here we show that bone marrow derived CA and AA macrophages have a distinct migratory capacity towards medium conditioned by various cell types of the CNS. AA macrophages were preferentially attracted by the low weight (< 10 kD) fraction of neuronal conditioned medium, while CA macrophages were attracted in higher numbers by astrocyte- and oligodendrocyte conditioned medium. Intrinsic motility was twice as high in AA macrophages compared to CA macrophages. The adhesion to extracellular matrix molecules (ECM) was significantly enhanced in CA macrophages compared to control and AA macrophages. The actin cytoskeleton was differentially organized between CA and AA macrophages, possibly due to greater activity of the GTPases RhoA and Rac in CA macrophages. Phagocytosis of myelin and neuronal fragments was increased in CA macrophages compared to AA macrophages. The increase in myelin phagocytosis was associated with higher expression of CR3/MAC-1 in CA macrophages. Conclusion In conclusion, since AA macrophages are more motile and are attracted by NCM, they are prone to migrate towards neurons in the CNS. CA macrophages have a lower motility and a stronger adhesion to ECM. In neuroinflammatory diseases the restricted migration and motility of CA macrophages might

  4. 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

    2016-04-08

    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.

  5. Mycobacterium tuberculosis ESAT6 and CPF10 Induce Adenosine Deaminase 2 mRNA Expression in Monocyte-Derived Macrophages

    PubMed Central

    Bae, Mi Jung; Ryu, Suyeon; Kim, Ha-Jeong; Cha, Seung Ick

    2017-01-01

    Background Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Methods Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. Results CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-γ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor α and interleukin 10, respectively. Conclusion ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis. PMID:28119750

  6. Mycobacterium tuberculosis ESAT6 and CPF10 Induce Adenosine Deaminase 2 mRNA Expression in Monocyte-Derived Macrophages.

    PubMed

    Bae, Mi Jung; Ryu, Suyeon; Kim, Ha-Jeong; Cha, Seung Ick; Kim, Chang Ho; Lee, Jaehee

    2017-01-01

    Delayed hypersensitivity plays a large role in the pathogenesis of tuberculous pleural effusion (TPE). Macrophages infected with live Mycobacterium tuberculosis (MTB) increase the levels of adenosine deaminase2 (ADA2) in the pleural fluid of TPE patients. However, it is as yet unclear whether ADA2 can be produced by macrophages when challenged with MTB antigens alone. This study therefore evaluated the levels of ADA2 mRNA expression, using monocyte-derived macrophages (MDMs) stimulated with MTB antigens. Purified monocytes from the peripheral blood mononuclear cells of healthy volunteers were differentiated into macrophages using granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). The MDMs were stimulated with early secretory antigenic target protein 6 (ESAT6) and culture filtrate protein 10 (CFP10). The mRNA expression levels for the cat eye syndrome chromosome region, candidate 1 (CECR1) gene encoding ADA2 were then measured. CECR1 mRNA expression levels were significantly higher in MDMs stimulated with ESAT6 and CFP10, than in the unstimulated MDMs. When stimulated with ESAT6, M-CSF-treated MDMs showed more pronounced CECR1 mRNA expression than GM-CSF-treated MDMs. Interferon-γ decreased the ESAT6- and CFP10-induced CECR1 mRNA expression in MDMs. CECR1 mRNA expression levels were positively correlated with mRNA expression of tumor necrosis factor α and interleukin 10, respectively. ADA2 mRNA expression increased when MDMs were stimulated with MTB antigens alone. This partly indicates that pleural fluid ADA levels could increase in patients with culture-negative TPE. Our results may be helpful in improving the understanding of TPE pathogenesis.

  7. Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL.

    PubMed Central

    Hamilton, J A; Jessup, W; Brown, A J; Whitty, G

    2001-01-01

    Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived "foam" cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo. PMID:11256965

  8. Effects of macrophage colony-stimulating factor (M-CSF) on the development, differentiation, and maturation of marginal metallophilic macrophages and marginal zone macrophages in the spleen of osteopetrosis (op) mutant mice lacking functional M-CSF activity.

    PubMed

    Takahashi, K; Umeda, S; Shultz, L D; Hayashi, S; Nishikawa, S

    1994-05-01

    Immunohistochemical techniques using an anti-mouse panmacrophage monoclonal antibody and anti-mouse monoclonal antibodies specific for marginal metallophilic macrophages or marginal zone macrophages were used to detect red pulp macrophages, marginal metallophilic macrophages, and marginal zone macrophages in the spleen of op/op mice. In the mutant mice, the red pulp macrophages were reduced to about 60% of those in the normal littermates and the marginal metallophilic macrophages and marginal zone macrophages were absent. After administration of recombinant human macrophage colony-stimulating factor (rhM-CSF), numbers of red pulp macrophages increased rapidly, reaching levels found in normal littermates 1 week later. In contrast, the marginal metallophilic macrophages as well as the marginal zone macrophages appeared slowly after rhM-CSF administration and their numbers were less than half of the baseline level of normal littermates even at 12 weeks of administration. The distribution of marginal metallophilic macrophages and marginal zone macrophages appearing after M-CSF administration was irregular in the spleen of the op/op mice. These splenic macrophage subpopulations differed in their responses to rhM-CSF, suggesting that distinct mechanisms may be involved in their development and differentiation. The splenic red pulp macrophages present in unmanipulated op/op mice are an M-CSF-independent macrophage population. Although the marginal metallophilic macrophages and marginal zone macrophages are thought to be M-CSF-dependent, their development and differentiation appear to be influenced by locally produced M-CSF or other cytokines.

  9. Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro.

    PubMed

    Zhang, Z; Harkiss, G D; Hopkins, J; Woodall, C J

    2002-08-01

    Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.

  10. GM-CSF Grown Bone Marrow Derived Cells Are Composed of Phenotypically Different Dendritic Cells and Macrophages

    PubMed Central

    Na, Yi Rang; Jung, Daun; Gu, Gyo Jeong; Seok, Seung Hyeok

    2016-01-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had MHCIIlowF4/80high as well as CD11c+CD11bhighCD80−CD64+MerTK+ phenotypes. In contrast, GM-BMDCs had MHCIIhighF4/80low and CD11chighCD8α− CD11b+CD80+CD64−MerTKlow phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing TNFα, IL-1β, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells. PMID:27788572

  11. Acute CD4+ T lymphocyte-dependent interleukin-1-driven arthritis selectively requires interleukin-2 and interleukin-4, joint macrophages, granulocyte-macrophage colony-stimulating factor, interleukin-6, and leukemia inhibitory factor.

    PubMed

    Lawlor, Kate E; Wong, Peter K K; Campbell, Ian K; van Rooijen, Nico; Wicks, Ian P

    2005-12-01

    To further investigate the effects of interleukin-1 (IL-1) in immune-mediated joint inflammation, we examined the role of IL-2, Th1 interferon-gamma (IFNgamma), and Th2 (IL-4) cytokines, joint macrophages, and macrophage-derived cytokines (IL-12 p40, IL-6, leukemia inhibitory factor [LIF], oncostatin M [OSM], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) in a CD4+ T lymphocyte-dependent model of acute arthritis. Methylated bovine serum albumin (mBSA)/IL-1-induced arthritis was elicited in wild-type, gene-knockout, and monoclonal antibody-treated mice. Synovial lining macrophages were selectively depleted by intraarticular injection of clodronate liposomes prior to disease induction. The severity of arthritis was assessed histologically. Mice deficient in IL-2 were almost completely protected from arthritis, and neutralization of IL-4 reduced the severity of disease. In contrast, arthritis severity and resolution appeared to be independent of IFNgamma. Synovial lining macrophage depletion markedly reduced arthritis severity. IL-6 or LIF deficiency was only modestly protective, although as previously reported, GM-CSF deficiency conferred profound disease resistance. IL-12 p40-deficient mice (which lack IL-12 and IL-23) and OSM receptor-deficient mice were susceptible to mBSA/IL-1-induced arthritis. Acute mBSA/IL-1-induced arthritis is dependent on IL-2 and IL-4, but not IFNgamma. In vivo, the Th1/Th2 paradigm may be distorted by the presence of macrophage-derived cytokines such as IL-1. Synovial lining macrophages are essential in mBSA/IL-1-induced arthritis. However, the requirement for macrophage-derived cytokines is selective; that is, IL-6, LIF, and especially GM-CSF are necessary, but IL-12, IL-23, and OSM are dispensable. IL-1 may therefore influence both adaptive and innate immune mechanisms in acute inflammatory arthritis.

  12. Roles of macrophage migration inhibitory factor in cartilage tissue engineering.

    PubMed

    Fujihara, Yuko; Hikita, Atsuhiko; Takato, Tsuyoshi; Hoshi, Kazuto

    2017-06-02

    To obtain stable outcomes in regenerative medicine, understanding and controlling immunological responses in transplanted tissues are of great importance. In our previous study, auricular chondrocytes in tissue-engineered cartilage transplanted in mice were shown to express immunological factors, including macrophage migration inhibitory factor (MIF). Since MIF exerts pleiotropic functions, in this study, we examined the roles of MIF in cartilage regenerative medicine. We made tissue-engineered cartilage consisting of auricular chondrocytes of C57BL/6J mouse, atellocollagen gel and a PLLA scaffold, and transplanted the construct subcutaneously in a syngeneic manner. Localization of MIF was prominent in cartilage areas of tissue-engineered cartilage at 2 weeks after transplantation, though it became less apparent by 8 weeks. Co-culture with RAW264 significantly increased the expression of MIF in chondrocytes, suggesting that the transplanted chondrocytes in tissue-engineered cartilage could enhance the expression of MIF by stimulation of surrounding macrophages. When MIF was added in the culture of chondrocytes, the expression of type II collagen was increased, indicating that MIF could promote the maturation of chondrocytes. Meanwhile, toluidine blue staining of constructs containing wild type (Mif+/+) chondrocytes showed increased metachromasia compared to MIF-knockout (Mif-/-) constructs at 2 weeks. However, this tendency was reversed by 8 weeks, suggesting that the initial increase in cartilage maturation in Mif+/+ constructs deteriorated by 8 weeks. Since the Mif+/+ constructs included more iNOS-positive inflammatory macrophages at 2 weeks, MIF might induce an M1 macrophage-polarized environment, which may eventually worsen the maturation of tissue-engineered cartilage in the long term. © 2017 Wiley Periodicals, Inc.

  13. Lipoprotein lipase is synthesized by macrophage-derived foam cells in human coronary atherosclerotic plaques.

    PubMed Central

    O'Brien, K D; Gordon, D; Deeb, S; Ferguson, M; Chait, A

    1992-01-01

    Lipoprotein lipase (LPL), hydrolyzes the core triglycerides of lipoproteins, thereby playing a role in their maturation. LPL may be important in the metabolic pathways that lead to atherosclerosis, since it is secreted in vitro by both of the predominant cell types of the atherosclerotic plaque, i.e., macrophages and smooth muscle cells. Because of uncertainty concerning the primary cellular source of LPL in atherosclerotic lesions, in situ hybridization assays for LPL mRNA were performed on 12 coronary arteries obtained from six cardiac allograft recipients. Macrophages and smooth muscle cells were identified on adjacent sections with cell-specific antibodies and foam cells were identified morphologically. LPL protein was localized using a polyclonal antibody. LPL mRNA was produced by a proportion of plaque macrophages, particularly macrophage-derived foam cells, but was not detected in association with any intimal or medial smooth muscle cells. These findings were confirmed by combined immunocytochemistry and in situ hybridization on the same tissue sections. LPL protein was detected in association with macrophage-derived foam cells, endothelial cells, adventitial adipocytes, and medial smooth muscle cells, and, to a lesser extent, in intimal smooth muscle cells and media underlying well-developed plaque. These results indicate that macrophage-derived foam cells are the primary source of LPL in atherosclerotic plaques and are consistent with a role for LPL in the pathogenesis of atherosclerosis. Images PMID:1569193

  14. Granulocyte, granulocyte–macrophage, and macrophage colony-stimulating factors can stimulate the invasive capacity of human lung cancer cells

    PubMed Central

    Pei, X-H; Nakanishi, Y; Takayama, K; Bai, F; Hara, N

    1999-01-01

    We and other researchers have previously found that colony-stimulating factors (CSFs), which generally include granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), promote invasion by lung cancer cells. In the present study, we studied the effects of these CSFs on gelatinase production, urokinase plasminogen activator (uPA) production and their activity in human lung cancer cells. Gelatin zymographs of conditioned media derived from human lung adenocarcinoma cell lines revealed two major bands of gelatinase activity at 68 and 92 kDa, which were characterized as matrix metalloproteinase (MMP)-2 and MMP-9 respectively. Treatment with CSFs increased the 68- and 92-kDa activity and converted some of a 92-kDa proenzyme to an 82-kDa enzyme that was consistent with an active form of the MMP-9. Plasminogen activator zymographs of the conditioned media from the cancer cells showed that CSF treatment resulted in an increase in a 48–55 kDa plasminogen-dependent gelatinolytic activity that was characterized as human uPA. The conditioned medium from the cancer cells treated with CSFs stimulated the conversion of plasminogen to plasmin, providing a direct demonstration of the ability of enhanced uPA to increase plasmin-dependent proteolysis. The enhanced invasive behaviour of the cancer cells stimulated by CSFs was well correlated with the increase in MMPs and uPA activities. These data suggest that the enhanced production of extracellular matrix-degrading proteinases by the cancer cells in response to CSF treatment may represent a biochemical mechanism which promotes the invasive behaviour of the cancer cells. © 1999 Cancer Research Campaign PMID:10408691

  15. Stimulation of macrophage protease secretion via liposomal delivery of muramyl dipeptide derivatives to intracellular sites.

    PubMed Central

    Mehta, K; Juliano, R L; Lopez-Berestein, G

    1984-01-01

    We have observed that murine macrophages can be activated for enhanced neutral protease secretion by exposing the cells to muramyl dipeptides (MDPs). A lipophilic derivative of nor-MDP is more efficacious than the parent hydrophilic nor-MDP. The efficacy and potency of the lipophilic and more prominently the hydrophilic drugs can be increased (10-10(3) fold) by encapsulating them in lipid vesicles (liposomes); however the encapsulation of drug causes a delay in the onset of activation. The enhanced effectiveness of liposomal MDPs seems in part, to be due to increased uptake, slow release and thus potentiated action of the drug at intracellular sites as emphasized by studies with [3H]-MDP. Appropriate distribution of the drug to intracellular compartments of the cell also seems to be an important factor in the activation process. The internalization of a relatively large amounts (greater than 5 ng/10(6) cells) of nor-MDP results in 'down regulation', that is reduced protease secretion, as compared to effects produced by internalization of lesser amounts of the drug. The macrophage activating effects of liposomal MDPs do not seem to require the processing of liposomes in the lysosomal compartment; thus lysosome-blocking agents, such as chloroquine and dextran sulphate, do not affect the induction of protease secretion. PMID:6365745

  16. Involvement of Macrophages in the Pathogenesis of Familial Amyloid Polyneuropathy and Efficacy of Human iPS Cell-Derived Macrophages in Its Treatment

    PubMed Central

    Komohara, Yoshihiro; Takamatsu, Koutaro; Kakuma, Tatsuyuki; Tasaki, Masayoshi; Misumi, Yohei; Ueda, Mitsuharu; Ito, Takaaki; Senju, Satoru; Ando, Yukio

    2016-01-01

    We hypothesized that tissue-resident macrophages in familial amyloid polyneuropathy (FAP) patients will exhibit qualitative or quantitative abnormalities, that may accelerate transthyretin (TTR)-derived amyloid deposition. To evaluate this, we examined the number and subset of tissue-resident macrophages in heart tissue from amyloid-deposited FAP and control patients. In both FAP and control patients, tissue-resident macrophages in heart tissue were all Iba+/CD163+/CD206+ macrophages. However, the number of macrophages was significantly decreased in FAP patients compared with control patients. Furthermore, the proportion of intracellular TTR in CD14+ monocytes was reduced in peripheral blood compared with healthy donors. Based on these results, we next examined degradation and endocytosis of TTR in human induced pluripotent stem (iPS) cell-derived myeloid lineage cells (MLs), which function like macrophages. iPS-MLs express CD163 and CD206, and belong to the inhibitory macrophage category. In addition, iPS-MLs degrade both native and aggregated TTR in a cell-dependent manner in vitro. Further, iPS-MLs endocytose aggregated, and especially polymerized, TTR. These results suggest that decreased tissue-localized macrophages disrupt clearance of TTR-derived amyloid deposits, leading to progression of a pathological condition in FAP patients. To improve this situation, clinical application of pluripotent stem cell-derived MLs may be useful as an approach for FAP therapy. PMID:27695122

  17. Activating transcription factor 1 directs Mhem atheroprotective macrophages through coordinated iron handling and foam cell protection.

    PubMed

    Boyle, Joseph J; Johns, Michael; Kampfer, Theresa; Nguyen, Aivi T; Game, Laurence; Schaer, Dominik J; Mason, Justin C; Haskard, Dorian O

    2012-01-06

    Intraplaque hemorrhage (IPH) drives atherosclerosis through the dual metabolic stresses of cholesterol-enriched erythrocyte membranes and pro-oxidant heme/iron. When clearing tissue hemorrhage, macrophages are typically seen storing either iron or lipid. We have recently defined hemorrhage-associated macrophages (HA-mac) as a plaque macrophage population that responds adaptively to IPH. This study aimed to define the key transcription factor(s) involved in HO-1 induction by heme. To address this question, we used microarray analysis and transfection with siRNA and plasmids. To maintain physiological relevance, we focused on human blood-derived monocytes. We found that heme stimulates monocytes through induction of activating transcription factor 1 (ATF-1). ATF-1 coinduces heme oxygenase-1 (HO-1) and Liver X receptor beta (LXR-β). Heme-induced HO-1 and LXR-β were suppressed by knockdown of ATF-1, and HO-1 and LXR-β were induced by ATF-1 transfection. ATF-1 required phosphorylation for full functional activity. Expression of LXR-β in turn led to induction of other genes central to cholesterol efflux, such as LXR-α and ABCA1. This heme-directed state was distinct from known macrophage states (M1, M2, Mox) and, following the same format, we have designated them Mhem. These results show that ATF-1 mediates HO-1 induction by heme and drives macrophage adaptation to intraplaque hemorrhage. Our definition of an ATF-1-mediated pathway for linked protection from foam cell formation and oxidant stress may have therapeutic potential.

  18. 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

  19. 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

    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. 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. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

  20. Expression profiling of TRIM protein family in THP1-derived macrophages following TLR stimulation

    PubMed Central

    Jiang, Mei-Xiu; Hong, Xuan; Liao, Bin-Bin; Shi, Shui-Zhen; Lai, Xiao-Fang; Zheng, Huai-Yu; Xie, Lin; Wang, Yuan; Wang, Xiao-Lei; Xin, Hong-Bo; Fu, Mingui; Deng, Ke-Yu

    2017-01-01

    Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage activation are not completely understood. Members of the tripartite motif (TRIM) family have recently emerged as important players in innate immunity and antivirus. Here, we systematically analyzed mRNA expressions of representative TRIM molecules in human THP1-derived macrophages activated by different toll-like receptor (TLR) ligands. Twenty-nine TRIM members were highly induced (>3 fold) by one or more TLR ligands, among which 19 of them belong to TRIM C-IV subgroup. Besides TRIM21, TRIM22 and TRIM38 were shown to be upregulated by TLR3 and TLR4 ligands as previous reported, we identified a novel group of TRIM genes (TRIM14, 15, 31, 34, 43, 48, 49, 51 and 61) that were significantly up-regulated by TLR3 and TLR4 ligands. In contrast, the expression of TRIM59 was down-regulated by TLR3 and TLR4 ligands in both human and mouse macrophages. The alternations of the TRIM proteins were confirmed by Western blot. Finally, overexpression of TRIM59 significantly suppressed LPS-induced macrophage activation, whereas siRNA-mediated knockdown of TRIM59 enhanced LPS-induced macrophage activation. Taken together, the study provided an insight into the TLR ligands-induced expressions of TRIM family in macrophages. PMID:28211536

  1. Expression of complement components and regulators by different subtypes of bone marrow-derived macrophages.

    PubMed

    Luo, Chang; Chen, Mei; Madden, Angelina; Xu, Heping

    2012-08-01

    Under inflammatory conditions, macrophages can differentiate into different functional subtypes. We show that bone marrow-derived macrophages constitutively express different levels of various complement-related genes. The relative expression levels are C1qb > Crry > CFH > C3 > C1r > CFB > DAF1 > CD59a > C2 > C1INH > C1s > C4. Upon activation, the expression of C1r, C1s, C3, C2, CFB, and C1INH was up-regulated, and CFH, CD59a, and DAF1, down-regulated in M1 (induced by interferon-γ + lipopolysaccharides (LPS)) and M2b (induced by immune complex + LPS) macrophages. The expression of C4 and CFH was slightly up-regulated in interleukin (IL)-10-induced M2c macrophages. Complement gene expression in IL-4-induced M2a macrophages was weakly down-regulated as compared to resting M0 macrophages. Higher levels of C3, C1INH, and CFB but lower levels of CFH expression in M1 and M2b macrophage suggests that they may be involved in the alternative pathway of complement activation during inflammation.

  2. Expression profiling of TRIM protein family in THP1-derived macrophages following TLR stimulation.

    PubMed

    Jiang, Mei-Xiu; Hong, Xuan; Liao, Bin-Bin; Shi, Shui-Zhen; Lai, Xiao-Fang; Zheng, Huai-Yu; Xie, Lin; Wang, Yuan; Wang, Xiao-Lei; Xin, Hong-Bo; Fu, Mingui; Deng, Ke-Yu

    2017-02-17

    Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage activation are not completely understood. Members of the tripartite motif (TRIM) family have recently emerged as important players in innate immunity and antivirus. Here, we systematically analyzed mRNA expressions of representative TRIM molecules in human THP1-derived macrophages activated by different toll-like receptor (TLR) ligands. Twenty-nine TRIM members were highly induced (>3 fold) by one or more TLR ligands, among which 19 of them belong to TRIM C-IV subgroup. Besides TRIM21, TRIM22 and TRIM38 were shown to be upregulated by TLR3 and TLR4 ligands as previous reported, we identified a novel group of TRIM genes (TRIM14, 15, 31, 34, 43, 48, 49, 51 and 61) that were significantly up-regulated by TLR3 and TLR4 ligands. In contrast, the expression of TRIM59 was down-regulated by TLR3 and TLR4 ligands in both human and mouse macrophages. The alternations of the TRIM proteins were confirmed by Western blot. Finally, overexpression of TRIM59 significantly suppressed LPS-induced macrophage activation, whereas siRNA-mediated knockdown of TRIM59 enhanced LPS-induced macrophage activation. Taken together, the study provided an insight into the TLR ligands-induced expressions of TRIM family in macrophages.

  3. EXAGGERATED INFLAMMATORY RESPONSES MEDIATED BY BURKHOLDERIA CENOCEPACIA IN HUMAN MACROPHAGES DERIVED FROM CYSTIC FIBROSIS

    PubMed Central

    Kopp, Benjamin T.; Abdulrahman, Basant A.; Khweek, Arwa A.; Kumar, Surender B.; Akhter, Anwari; Montione, Richard; Tazi, Mia F.; Caution, Kyle; McCoy, Karen; Amer, Amal O.

    2012-01-01

    Cystic Fibrosis (CF) is accompanied with heightened inflammation worsened by drug resistant Burkholderia cenocepacia. Human CF macrophage responses to B. cenocepacia are poorly characterized and variable in the literature. Therefore, we examined human macrophage responses to the epidemic B. cenocepacia J2315 strain in order to identify novel anti-inflammatory targets. Peripheral blood monocyte derived macrophages were obtained from 23 CF and 27 non-CF donors. Macrophages were infected with B. cenocepacia J2315 and analyzed for cytokines, cytotoxicity, and microscopy. CF macrophages demonstrated significant increases in IL-1β, IL-10, MCP-1, and IFN-γ production in comparison to non-CF controls. CF patients on prednisone exhibited globally diminished cytokines compared to controls and other CF patients. CF macrophages also displayed increased bacterial burden and cell death. In conclusion, CF macrophages demonstrate exaggerated IL-1β, IL-10, MCP-1, and IFN-γ production and cell death during B. cenocepacia infection. Treatment with corticosteroids acutely suppressed cytokine responses. PMID:22728038

  4. In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages.

    PubMed

    McCanna, David Joseph; Barthod-Malat, Aurore V; Gorbet, Maud B

    2015-01-01

    Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

  5. Intraphagosomal Peroxynitrite as a Macrophage-derived Cytotoxin against Internalized Trypanosoma cruzi

    PubMed Central

    Alvarez, María Noel; Peluffo, Gonzalo; Piacenza, Lucía; Radi, Rafael

    2011-01-01

    Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO− + ONOOH) formation, a strong oxidant arising from the reaction of •NO with superoxide radical (O2˙̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O2˙̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained •NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when •NO and O2˙̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite. PMID:21098483

  6. 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, and N-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

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

    PubMed Central

    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

    2016-01-01

    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, and N-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

  8. Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells

    PubMed Central

    1996-01-01

    Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose- specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM- CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration. PMID:8601605

  9. Unlocking bat immunology: establishment of Pteropus alecto bone marrow-derived dendritic cells and macrophages.

    PubMed

    Zhou, Peng; Chionh, Yok Teng; Irac, Sergio Erdal; Ahn, Matae; Jia Ng, Justin Han; Fossum, Even; Bogen, Bjarne; Ginhoux, Florent; Irving, Aaron T; Dutertre, Charles-Antoine; Wang, Lin-Fa

    2016-12-09

    Bats carry and shed many emerging infectious disease agents including Ebola virus and SARS-like Coronaviruses, yet they rarely display clinical symptoms of infection. Bat epithelial or fibroblast cell lines were previously established to study the bat immune response against viral infection. However, the lack of professional immune cells such as dendritic cells (DC) and macrophages has greatly limited the significance of current investigations. Using Pteropus alecto (P. alecto) GM-CSF plus IL4, FLT3L and CSF-1, we successfully generated bat bone marrow-derived DC and macrophages. Cells with the phenotype, morphology and functional features of monocyte-derived DC, bona fide DC or macrophages were obtained in GM-CSF/IL4, FLT3L or CSF-1 cultures, respectively. The successful generation of the first bat bone marrow-derived immune cells paves the way to unlocking the immune mechanisms that confer host resilience to pathogens in bats.

  10. Unlocking bat immunology: establishment of Pteropus alecto bone marrow-derived dendritic cells and macrophages

    PubMed Central

    Zhou, Peng; Chionh, Yok Teng; Irac, Sergio Erdal; Ahn, Matae; Jia Ng, Justin Han; Fossum, Even; Bogen, Bjarne; Ginhoux, Florent; Irving, Aaron T; Dutertre, Charles-Antoine; Wang, Lin-Fa

    2016-01-01

    Bats carry and shed many emerging infectious disease agents including Ebola virus and SARS-like Coronaviruses, yet they rarely display clinical symptoms of infection. Bat epithelial or fibroblast cell lines were previously established to study the bat immune response against viral infection. However, the lack of professional immune cells such as dendritic cells (DC) and macrophages has greatly limited the significance of current investigations. Using Pteropus alecto (P. alecto) GM-CSF plus IL4, FLT3L and CSF-1, we successfully generated bat bone marrow-derived DC and macrophages. Cells with the phenotype, morphology and functional features of monocyte-derived DC, bona fide DC or macrophages were obtained in GM-CSF/IL4, FLT3L or CSF-1 cultures, respectively. The successful generation of the first bat bone marrow-derived immune cells paves the way to unlocking the immune mechanisms that confer host resilience to pathogens in bats. PMID:27934903

  11. Investigating the Human Immunodeficiency Virus Type One-Infected Monocyte-Derived Macrophage Secretome

    PubMed Central

    Ciborowski, Pawel; Kadiu, Irena; Rozek, Wojciech; Smith, Lynette; Bernhardt, Kristen; Fladseth, Melissa; Ricardo-Dukelow, Mary; Gendelman, Howard E.

    2007-01-01

    Mononuclear phagocytes (bone marrow monocyte-derived macrophages, alveolar macrophages, perivascular macrophages, and microglia) are reservoirs and vehicles of dissemination for the human immunodeficiency virus type-1 (HIV-1). How virus alters mononuclear phagocyte immunoregulatory activities to complete its life cycle and influence disease is incompletely understood. In attempts to better understanding the influence of virus on macrophage functions, we used one-dimensional electrophoresis, and liquid chromatography tandem mass spectrometry to analyze the secretome of HIV-1 infected human monocyte-derived macrophages. We identified 111 proteins in culture supernatants of control (uninfected) and virus-infected cells. Differentially expressed cytoskeletal, enzymes, redox, and immunoregulatory protein classes were discovered and validated by Western-blot tests. These included, but were not limited to, cystatin C, cystatin B, chitinase 3-like 1 protein, cofilin-1, L-plastin, superoxide dismutase, leukotriene A4 hydrolase, and α-enolase. This study, through the use of a unique proteomics platform, provides novel insights into virus-host cell interactions that affect the functional role of macrophages in HIV disease. PMID:17320137

  12. Microbicidal activity of monocyte derived macrophages in AIDS and related disorders.

    PubMed Central

    Eales, L J; Moshtael, O; Pinching, A J

    1987-01-01

    We have examined the ability of monocyte-derived macrophages from patients with AIDS and other HIV-related disorders to kill the intracellular pathogen Toxoplasma gondii. We have also examined the capacity of peripheral blood mononuclear cells from these patients to produce macrophage-activating and other lymphokines. The capacity to produce interleukin 2 and gamma interferon decreases from controls through asymptomatic seropositive subjects and lymphadenopathy groups A (benign) and B (prodromal) to AIDS. The decrease did not correlate precisely with the decrease in CD4+ cells in these patients. Monocyte-derived macrophages from asymptomatic HIV-infected subjects and lymphadenopathy patients showed a decreased ability to kill T. gondii after activation with recombinant gamma interferon; paradoxically, this was most striking for PGL group A. The defect was largely overcome by using Concanavalin A stimulated autologous supernatants. It was notable that macrophages from AIDS patients showed normal killing with recombinant gamma interferon, but that the supernatants from AIDS patients had reduced activity with normal macrophages. These studies confirm that functional defects of both lymphocytes and macrophages are found in HIV-infected subjects; they serve to emphasize the heterogeneity of the clinical and biological responses to this retrovirus, responses which have important implications in the pathogenesis and treatment of the immunodeficiency. PMID:3111759

  13. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha.

    PubMed

    Leibovich, S J; Polverini, P J; Shepard, H M; Wiseman, D M; Shively, V; Nuseir, N

    Macrophages are important in the induction of new blood vessel growth during wound repair, inflammation and tumour growth. We show here that tumour necrosis factor-alpha (TNF-alpha), a secretory product of activated macrophages that is believed to mediate tumour cytotoxicity, is a potent inducer of new blood vessel growth (angiogenesis). In vivo, TNF-alpha induces capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membrane at very low doses. In vitro, TNF-alpha stimulates chemotaxis of bovine adrenal capillary endothelial cells and induces cultures of these cells grown on type-1 collagen gels to form capillary-tube-like structures. The angiogenic activity produced by activated murine peritoneal macrophages is completely neutralized by a polyclonal antibody to TNF-alpha, suggesting immunological features are common to TNF-alpha and the protein responsible for macrophage-derived angiogenic activity. In inflammation and wound repair, TNF-alpha could augment repair by stimulating new blood vessel growth; in tumours, TNF-alpha might both stimulate tumour development by promoting vessel growth and participate in tumour destruction by direct cytotoxicity.

  14. Macrophage-induced angiogenesis is mediated by tumour necrosis factor

    NASA Astrophysics Data System (ADS)

    Leibovich, S. Joseph; Polverini, Peter J.; Shepard, H. Michael; Wiseman, David M.; Shively, Vera; Nuseir, Nureddin

    1987-10-01

    Macrophages are important in the induction of new blood vessel growth during wound repair, inflammation and tumour growth1-4. We show here that tumour necrosis factor-α (TNF-α), a secretory product of activated macrophages that is believed to mediate tumour cytotoxicity5-9, is a potent inducer of new blood vessel growth (angiogenesis). In vivo, TNF-α induces capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membrane at very low doses. In vitro, TNF-α stimulates chemotaxis of bovine adrenal capillary endothelial cells and induces cultures of these cells grown on type-1 collagen gels to form capillary-tube-like structures. The angiogenic activity produced by activated murine peritoneal macrophages is completely neutralized by a polyclonal antibody to TNF-α, suggesting immunological features are common to TNF-α and the protein responsible for macrophage-derived angiogenic activity. In inflammation and wound repair, TNF-α could augment repair by stimulating new blood vessel growth; in tumours, TNF-α might both stimulate tumour development by promoting vessel growth and participate in tumour destruction by direct cytotoxicity10-12.

  15. 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

  16. Urine macrophage migration inhibitory factor in pediatric systemic lupus erythematosus.

    PubMed

    Otukesh, Hasan; Chalian, Majid; Hoseini, Rozita; Chalian, Hamid; Hooman, Nakysa; Bedayat, Arash; Yazdi, Reza Salman; Sabaghi, Saeed; Mahdavi, Saeed

    2007-12-01

    We reported a series of ten patients with lupus nephritis (five patients in the relapse phase and five in the remission phase) and measured the macrophage migration inhibitory factor (MIF), an important pro-inflammatory cytokine with probable role in the pathogenesis of many inflammatory diseases, in their urine samples. MIF/creatinine (Cr) ratio directly correlated with disease activity and it does not have any significant difference between inactive disease and normal ones. We found that the urine MIF/Cr ratio not only differentiates active disease from inactive disease and normal ones but also correlates with the activity indices of renal pathology.

  17. 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.

  18. Platelet-activating factor increases reactive oxygen species-mediated microbicidal activity of human macrophages infected with Leishmania (Viannia) braziliensis.

    PubMed

    Borges, Arissa Felipe; Morato, Camila Imai; Gomes, Rodrigo Saar; Dorta, Miriam Leandro; de Oliveira, Milton Adriano Pelli; Ribeiro-Dias, Fátima

    2017-09-29

    Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. NDRG2 induced by oxidized LDL in macrophages antagonizes growth factor productions via selectively inhibiting ERK activation.

    PubMed

    Liu, Shumei; Yang, Pengyuan; Kang, Hui; Lu, Ling; Zhang, Yuefan; Pan, Jingwei; Rui, Yao-Cheng

    2010-02-01

    During atherogenesis, macrophage foam cells produce prodigious growth factors, cytokines, and chemokines, which play the central roles in inflammatory process in atherosclerotic plaque formation. In the present study, we identified a new protein marker, N-Myc downstream-regulated protein 2 (NDRG2), which is significantly up-regulated in oxidized low density lipoprotein (oxLDL) treated macrophages and in human atherosclerotic plaques. Over-expression and siRNA knockdown studies showed that NDRG2 is a negative regulator of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) productions in macrophages. Furthermore, we investigated the effects of NDRG2 on MAPK signal activation. Our results showed ERK1/2 activation, but not P38 or JNK1/2 activation, is responsible for regulation of NDRG2 on VEGF and PDGF productions. Consistent with the PDGF levels, the vascular smooth muscle cell (VSMC) proliferation was also regulated by the conditional medium of the oxLDL treated macrophages with NDRG2 knockdown or over-expression. Neutralizing anti-PDGF antibody can significantly inhibit the enhanced VSMC proliferation by macrophage medium with NDRG2 knockdown. Our present results demonstrate that NDRG2 participates in oxLDL-induced macrophage activation and modulates ERK1/2-dependent PDGF and VEGF production, which has potential application in atherogenesis. 2009 Elsevier B.V. All rights reserved.

  20. Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

    PubMed

    Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

    2015-04-01

    Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. 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.

  2. Induction of prostaglandin E synthesis in normal and neoplastic macrophages: role for colony-stimulating factor(s) distinct from effects on myeloid progenitor cell proliferation.

    PubMed Central

    Kurland, J I; Pelus, L M; Ralph, P; Bockman, R S; Moore, M A

    1979-01-01

    The biosynthesis of prostaglandin E (PGE) by normal and neoplastic macrophages is intrinsically linked to their synthesis of, and exposure to, myeloid colony-stimulating factors (CS-factors). The defect in responsiveness to endotoxin lipopolysaccharide (LPS) by macrophages from C3H/HeJ mice extends equally to the synthesis of CS-factor and PGE. However, C3H/HeJ macrophages can be stimulated to synthesize PGE by treatment with agents other than LPS [zymosan, tuberculin purified protein derivative, concanavalin A, poly(I).poly(C)], which also stimulate CS-factor production, or by the addition of various preparations of soluble CS-factor. In peritoneal wash preparations, constitutive PGE synthesis occurred in rapidly sedimenting macrophage cells, whereas constitutive CS-factor production and inducible PGE synthesis occurred in slower sedimenting adherent cells. A similar functional heterogeneity in CS-factor and PGE production was found in neoplastic macrophagae cell lines. The association of elevated CS-factor levels and PGE synthesis by macrophages suggests a role for CS-factor in many of the physiological responses heretofore associated with elevated tissue levels of the E type prostaglandins. PMID:313054

  3. Trypanosoma cruzi: the immunological induction of macrophage plasminogen activator requires thymus-derived lymphocytes

    PubMed Central

    1977-01-01

    In this article we describe methods in which unstimulated mouse peritoneal macrophages were induced to secrete high livels of plasminogen activator under in vitro conditions. The exposure of sensitized peritoneal or spleen cell populations from Trypanosoma cruzi- infected animals to either viable or heat-killed trypanosomes lead to the release of an inducing factor(s). Maximal levels of plasminogen activator secretion are achieved by the incubation of such factors (s) with unstimulated macrophages for 48 h. A significant increase in enzyme secretion was already observed after a 24 h incubation. The production of the inducing factor(s) by sensitized cells was immunologically specific and unrelated antigens did not stimulate the production of the factor(s) by sensitized peritoneal or spleen cell populations. The inducing factor(s) was produced by nylon-wool- fractionated spleen and peritoneal cells which had been depleted of marcrophages. Pretreatment of sensitized spleen cells with anti-theta serum and C abolished the production of the activating factor(s). The active supernatant fluids were able to induce secretion of macrophage plasminogen activator across H-2 barriers. Attempts to induce trypanocidal activity in unstimulated macrophages have not been successful. PMID:327013

  4. 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.

  5. Functional evaluation of gene silencing on macrophages derived from U937 cells using interference RNA (shRNA) in a model of macrophages infected with Leishmania (Viannia) braziliensis.

    PubMed

    Ovalle-Bracho, Clemencia; Londoño-Barbosa, Diana A; Franco-Muñoz, Carlos; Clavijo-Ramírez, Carlos

    2015-12-01

    Leishmaniasis development is multifactorial; nonetheless, the establishment of the infection, which occurs by the survival and replication of the parasite inside its main host cell, the macrophage, is mandatory. Thus, the importance of studying the molecular mechanisms involved in the Leishmania-macrophage interaction is highlighted. The aim of this study was to characterize a cellular model of macrophages derived from U937 cells that would allow for the identification of infection phenotypes induced by genetic silencing with interference RNA in the context of macrophages infected with Leishmania (Viannia) braziliensis. The model was standardized by silencing an exogenous gene (gfp), an endogenous gene (lmna) and a differentially expressed gene between infected and non-infected macrophages (gro-β). The silencing process was successful for the three genes studied, obtaining reductions of 88·9% in the GFP levels, 87·5% in LMNA levels and 74·4% for Gro-β with respect to the corresponding control cell lines. The cell model revealed changes in the infection phenotype of the macrophages in terms of number of amastigotes per infected macrophage, number of amastigotes per sampled macrophage and percentage of infected macrophages as a result of gene silencing. Thus, this cell model constitutes a research platform for the study of parasite-host interactions and for the identification of potentially therapeutic targets.

  6. Oral contraceptives modify DNA methylation and monocyte-derived macrophage function

    PubMed Central

    2012-01-01

    Background Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or non-androgenic properties of progestin. Methods Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells. Results As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, follicle-stimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA

  7. Macrophage-Derived Angiopoietin-Like Protein 2 Exacerbates Brain Damage by Accelerating Acute Inflammation after Ischemia-Reperfusion.

    PubMed

    Amadatsu, Toshihiro; Morinaga, Jun; Kawano, Takayuki; Terada, Kazutoyo; Kadomatsu, Tsuyoshi; Miyata, Keishi; Endo, Motoyoshi; Kasamo, Daiki; Kuratsu, Jun-Ichi; Oike, Yuichi

    2016-01-01

    Ischemic stroke is a leading cause of death and disability worldwide. Several reports suggest that acute inflammation after ischemia-reperfusion exacerbates brain damage; however, molecular mechanisms underlying this effect remain unclear. Here, we report that MAC-3-positive immune cells, including infiltrating bone marrow-derived macrophages and activated microglia, express abundant angiopoietin-like protein (ANGPTL) 2 in ischemic mouse brain in a transient middle cerebral artery occlusion (MCAO) model. Both neurological deficits and infarct volume decreased in transient MCAO model mice established in Angptl2 knockout (KO) relative to wild-type mice. Acute brain inflammation after ischemia-reperfusion, as estimated by expression levels of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor alpha (TNF)-α, was significantly suppressed in Angptl2 KO compared to control mice. Moreover, analysis employing bone marrow chimeric models using Angptl2 KO and wild-type mice revealed that infiltrated bone marrow-derived macrophages secreting ANGPTL2 significantly contribute to acute brain injury seen after ischemia-reperfusion. These studies demonstrate that infiltrating bone marrow-derived macrophages promote inflammation and injury in affected brain areas after ischemia-reperfusion, likely via ANGPTL2 secretion in the acute phase of ischemic stroke.

  8. Modulation of virulence factors in Francisella tularensis determines human macrophage responses

    PubMed Central

    Carlson, Paul E.; Carroll, James A.; O’Dee, Dawn M.; Nau, Gerard J.

    2009-01-01

    Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis Live Vaccine Strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially-expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a σ54 modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically-defined media. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses. PMID:17369012

  9. Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis

    PubMed Central

    DeFalco, Tony; Bhattacharya, Indrashis; Williams, Alyna V.; Sams, Dustin M.; Capel, Blanche

    2014-01-01

    Organogenesis of the testis is initiated when expression of Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. The cells in the early bipotential gonad undergo de novo organization to form testis cords that enclose germ cells inside tubules lined by epithelial Sertoli cells. Although Sertoli cells are a driving force in the de novo formation of testis cords, recent studies in mouse showed that reorganization of the vasculature and of interstitial cells also play critical roles in testis cord morphogenesis. However, the mechanism driving reorganization of the vasculature during fetal organogenesis remained unclear. Here we demonstrate that fetal macrophages are associated with nascent gonadal and mesonephric vasculature during the initial phases of testis morphogenesis. Macrophages mediate vascular reorganization and prune errant germ cells and somatic cells after testis architecture is established. We show that gonadal macrophages are derived from primitive yolk-sac hematopoietic progenitors and exhibit hallmarks of M2 activation status, suggestive of angiogenic and tissue remodeling functions. Depletion of macrophages resulted in impaired vascular reorganization and abnormal cord formation. These findings reveal a previously unappreciated role for macrophages in testis morphogenesis and suggest that macrophages are an intermediary between neovascularization and organ architecture during fetal organogenesis. PMID:24912173

  10. Nifedipine inhibits ox-LDL-induced lipid accumulation in human blood-derived macrophages.

    PubMed

    Zhang, Qian; Sha Ma, A Zhi; Wang, Chan; Tang, Wei-Qing; Song, Zhi-Yuan

    2015-02-13

    Studies have shown that nifedipine, an anti-hypertensive drug, protects against atherosclerotic progression, but the underlying mechanisms remain elusive. Oxidized low-density lipoprotein (ox-LDL) is critically implicated in macrophage lipid deposition seen in atherosclerosis. In this study, we examined the effects of nifedipine on some ox-LDL-associated changes in human blood-derived macrophages. We isolated monocytes from normal human blood and differentiated them into macrophages. We then treated these human macrophages with ox-LDL and/or nifedipine, and examined lipid accumulation and expression levels of two scavenge receptors CD36 and SR-A as well as a protein kinase PKC-θ. Nifedipine treatment substantially reduced lipid accumulation and the expression of CD36, SR-A, and protein kinase C (PKC)-θ in human macrophages treated with ox-LDL. Silencing of PKC-θ using siRNA also reduced the expression of CD36 and SR-A in these cells. Our results thus suggest that nifedipine may inhibit atherosclerosis by reducing ox-LDL-induced lipid deposition through suppression of the CD36/SR-A-mediated uptake of ox-LDL by macrophages via a PKC-θ-dependent mechanism. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Yolk-sac-derived macrophages regulate fetal testis vascularization and morphogenesis.

    PubMed

    DeFalco, Tony; Bhattacharya, Indrashis; Williams, Alyna V; Sams, Dustin M; Capel, Blanche

    2014-06-10

    Organogenesis of the testis is initiated when expression of Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. The cells in the early bipotential gonad undergo de novo organization to form testis cords that enclose germ cells inside tubules lined by epithelial Sertoli cells. Although Sertoli cells are a driving force in the de novo formation of testis cords, recent studies in mouse showed that reorganization of the vasculature and of interstitial cells also play critical roles in testis cord morphogenesis. However, the mechanism driving reorganization of the vasculature during fetal organogenesis remained unclear. Here we demonstrate that fetal macrophages are associated with nascent gonadal and mesonephric vasculature during the initial phases of testis morphogenesis. Macrophages mediate vascular reorganization and prune errant germ cells and somatic cells after testis architecture is established. We show that gonadal macrophages are derived from primitive yolk-sac hematopoietic progenitors and exhibit hallmarks of M2 activation status, suggestive of angiogenic and tissue remodeling functions. Depletion of macrophages resulted in impaired vascular reorganization and abnormal cord formation. These findings reveal a previously unappreciated role for macrophages in testis morphogenesis and suggest that macrophages are an intermediary between neovascularization and organ architecture during fetal organogenesis.

  12. Recombinant human lactoferrin modulates human PBMC derived macrophage responses to BCG and LPS.

    PubMed

    Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

    2016-12-01

    Lactoferrin, an iron-binding glycoprotein found in mammalian mucosal secretions and granules of neutrophils, possesses several immune modulatory properties. Published reports indicate that lactoferrin enhances the efficacy of the tuberculosis vaccine, BCG (Bacillus Calmette Guerin), both by increasing macrophage and dendritic cell ability to stimulate receptive T cells and by modulating the inflammatory response. This report is the first to demonstrate the effects of a recombinant human lactoferrin (10 μg/mL) on human PBMC derived CD14(+) and CD16(+) macrophages stimulated with a strong (LPS, 10 ng/mL) or weaker (BCG, MOI 1:1) stimulator of inflammation. After 3 days culture, LPS and human lactoferrin treated CD14(+) cells significantly increased production of IL-10, IL-6, and MCP-1 compared to the LPS only group. In contrast, similarly treated CD16(+) macrophages increased production of IL-12p40 and IL-10 and decreased TNF-α. Limited changes were observed in BCG stimulated CD14(+) and CD16(+) macrophages with and without lactoferrin. Analysis of surface expression of antigen presentation and co-stimulatory molecules demonstrated that CD14(+) macrophages, when stimulated with BCG or LPS and cultured with lactoferrin, increased expression of CD86. CD16(+) macrophages treated with lactoferrin showed a similar trend of increase in CD86 expression, but only when stimulated with BCG.

  13. Methamphetamine and HIV-1 gp120 effects on lipopolysaccharide stimulated matrix metalloproteinase-9 production by human monocyte-derived macrophages.

    PubMed

    Reynolds, Jessica L; Mahajan, Supriya D; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E; Schwartz, Stanley A

    2011-01-01

    Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS.

  14. Methamphetamine and HIV-1 gp120 Effects on Lipopolysaccharide Stimulated Matrix Metalloproteinase-9 Production by Human Monocyte-Derived Macrophages

    PubMed Central

    Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E.; Schwartz, Stanley A.

    2011-01-01

    Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS. PMID:21425912

  15. Tumor-derived interleukin-1 promotes lymphangiogenesis and lymph node metastasis through M2-type macrophages.

    PubMed

    Watari, Kosuke; Shibata, Tomohiro; Kawahara, Akihiko; Sata, Ken-ichi; Nabeshima, Hiroshi; Shinoda, Ai; Abe, Hideyuki; Azuma, Koichi; Murakami, Yuichi; Izumi, Hiroto; Takahashi, Takashi; Kage, Masayoshi; Kuwano, Michihiko; Ono, Mayumi

    2014-01-01

    Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.

  16. 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

  17. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide.

    PubMed

    Kwon, N S; Stuehr, D J; Nathan, C F

    1991-10-01

    Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but

  18. 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. Copyright © 2015 John Wiley & Sons, Ltd.

  19. Lipopolysaccharide downregulates macrophage-derived IL-22 to modulate alcohol-induced hepatocyte cell death.

    PubMed

    Liu, Yaming; Verma, Vikas K; Malhi, Harmeet; Gores, Greg J; Kamath, Patrick S; Sanyal, Arun; Chalasani, Naga; Gao, Bin; Shah, Vijay H

    2017-09-01

    Interleukin-22 (IL-22) is a Th17 cell hepatoprotective cytokine that is undergoing clinical trials to treat patients with alcoholic hepatitis (AH). Lipopolysaccharide (LPS) activation of macrophage is implicated in hepatocyte cell death and pathogenesis of AH. The role of IL-22 production from macrophage, its regulation by LPS, and effects on alcohol-induced hepatocyte cell death are unexplored and were examined in this study. Low levels of IL-22 mRNA/protein were detected in macrophage but were significantly upregulated by 6.5-fold in response to the tissue reparative cytokine IL-10. Conversely, LPS significantly decreased IL-22 mRNA levels in a temporal and concentration-dependent manner with a maximum reduction of 5-fold. LPS downregulation of IL-22 mRNA levels was rescued in the presence of a pharmacological inhibitor of c-Jun NH2-terminal kinase (JNK) and by JNK knockdown. Next, we explored whether macrophage-derived IL-22 regulated ethanol-induced hepatocyte death. Conditioned media from IL-10-stimulated macrophages attenuated ethanol-induced hepatocyte caspase-3/7 activity, and apoptosis as assessed by fluorometric assay and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. This effect was diminished in conditioned media from macrophages with IL-22 knockdown. Cytokine analysis in sera samples of patients with AH revealed that IL-22 levels were significantly elevated compared with healthy controls and heavy-drinking controls, implying a state of IL-22 resistance in human AH. Macrophage-derived IL-22 protects hepatocytes from ethanol-induced cell death. IL-22 downregulation is a new regulatory target of LPS in the pathogenesis of AH.

  20. Cord Blood-Derived Macrophage-Lineage Cells Rapidly Stimulate Osteoblastic Maturation in Mesenchymal Stem Cells in a Glycoprotein-130 Dependent Manner

    PubMed Central

    Fernandes, Tania J.; Hodge, Jason M.; Singh, Preetinder P.; Eeles, Damien G.; Collier, Fiona M.; Holten, Ian; Ebeling, Peter R.

    2013-01-01

    In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC) generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors) induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate) these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF) to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL) to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM) greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM), and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2) actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation) or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells, including

  1. Macrophage-derived IL-1β enhances monosodium urate crystal-triggered NET formation.

    PubMed

    Sil, Payel; Wicklum, Haley; Surell, Chandler; Rada, Balázs

    2017-03-01

    . We found that recombinant IL-1β strongly promoted MSU crystal-induced NET formation in human neutrophils. Interestingly, IL-1β alone did not induce any NET release. We also found that clinical grade anakinra, an IL-1 receptor blocker, strongly reduced the NETosis-enhancing effect of macrophage supernatants indicating that IL-1β is mainly responsible for this effect. Macrophage-derived IL-1β enhances MSU crystal-induced NET release in neutrophils. We identified a new mechanism by which macrophages and IL-1β affect neutrophil functions, and could contribute to the inflammatory conditions present in gout. Our results also revealed a new anti-inflammatory mechanism of anakinra.

  2. 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

  3. Macrophage migration inhibitory factor (MIF): a glucocorticoid counter-regulator within the immune system.

    PubMed

    Calandra, T; Bucala, R

    1997-01-01

    Originally described as a T lymphocyte-derived factor that inhibited the random migration of macrophages, the protein known as macrophage migration inhibitory factor (MIF) was an enigmatic cytokine for almost 3 decades. In recent years, the discovery of MIF as a product of the anterior pituitary gland and the cloning and expression of bioactive, recombinant MIF protein have led to the definition of its critical biological role in vivo. MIF has the unique property of being released from macrophages and T lymphocytes that have been stimulated by glucocorticoids. Once released, MIF overcomes the inhibitory effects of glucocorticoids on TNF alpha, IL-1 beta, IL-6, and IL-8 production by LPS-stimulated monocytes in vitro and suppresses the protective effects of steroids against lethal endotoxemia in vivo. MIF also antagonizes glucocorticoid inhibition of T-cell proliferation in vitro by restoring IL-2 and IFN-gamma production. This observation has identified a pivotal role for MIF within the immune system and fills an important gap in our understanding of the control of inflammatory and immune responses. Glucocorticoids have long been considered to be an integral component of the stress response to infection or tissue invasion and serve to modulate inflammatory and immune responses. MIF is the first mediator to be identified that can counter-regulate the inhibitory effects of glucocorticoids and thus plays a critical role in the host control of inflammation and immunity.

  4. 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.

  5. 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.

  6. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    PubMed

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.

  7. Bone marrow derived macrophages fuse with intestine stromal cells and contribute to chronic fibrosis after radiation.

    PubMed

    Yeh, Ming-Han; Chang, Ya-Hui; Tsai, Yi-Chih; Chen, Su-Liang; Huang, Tze-Sing; Chiu, Jeng-Fong; Ch'ang, Hui-Ju

    2016-05-01

    Bone marrow-derived cells (BMDC) have been demonstrated to play a critical role in intestine regeneration. However, organ fibrosis was one of the major side effects of bone marrow (BM) transplantation. It warrants further investigation on the mechanisms of BM cell therapy in radiation induced intestine damage. We established three murine models to evaluate BMDC within intestines after radiation, including cre-loxP system of transgenic mice. In vitro co-culture between murine BM with human intestine stromal cells was also performed to measure the level of fusion and fibrosis after treatment with anti-fibrotic agents or after macrophage depletion. Despite complete recovery of epithelial mucosa from radiation damage, we found persistent proliferation and repopulation of BMDC within the lamina propria. Fusion between BM derived monocytic and intestine stromal cells correlated with the level of fibrosis and proliferation index. Depleting macrophages genetically using CD11b-DTR mouse model or pharmacologically using clodronate liposome reduced the level of cell fusion and intestine fibrosis. Fibrotic cues from intestine enhance fusion between BM-derived monocytes/macrophages with intestine stromal cells. The fusion hybrids promote cell cycle re-entry, proliferation and reinforce fibrosis signal. Depleting macrophages interferes with cell fusion and ameliorates radiation-induced intestine fibrosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

    PubMed

    Marton, Annamaria; Vizler, Csaba; Kusz, Erzsebet; Temesfoi, Viktoria; Szathmary, Zsuzsa; Nagy, Krisztina; Szegletes, Zsolt; Varo, Gyorgy; Siklos, Laszlo; Katona, Robert L; Tubak, Vilmos; Howard, O M Zack; Duda, Erno; Minarovits, Janos; Nagy, Katalin; Buzas, Krisztina

    2012-01-01

    To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

  9. Differential clearance and immune responses to tick cell-derived versus macrophage culture-derived Ehrlichia chaffeensis in mice.

    PubMed

    Ganta, Roman R; Cheng, Chuanmin; Miller, Elizabeth C; McGuire, Bridget L; Peddireddi, Lalitha; Sirigireddy, Kamesh R; Chapes, Stephen K

    2007-01-01

    Human monocytic ehrlichiosis is caused by a tick-transmitted rickettsia, Ehrlichia chaffeensis. We recently reported that E. chaffeensis grown in tick cells expresses different proteins than bacteria grown in macrophages. Therefore, we tested the hypothesis that immune responses against E. chaffeensis would be different if the mice are challenged with bacteria grown in macrophages or tick cells. We assessed the E. chaffeensis clearance from the peritoneum, spleen, and liver by C57BL/6J mice using a TaqMan-based real-time reverse transcription-PCR assay. Macrophage-grown E. chaffeensis was cleared in 2 weeks from the peritoneum, whereas the pathogen from tick cells persisted for nine additional days and included three relapses of increasing bacterial load separated by three-day intervals. Tick cell-grown bacteria also persisted in the livers and spleens with higher bacterial loads compared to macrophage-grown bacteria and fluctuated over a period of 35 days. Three-day periodic cycles were detected in T-cell CD62L/CD44 ratios in the spleen and bone marrow in response to infections with both tick cell- and macrophage-grown bacteria and were accompanied by similar periodic cycles of spleen cell cytokine secretions and nitric oxide and interleukin-6 by peritoneal macrophages. The E. chaffeensis-specific immunoglobulin G response was considerably higher and steadily increased in mice infected with the tick cell-derived E. chaffeensis compared to DH82-grown bacteria. In addition, antigens detected by the immunoglobulins were significantly different between mice infected with the E. chaffeensis originating from tick cells or macrophages. The differences in the immune response to tick cell-grown bacteria compared to macrophage-grown bacteria reflected a delay in the shift of gene expression from the tick cell-specific Omp 14 gene to the macrophage-specific Omp 19 gene. These data suggest that the host response to E. chaffeensis depends on the source of the bacteria and that

  10. Adrenergic modulation of cytokine release in bone marrow progenitor-derived macrophage following polymicrobial sepsis.

    PubMed

    Muthu, Kuzhali; Deng, Jiangping; Gamelli, Richard; Shankar, Ravi; Jones, Stephen B

    2005-01-01

    Catecholamines may impact on the pathophysiology of sepsis by attenuating proinflammatory cytokine and augmenting antiinflammatory cytokine production by macrophages. We tested this premise in bone marrow monocyte progenitor-derived macrophages. Polymicrobial sepsis was induced in mice through cecal ligation and puncture. ER-MP 12 monocyte progenitors were isolated and differentiated into macrophages in vitro 72 hr later. Lipopolysaccharide (LPS)-stimulated cytokine production was measured with and without epinephrine, IL-10 and anti-IL-10 antibody. Epinephrine significantly increased IL-10 production, but attenuated TNF-alpha release exclusively through beta2 adrenergic receptors, and is independent of IL-10 production. Together, these results suggest that epinephrine can promote a potent antiinflammatory response in sepsis.

  11. Brief report: Granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and NLRP3 inflammasome up-regulation in an in vivo mouse model.

    PubMed

    Shaw, Odette M; Steiger, Stefanie; Liu, Xiao; Hamilton, John A; Harper, Jacquie L

    2014-09-01

    To determine the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of inflammatory macrophages in an in vivo model of monosodium urate monohydrate (MSU) crystal-induced inflammation. C57BL/6J mice were treated with either clodronate liposomes to deplete peritoneal macrophages or GM-CSF antibody and were then challenged by intraperitoneal injection of MSU crystals. Peritoneal lavage fluid was collected, and cellular infiltration was determined by flow cytometry. Purified resident and MSU crystal-recruited monocyte/macrophages were stimulated ex vivo with MSU crystals. The interleukin-1β (IL-1β) levels in lavage fluids and ex vivo assay supernatants were measured. GM-CSF-derived and macrophage colony-stimulating factor (M-CSF)-derived macrophages were generated in vitro from bone marrow cells. Protein expression of IL-1β, caspase 1, NLRP3, and ASC by in vitro- and in vivo-generated monocyte/macrophages was analyzed by Western blotting. Depletion of resident macrophages lowered MSU crystal-induced IL-1β and GM-CSF levels in vivo as well as IL-1β production by MSU crystal-recruited monocytes stimulated ex vivo. GM-CSF neutralization in vivo decreased MSU crystal-induced IL-1β levels and neutrophil infiltration. MSU crystal-recruited monocyte/macrophages from GM-CSF-neutralized mice expressed lower levels of the macrophage marker CD115 and produced less IL-1β following ex vivo stimulation. These monocytes exhibited decreased expression of NLRP3, pro/active IL-1β, and pro/active caspase 1. In vitro-derived GM-CSF-differentiated macrophages expressed higher levels of NLRP3, pro/active IL-1β, and pro/active caspase 1 compared to M-CSF-differentiated macrophages. GM-CSF plays a key role in the differentiation of MSU crystal-recruited monocytes into proinflammatory macrophages. GM-CSF production may therefore contribute to the exacerbation of inflammation in gout. Copyright © 2014 by the American College of Rheumatology.

  12. Macrophage migration inhibitory factor (MIF): a promising biomarker

    PubMed Central

    Grieb, Gerrit; Merk, Melanie; Bernhagen, Jürgen; Bucala, Richard

    2011-01-01

    Macrophage migration inhibitory (MIF) factor is an immunoregulatory cytokine whose effect on arresting random immune cell movement was recognized several decades ago. Despite its historic name, MIF also has a direct chemokine-like function and promotes cell recruitment. Multiple clinical studies have pointed to the utility of MIF as a biomarker for different diseases that have an inflammatory component; these include systemic infections and sepsis, autoimmune diseases, cancer, and metabolic disorders such as type 2 diabetes and obesity. The identification of functional promoter polymorphisms in the MIF gene (MIF) and their association with the susceptibility or severity of different diseases has served not only to validate MIF’s role in disease development but opened the possibility of using MIF genotype information to better predict risk and outcome. In this article, we review the clinical data of MIF and discuss its potential as a biomarker for different disease applications. PMID:20520854

  13. Impaired phagocytosis of apoptotic cells causes accumulation of bone marrow-derived macrophages in aged mice

    PubMed Central

    Kim, Ok-Hee; Kim, Hyojung; Kang, Jinku; Yang, Dongki; Kang, Yu-Hoi; Lee, Dae Ho; Cheon, Gi Jeong; Park, Sang Chul; Oh, Byung-Chul

    2017-01-01

    Accumulation of tissue macrophages is a significant characteristic of disease-associated chronic inflammation, and facilitates the progression of disease pathology. However, the functional roles of these bone marrow-derived macrophages (BMDMs) in aging are unclear. Here, we identified age-dependent macrophage accumulation in the bone marrow, showing that aging significantly increases the number of M1 macrophages and impairs polarization of BMDMs. We found that age-related dysregulation of BMDMs is associated with abnormal overexpression of the anti-inflammatory interleukin-10. BMDM dysregulation in aging impairs the expression levels of pro-inflammatory cytokines and genes involved in B-cell maturation and activation. Phagocytosis of apoptotic Jurkat cells by BMDMs was reduced because of low expression of phagocytic receptor CD14, indicating that increased apoptotic cells may result from defective phagocytosis of apoptotic cells in the BM of aged mice. Therefore, CD14 may represent a promising target for preventing BMDM dysregulation, and macrophage accumulation may provide diagnostic and therapeutic clues. PMID:27866511

  14. The improvement of M1 polarization in macrophages by glycopeptide derived from Ganoderma lucidum.

    PubMed

    Sun, Li-Xin; Lin, Zhi-Bin; Lu, Jie; Li, Wei-Dong; Niu, Yan-Dong; Sun, Yu; Hu, Chen-Yang; Zhang, Guo-Qiang; Duan, Xin-Suo

    2017-01-26

    Ganoderma lucidum (Fr.) Karst (Ganodermataceae) is a medicinal mushroom that has been extensively used in China for centuries to promote longevity and improve vigor without significant adverse effects. There is continuous interest in the bioactive properties of G. lucidum in view of its newly developed popularity in other regions besides Asia, such as Europe. Glycopeptide derived from G. lucidum (Gl-PS) is one of the main effective components isolated from this mushroom. The Gl-PS has been demonstrated pleiotropic with many bioactivities including immunomodulatory and antitumor effects. Macrophages are important cells involved in innate and adaptive immunity. Classically activated macrophages (M1) and alternatively activated macrophages (M2), with their different roles, display distinct cytokine profiles: M1 preferentially produces TNF-α, IL-6, and IL-12; conversely, M2 generates more IL-10 and arginase. Gl-PS might have the potential to promote macrophage M1 polarization by lipopolysaccharide (LPS). In this study, LPS was used to induce the M1 polarization. It was shown that the level of the TNF-α, IL-6, and IL-12 were increased and the IL-10 and arginase I were decreased in the polarized M1 macrophages after application of Gl-PS compared to the control. The results indicated the potential of Gl-PS to promote M1 polarization vs M2, with the health beneficial understanding of the bioactivities of Gl-PS.

  15. Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

    PubMed Central

    Tilstam, Pathricia V.; Springenberg-Jung, Katrin; Boecker, Arne Hendrick; Schmitz, Corinna; Heinrichs, Daniel; Hwang, Soo Seok; Stromps, Jan Philipp; Ganse, Bergita; Kopp, Ruedger; Knobe, Matthias; Bernhagen, Juergen

    2017-01-01

    Background Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. Methods In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Results Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged. Discussion This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds. PMID:28070458

  16. Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer.

    PubMed

    Subbannayya, Tejaswini; Leal-Rojas, Pamela; Barbhuiya, Mustafa A; Raja, Remya; Renuse, Santosh; Sathe, Gajanan; Pinto, Sneha M; Syed, Nazia; Nanjappa, Vishalakshi; Patil, Arun H; Garcia, Patricia; Sahasrabuddhe, Nandini A; Nair, Bipin; Guerrero-Preston, Rafael; Navani, Sanjay; Tiwari, Pramod K; Santosh, Vani; Sidransky, David; Prasad, T S Keshava; Gowda, Harsha; Roa, Juan Carlos; Pandey, Akhilesh; Chatterjee, Aditi

    2015-11-04

    Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.

  17. Chronicity following ischaemia-reperfusion injury depends on tubular-macrophage crosstalk involving two tubular cell-derived CSF-1R activators: CSF-1 and IL-34.

    PubMed

    Sanchez-Niño, Maria Dolores; Sanz, Ana Belen; Ortiz, Alberto

    2016-09-01

    Two structurally unrelated ligands activate the macrophage colony stimulating factor receptor (CSF-1R, c-fms, CD115): M-CSF/CSF-1 and interleukin-34 (IL-34). Both ligands promote macrophage proliferation, survival and differentiation. IL-34 also activates the protein-tyrosine phosphatase ζ receptor (PTP-ζ, PTPRZ1). Both receptors and cytokines are increased during acute kidney injury. While tubular cell-derived CSF-1 is required for kidney repair, Baek et al (J Clin Invest 2015; 125: 3198-3214) have now identified tubular epithelial cell-derived IL-34 as a promoter of kidney neutrophil and macrophage infiltration and tubular cell destruction during experimental kidney ischaemia-reperfusion, leading to chronic injury. IL-34 promoted proliferation of both intrarenal macrophages and bone marrow cells, increasing circulating neutrophils and monocytes and their kidney recruitment. Thus, injured tubular cells release two CSF-1R activators, one (CSF-1) that promotes tubular cell survival and kidney repair and another (IL-34) that promotes chronic kidney damage. These results hold promise for the development of IL-34-targeting strategies to prevent ischaemia-reperfusion kidney injury in contexts such as kidney transplantation. However, careful consideration should be given to the recent characterization by Bezie et al. (J Clin Invest 2015; 125: 3952-3964) of IL-34 as a T regulatory cell (Treg) cytokine that modulates macrophage responses so that IL-34-primed macrophages potentiate the immune suppressive capacity of Tregs and promote graft tolerance.

  18. Quantitative proteomics reveals the induction of mitophagy in tumor necrosis factor-α-activated (TNFα) macrophages.

    PubMed

    Bell, Christina; English, Luc; Boulais, Jonathan; Chemali, Magali; Caron-Lizotte, Olivier; Desjardins, Michel; Thibault, Pierre

    2013-09-01

    Macrophages play an important role in innate and adaptive immunity as professional phagocytes capable of internalizing and degrading pathogens to derive antigens for presentation to T cells. They also produce pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that mediate local and systemic responses and direct the development of adaptive immunity. The present work describes the use of label-free quantitative proteomics to profile the dynamic changes of proteins from resting and TNF-α-activated mouse macrophages. These analyses revealed that TNF-α activation of macrophages led to the down-regulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the down-regulation of mitochondria proteins occurred through mitophagy and was specific to TNF-α, as other cytokines such as IL-1β and IFN-γ had no effect on mitochondria degradation. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-α enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules. These findings highlight an unsuspected role of TNF-α in mitophagy and expanded our understanding of the mechanisms responsible for MHC presentation of self-antigens.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. Role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in gastric ulcer healing in mice.

    PubMed

    Kawahara, Y; Nakase, Y; Isomoto, Y; Matsuda, N; Amagase, K; Kato, S; Takeuchi, K

    2011-08-01

    We examined the role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in the healing of gastric ulcers in mice. Male M-CSF-deficient (op/op) and M-CSF-expressing heterozygote (+/?) mice were used. Gastric ulcers were induced by thermal cauterization under ether anesthesia, and healing was observed for 14 days after ulceration. The numbers of macrophages and microvessels in the gastric mucosa were determined immunohistochemically with anti-CD68 and anti-CD31 antibodies, respectively. Expression of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF) mRNA was determined via real-time reverse transcription-polymerase chain reaction (RT-PCR), and the mucosal content of prostaglandin (PG) E(2) was determined via enzyme immunoassay on day 10 after ulceration. The healing of gastric ulcers was significantly delayed in op/op mice compared with +/? mice. Further, significantly fewer macrophages were observed in the normal gastric mucosa of op/op mice than in +/? mice. Ulcer induction caused a marked accumulation of macrophages around the ulcer base in +/? mice, but this response was attenuated in op/op mice. The mucosal PGE(2) content as well as the expression of COX-2, VEGF, and TNF-α mRNA were all upregulated in the ulcerated area of +/? mice but significantly suppressed in op/op mice. The degree of vascularization in the ulcerated area was significantly lower in op/op mice than in +/? mice. Taken together, these results suggest that M-CSF-dependent macrophages play an important role in the healing of gastric ulcers, and that this action may be associated with angiogenesis promoted by upregulation of COX-2/PGE(2) production.

  1. 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

  2. Influence of cadmium, lead, and zinc on the ability of guinea pig macrophages to interact with macrophage migration inhibitory factor

    SciTech Connect

    Kiremidjian-Schumacher, L.; Stotzky, G.; Dickstein, R.A.; Schwartz, J.

    1981-02-01

    The effects of cadmium, lead, and zinc on the ability of guinea pig macrophages to migrate and to interact with migration inhibitory factor (MIF) were studied by cell electrophoresis and the indirect migration inhibition assay. The metals significantly inhibited the motility of the cells and decreased or abolished the effect of MIF on their migration.

  3. Regulation of Alternative Macrophage Activation in the Liver following Acetaminophen Intoxication by Stem Cell-Derived Tyrosine Kinase

    PubMed Central

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

    2012-01-01

    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−/− 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 hr 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−/− mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK −/− mice. Whereas F4/80+ macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK−/− 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−/− mice treated with acetaminophen. These data

  4. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells.

    PubMed

    Godiska, R; Chantry, D; Raport, C J; Sozzani, S; Allavena, P; Leviten, D; Mantovani, A; Gray, P W

    1997-05-05

    A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophage-derived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin-Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2-activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

  6. Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

    PubMed

    Cerri, Chiara; Chimenti, Daniele; Conti, Ilaria; Neri, Tommaso; Paggiaro, Pierluigi; Celi, Alessandro

    2006-08-01

    Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

  7. Kinetics of tumor necrosis factor production by photodynamic-therapy-activated macrophages

    NASA Astrophysics Data System (ADS)

    Pass, Harvey I.; Evans, Steven; Perry, Roger; Matthews, Wilbert

    1990-07-01

    The ability of photodynamic therapy (PDT) to activate macrophages and produce cytokines, specifically tumor necrosis factor (TNF), is unknown. Three day thioglycolate elicited macrophages were incubated with 25 ug/mi Photofrin II (P11) for 2 hour, after which they were subjected to 630 nm light with fluences of 0-1800 J/m. The amount of TNF produced in the system as well as macrophage viability was measured 1, 3, 6, and 18 hours after POT. The level of TNF produced by the macrophages was significantly elevated over control levels 6 hours after POT and the absolute level of tumor necrosis factor production was influenced by the treatment energy and the resulting macrophage cytotoxicity. These data suggest that POT therapy induced cytotoxicity in vivo may be amplified by macrophage stimulation to secrete cytokines and these cytokines may also participate in other direct/indirect photodynamic therapy effects, i.e. immunosuppression, vascular effects.

  8. Production of macrophage migration inhibitory factor by human and murine neuroblastoma.

    PubMed

    Bin, Qian; Johnson, Bryon D; Schauer, Dennis W; Casper, James T; Orentas, Rimas J

    2002-01-01

    Tumor cells avoid immune recognition by subverting the ability of the immune system to mount an inflammatory response that generates cytotoxic effector cells. This can be achieved through cytokine production by the tumor itself. Our objective was to determine the cytokine profile of neuroblastoma (NB) lesions in tumor vaccine models. We found that the murine NB cell line, Neuro2a, secretes macrophage migration inhibitory factor, MIF, a multifunctional cytokine with the potential to block effective immune responses to a tumor. Patient-derived NB cell lines were also found to produce MIF. MIF production by NB was documented at the level of RNA by RNAse protection, soluble cytokine production by ELISA, and in a macrophage migration assay. Our studies also confirmed reports of IL-6 production by human NB cell lines. NB culture-derived MIF was also shown to activate tumor cell migration. This supports the hypothesis that MIF is a tumor-derived cytokine that may play a role in NB aggressiveness and evasion of immune recognition. Copyright 2002 S. Karger AG, Basel

  9. 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

  10. Immunoregulation of bovine macrophages by factors in the salivary glands of Rhipicephalus microplus

    PubMed Central

    2012-01-01

    Background Alternative strategies are required to control the southern cattle tick, Rhipicephalus microplus, due to evolving resistance to commercially available acaricides. This invasive ectoparasite is a vector of economically important diseases of cattle such as bovine babesiosis and anaplasmosis. An understanding of the biological intricacies underlying vector-host-pathogen interactions is required to innovate sustainable tick management strategies that can ultimately mitigate the impact of animal and zoonotic tick-borne diseases. Tick saliva contains molecules evolved to impair host innate and adaptive immune responses, which facilitates blood feeding and pathogen transmission. Antigen presenting cells are central to the development of robust T cell responses including Th1 and Th2 determination. In this study we examined changes in co-stimulatory molecule expression and cytokine response of bovine macrophages exposed to salivary gland extracts (SGE) obtained from 2-3 day fed, pathogen-free adult R. microplus. Methods Peripheral blood-derived macrophages were treated for 1 hr with 1, 5, or 10 μg/mL of SGE followed by 1, 6, 24 hr of 1 μg/mL of lipopolysaccharide (LPS). Real-time PCR and cytokine ELISA were used to measure changes in co-stimulatory molecule expression and cytokine response. Results Changes were observed in co-stimulatory molecule expression of bovine macrophages in response to R. microplus SGE exposure. After 6 hrs, CD86, but not CD80, was preferentially up-regulated on bovine macrophages when treated with 1 μg/ml SGE and then LPS, but not SGE alone. At 24 hrs CD80, CD86, and CD69 expression was increased with LPS, but was inhibited by the addition of SGE. SGE also inhibited LPS induced upregulation of TNFα, IFNγ and IL-12 cytokines, but did not alter IL-4 or CD40 mRNA expression. Conclusions Molecules from the salivary glands of adult R. microplus showed bimodal concentration-, and time-dependent effects on differential up-regulation of CD

  11. Simultaneous labeling of lipoprotein intracellular trafficking in pigeon monocyte-derived macrophages.

    PubMed Central

    Jones, N. L.

    1997-01-01

    Macrophage foam cell formation resulting from the accumulation of cholesterol and cholesterol esters derived from plasma lipoproteins is important for progression of atherosclerosis. Hypothetically, intracellular processing of lipoproteins that stimulate foam cell formation differs from processing of lipoproteins that do not. To test this, we examined simultaneous subcellular trafficking of lipoproteins in pigeon monocyte-derived macrophages. Pigeon beta-very-low-density lipoprotein (beta-VLDL), low-density lipoprotein (LDL), and acetylated low-density lipoprotein (Ac-LDL), differentially labeled with colloidal gold, were added in pairs to cells at 4 degrees C for 2 hours before uptake at 18 degrees C, 22 degrees C, or 37 degrees C for either 30 minutes or 2 hours. The colloidal gold distribution and percent co-labeling as observed by transmission electron microscopy were determined for organelles of the endocytic pathway. Incubations at 18 degrees C and 22 degrees C blocked lipoprotein trafficking to lysosomes. Incubation at 18 degrees C increased the percent distribution of lipoproteins in the endocytic pathway up to the early cisternal endosomes. Incubations at 22 degrees C resulted in a greater distribution of lipoproteins in the spherical late endosomes and late endosomal-prelysosomal tubular reticular compartment. The distribution in the endocytic pathway was a factor of time and temperature rather than lipoprotein type. The percentage of co-labeling of organelles for the three pairs of lipoproteins examined, Ac-LDL plus beta-VLDL, LDL plus beta-VLDL, and LDL plus Ac-LDL, was similar. Fewer noncoated and clathrin-coated pits and vesicles were co-labeled (average of 6%, maximum of 17%) than the rest of the endocytic pathway, early cisternal endosomes, spherical late endosomes, late endosomal-prelysosomal tubuloreticular compartment, and spherical lysosomes (average of 36%, maximum of 47%). The 36% of co-labeled later endocytic organelles contained an average

  12. Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity.

    PubMed

    Wintergerst, E S; Jelk, J; Asmis, R

    1998-09-01

    Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36+CD14-LDL-R-(58+/-12%), CD36+CD14+LDL-R+(18+/-5%), the remaining cells being CD36-CD14- LDL-R-. The first two subsets decreased in size during further differentiation (51+/-12% and 8+/-3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis.

  13. Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages

    PubMed Central

    Bannantine, John P.; Mack, Victoria; Fry, Lindsay M.; Davis, William C.

    2016-01-01

    Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2), plasmacytoid DC (pDC), and monocyte derived DC (MoDC). DC of Artiodactyla (pigs and ruminants) can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC). Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb) to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ). Functional analysis revealed each of these populations can take up and process antigens (Ags), present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo. PMID:27764236

  14. Small Heat-Shock Proteins, IbpAB, Protect Non-Pathogenic Escherichia coli from Killing by Macrophage-Derived Reactive Oxygen Species

    PubMed Central

    Goeser, Laura; Fan, Ting-Jia; Tchaptchet, Sandrine; Stasulli, Nikolas; Goldman, William E.; Sartor, R. Balfour; Hansen, Jonathan J.

    2015-01-01

    Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn’s disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox-/-) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox-/- macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains. PMID:25798870

  15. 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.

  16. Krüppel Like Factor 4 Promoter Undergoes Active Demethylation during Monocyte/Macrophage Differentiation

    PubMed Central

    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

  17. HIV-1 Nef activates STAT1 in human monocytes/macrophages through the release of soluble factors.

    PubMed

    Federico, M; Percario, Z; Olivetta, E; Fiorucci, G; Muratori, C; Micheli, A; Romeo, G; Affabris, E

    2001-11-01

    Monocytes/macrophages play a predominant role in the immunologic network by secreting and reacting to a wide range of soluble factors. Human immunodeficiency virus (HIV) infection leads to deep immunologic dysfunctions, also as a consequence of alterations in the pattern of cytokine release. Recent studies on in vivo models demonstrated that the expression of HIV Nef alone mimics many pathogenetic effects of HIV infection. In particular, Nef expression in monocytes/macrophages has been correlated with remarkable modifications in the pattern of secreted soluble factors, suggesting that the interaction of Nef with monocytes/macrophages plays a role in the pathogenesis of acquired immunodeficiency syndrome (AIDS). This study sought to define possible alterations in intracellular signaling induced by Nef in monocytes/macrophages. Results demonstrate that HIV-1 Nef specifically activates both alpha and beta isoforms of the signal transducer and activator of transcription 1 (STAT1). This was observed both by infecting human monocyte-derived macrophages (MDMs) with HIV-1 deletion mutants, and by exploiting the ability of MDMs to internalize soluble, recombinant Nef protein (rNef). STAT1-alpha activation occurs on phosphorylation of both C-terminal Tyr701 and Ser727 and leads to a strong binding activity. Nef-dependent STAT1 activation is followed by increased expression of both STAT1 and interferon regulatory factor-1, a transcription factor transcriptionally regulated by STAT1 activation. It was also established that Nef-induced STAT1- alpha/beta activation occurs through the secretion of soluble factors. Taken together, the results indicate that HIV-1 Nef could interfere with STAT1-governed intracellular signaling in human monocytes/macrophages.

  18. A Rapid Increase in Macrophage-Derived Versican and Hyaluronan in Infectious Lung Disease

    PubMed Central

    Chang, Mary Y.; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G.; Chan, Christina K.; Johnson, Pamela Y.; Wight, Thomas N.; Frevert, Charles W.

    2014-01-01

    The goals of this study were to characterize the changes to chondroitin sulfate proteoglycans and hyaluronan in lungs in the acute response to gram-negative bacterial infection, and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection. PMID:24472738

  19. A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

    PubMed Central

    Chang, Mary Y.; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G.; Chan, Christina K.; Johnson, Pamela Y.; Wight, Thomas N.; Frevert, Charles W.

    2014-01-01

    The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection. PMID:24727035

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

    PubMed Central

    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

    2016-01-01

    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

  1. Macrophage derived cystatin B/cathepsin B in HIV replication and neuropathogenesis

    PubMed Central

    Rivera, Linda E; Colón, Krystal; Cantres-Rosario, Yisel M; Zenón, Frances M; Meléndez, Loyda M

    2013-01-01

    Mononuclear phagocytes including monocytes and macrophages, are important defense components of innate immunity, but can be detrimental in HIV-1 infection by serving as the principal reservoirs of virus in brain and triggering a strong immune response. These viral reservoirs represent a challenge to HIV-1 eradication since they continue producing virus in tissue despite antiretroviral therapy. HIV-1 associated neurocognitive disorders (HAND) involves alterations to the blood-brain barrier and migration of activated HIV-1 infected monocytes to the brain with subsequent induced immune activation response. Our group recently showed that HIV replication in monocyte-derived macrophages (MDM) is associated with increased cystatin B. This cysteine protease inhibitor also inhibits the interferon-induced antiviral response by decreasing levels of tyrosine phosphorylated STAT-1. These recent discoveries reveal novel mechanisms of HIV persistence that could be targeted by new therapeutic approaches to eliminate HIV in macrophage reservoirs. However, cystatin B has been also associated with neuroprotection. Cystatin B is an inhibitor of the cysteine protease cathepsin B, a potent neurotoxin. During HIV-1 infection cystatin B and cathepsin B are upregulated in macrophages, meanwhile there is a reduction in cystatin/cathepsin interactions, leading to increased cathepsin B secretion and activity that induce neuronal apoptosis. Increased intracellular expression of both proteins was recently found in monocytes from Hispanic women with HAND. These findings provide new evidence for the role of cathepsin/cystatin system in the neuropathogenesis induced by HIV-infected macrophages. We summarize recent research on cystatin B and one of its substrates, cathepsin B, in HIV replication in macrophages and neuropathogenesis. PMID:24862331

  2. A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

    PubMed

    Chang, Mary Y; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G; Chan, Christina K; Johnson, Pamela Y; Wight, Thomas N; Frevert, Charles W

    2014-02-01

    The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

  3. Reprint of: A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

    PubMed

    Chang, Mary Y; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G; Chan, Christina K; Johnson, Pamela Y; Wight, Thomas N; Frevert, Charles W

    2014-04-01

    The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

  4. Actinobacillus pleuropneumoniae serotype 10 derived ApxI induces apoptosis in porcine alveolar macrophages.

    PubMed

    Chien, Maw-Sheng; Chan, You-Yu; Chen, Zeng-Weng; Wu, Chi-Ming; Liao, Jiunn-Wang; Chen, Ter-Hsin; Lee, Wei-Cheng; Yeh, Kuang-Sheng; Hsuan, Shih-Ling

    2009-03-30

    Actinobacillus pleuropneumoniae (AP) is the causative agent of swine pleuropneumonia, a fibrinous, exudative, hemorrhagic, necrotizing pleuropneumonia affecting all ages of pigs. Actinobacillus pleuropneumoniae exotoxins (Apx) are one of the major virulence factors of AP. Due to the complex nature of Apx toxins produced by AP, little is known regarding the interactions of individual species of Apx toxin with target cells. The objective of this study was to examine whether AP serotype 10-derived exotoxin, ApxI, caused apoptosis in porcine alveolar macrophages (PAMs) and to delineate the underlying signaling pathways. Isolated PAMs were stimulated with different concentrations of native ApxI and monitored for apoptosis using Hoechst staining, TUNEL, and DNA laddering assays. The ApxI-stimulated PAMs exhibited typical morphological features of apoptosis, including condensation of chromatin, formation of apoptotic bodies and DNA laddering. ApxI-induced apoptosis in a concentration- and time-dependent manner. Furthermore, to delineate the signaling events involved in ApxI-induced apoptosis, it was observed that caspase 3 was activated in ApxI-stimulated PAMs. Ablation of caspase 3 activity via specific inhibitors protected PAMs from apoptosis by ApxI. This study is the first to demonstrate that native ApxI causes apoptosis in PAMs at low concentrations and that these apoptotic events are mediated via a caspase 3-dependent pathway. These findings suggest a role of ApxI in AP infection as it might impair the host defense system through the induction of apoptosis in PAMs.

  5. Fucoidan modulates cytokine production and migration of THP‑1‑derived macrophages via colony‑stimulating factor‑1.

    PubMed

    Li, Peng; Wang, Huayang; Shao, Qianqian; Kong, Beihua; Qu, Xun

    2017-04-01

    Fucoidan is known for its various biological activities, including immunomodulatory effects on immune cells. However, the effect of fucoidan on the functions of macrophages remains to be elucidated. The present study examined the effects of fucoidan on cytokine production and migration of THP‑1‑derived macrophages and its potential mechanisms. Fucoidan was added during the differentiation process of THP‑1‑derived macrophages along with lipopolysaccharide and interferon‑γ for 42 h, and then macrophages were harvested for functional assays. Fucoidan altered the morphology of THP‑1‑derived macrophages, and also attenuated their migration activity and pro‑inflammatory cytokine production. Additionally, THP‑1‑derived macrophages intensively produced colony‑stimulating factor‑1 (CSF‑1), which was significantly decreased by fucoidan. CSF‑1 neutralizing antibody attenuated the basic production level of pro‑inflammatory cytokines in macrophages. Furthermore, when recombinant human CSF‑1 was added along with fucoidan, the attenuating effects of fucoidan on migration and cytokine production were significantly reversed. In conclusion, the present study suggests that macrophages appear to be a potential target in the immunomodulatory action of fucoidan, and CSF‑1 may be involved in this modulation.

  6. Interaction of Salmonella typhi strains with cultured human monocyte-derived macrophages.

    PubMed Central

    Sizemore, D R; Elsinghorst, E A; Eck, L C; Branstrom, A A; Hoover, D L; Warren, R L; Rubin, F A

    1997-01-01

    Human monocyte-derived macrophages (MDM) provided this laboratory with a tool to develop a primary-cell assay for evaluating the relative virulence of newly constructed Salmonella typhi carrier strains. In this study, the interaction with and survival within MDM were compared for delta aroA143-attenuated strains, wild-type virulent strains, and the current oral-vaccine strain, Ty21a. PMID:8975929

  7. Placental growth factor is a survival factor for tumor endothelial cells and macrophages.

    PubMed

    Adini, Avner; Kornaga, Tad; Firoozbakht, Farshid; Benjamin, Laura E

    2002-05-15

    The vascular endothelial growth factor (VEGF)-related factor, placental growth factor (PlGF),has been shown recently to play an important role in pathological VEGF-driven angiogenesis. In this study, we examine the effects of mPlGF/PlGF-2 overexpression in tumors grown from glioma cells containing a tetracycline-regulated mPlGF cDNA. Overexpression of mPlGF leads to increased tumor growth and vascular survival. When tetracycline is used to abruptly withdraw mPlGF overexpression, we see increased apoptosis in both vascular cells and macrophages. In addition, PlGF-2 induces survival gene expression and inhibits apoptosis in vitro. Thus, we propose that PlGF-2 contributes to tumor angiogenesis by providing increased survival function to endothelial cells and macrophages.

  8. 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

  9. Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

    PubMed Central

    Vendrame, Celia Maria Vieira; Carvalho, Marcia Dias Teixeira; Tempone, Andre Gustavo

    2014-01-01

    Leishmania (Leishmania) amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF)-I on interactions between L. (L.) amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS) exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L.) amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms. PMID:25294956

  10. Effect of Tinospora cordifolia on the antitumor activity of tumor-associated macrophages-derived dendritic cells.

    PubMed

    Singh, Nisha; Singh, Sukh Mahendra; Shrivastava, Pratima

    2005-01-01

    We and others previously have reported that extract prepared from medicinal plant Tinospora cordifolia shows a wide spectrum of immunoaugmentary effects. Tinospora cordifolia was shown to upregulate antitumor activity of tumor-associated macrophages (TAM). In this article we present evidence to show that an alcoholic extract of Tinospora cordifolia (ALTC) enhances the differentiation of TAM to dendritic cells (DC) in response to granulocyte/macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor. DC differentiated in vitro from TAM that were harvested from tumor-bearing mice after i.p. administration of ALTC (200 mg/kg body weight) 2 days posttumor transplantation shows an enhanced tumor cytotoxicity and production of tumoricidal soluble molecules like TNF, IL-1, and NO. Adoptive transfer of these TAM-derived DC to Dalton's lymphoma-bearing mice resulted in prolongation of survival of tumor-bearing mice. This is the first report regarding the differentiation and antitumor functions of TAM-derived DC obtained from tumor-bearing host administered with ALTC. The possible mechanisms involved also are discussed.

  11. Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro.

    PubMed

    Heinrich, Franziska; Lehmbecker, Annika; Raddatz, Barbara B; Kegler, Kristel; Tipold, Andrea; Stein, Veronika M; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner; Spitzbarth, Ingo

    2017-01-01

    Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future

  12. Piperine inhibits ABCA1 degradation and promotes cholesterol efflux from THP‐1‐derived macrophages

    PubMed Central

    Wang, Limei; Palme, Veronika; Rotter, Susanne; Schilcher, Nicole; Cukaj, Malsor; Wang, Dongdong; Ladurner, Angela; Heiss, Elke H.; Stangl, Herbert; Dirsch, Verena M.

    2016-01-01

    1 Scope Increased macrophage cholesterol efflux (ChE) is considered to have anti‐atherosclerotic effect counteracting cardiovascular disease. The principle pungent ingredient of the fruits of Piper nigrum, piperine, is identified in this study as a ChE inducer in THP‐1‐derived macrophages, and mechanisms underlying this effect are explored. 2 Methods and results Without affecting cell viability, piperine concentration‐dependently enhances ChE in THP‐1‐derived macrophages from 25 to 100 μM. The expression level of the key cholesterol transporter protein ATP‐binding cassette transporter A1 (ABCA1) is significantly upregulated by piperine, as revealed by western blot analyses. However, two other ChE‐mediating transporter proteins, ATP‐binding cassette transporter G1 (ABCG1) and scavenger receptor class B member 1 (SR‐B1), remain unaffected. Piperine exerts no influence on ABCA1 mRNA levels, but significantly inhibits the degradation of ABCA1, as evident by an increased half‐life of the protein in the presence of cycloheximide. Furthermore, it is found that piperine likely interferes with the calpain‐mediated ABCA1 degradation pathway and exhibits significant inhibition of calpain activity. 3 Conclusion Our findings suggest that piperine promotes ChE in THP‐1‐derived macrophages by upregulation of ABCA1, which might be mediated by inhibition of calpain activity. This novel bioactivity makes the dietary constituent piperine a good candidate to be further explored for therapeutic or preventive applications in the context of atherosclerosis. PMID:27862930

  13. Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization.

    PubMed Central

    Broide, D H; Firestein, G S

    1991-01-01

    Airway inflammation is thought to play an important role in the pathogenesis of asthma. We have used in situ hybridization and an immunoassay to determine whether granulocyte macrophage colony-stimulating factor (GM-CSF) (a cytokine capable of eosinophil activation) is present in the airway of asthmatics (n = 6) who have 37.0 +/- 15.1% airway eosinophilia after endobronchial allergen challenge. Levels of immunoreactive GM-CSF (less than 4 pg/ml pre-allergen versus 180.5 +/- 46.9 pg/ml post-allergen) increased significantly 24 h after endobronchial allergen stimulation. The cellular source of bronchoalveolar lavage (BAL) GM-CSF, as determined by in situ hybridization and immunoperoxidase staining, was derived predominantly from UCHL-1 positive BAL lymphocytes, as well as from a smaller population of alveolar macrophages. Before local endobronchial allergen challenge, less than 1% of lymphocytes and alveolar macrophages recovered by BAL expressed GM-CSF mRNA, whereas after allergen stimulation 92.6 +/- 3.4% of lymphocytes and 17.5 +/- 22.7% of alveolar macrophages expressed GM-CSF mRNA. This study provides evidence that in an experimental model of allergen-induced asthma, activation of the immune and inflammatory response (BAL lymphocyte and alveolar macrophage production of GM-CSF) is temporally associated with an inflammatory cell influx of eosinophils into the airway. Images PMID:1885766

  14. Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction.

    PubMed

    Paiva, Claudia N; Arras, Rosa H; Magalhães, Elisabeth S; Alves, Letícia S; Lessa, Luiz Paulo; Silva, Maria Helena; Ejzemberg, Regina; Canetti, Cláudio; Bozza, Marcelo T

    2009-05-01

    Deposition of immune complexes (IC) triggers Fc gamma R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif-/- mice produced smaller amounts of TNF when stimulated with IgG-opsonized erythrocytes than wild-type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC-induced inflammation, we demonstrated that Mif-/- mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP-2, and keratinocyte-derived chemokine compared with WT controls. Adoptive transfer of alveolar macrophages from WT to Mif-/- mice rescued pulmonary neutrophil recruitment and TNF production upon passive reverse Arthus reaction. Our study indicates that Arthus inflammatory reaction is largely dependent on MIF and poses macrophages as a source of the MIF released upon IC recognition. These results give experimental support to the proposition that blockade of MIF might constitute an adjunctive, therapeutic approach to IC disease.

  15. Cloning the human gene for macrophage migration inhibitory factor (MIF)

    SciTech Connect

    Paralkar, V.; Wistow, G. )

    1994-01-01

    Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. However, recent work strongly suggests a wider role for MIF beyond the immune system. It is expressed specifically in the differentiating cells of the immunologically privileged eye lens and brain, is a delayed early response gene in fibroblasts, and is expressed in many tissues. Here, the authors report the structure of the remarkably small gene for human MIF that has three exons separated by introns of only 189 and 95 bp and covers less than 1 kb. The cloned sequence also includes 1 kb of 5[prime] flanking region. Primer extension and 5[prime] rapid amplification of cDNA ends (RACE) of human brain RNA both indicate the presence of a single transcription start site in a TATA-less promoter. Northern blot analysis shows a single size of MIF mRNA (about 800 nt) in all human tissues examined. In contrast to previous reports, they find no evidence for multiple genes for MIF in the human genome. 20 refs., 3 figs.

  16. 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.

  17. 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.

  18. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

    PubMed

    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

    2012-08-31

    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.

  19. Expression of macrophage migration inhibitory factor in diffuse systemic sclerosis

    PubMed Central

    Selvi, E; Tripodi, S; Catenaccio, M; Lorenzini, S; Chindamo, D; Manganelli, S; Romagnoli, R; Ietta, F; Paulesu, L; Miracco, C; Cintorino, M; Marcolongo, R

    2003-01-01

    Objective: To evaluate whether, in patients with the diffuse form of systemic sclerosis (dSSc), macrophage migration inhibitory factor (MIF) production is dysregulated. Methods: 10 patients with dSSc and 10 healthy controls, matched for age and sex, were studied. MIF expression was evaluated by immunohistochemistry on formalin fixed skin biopsies of patients with dSSc and controls. MIF levels were assayed in the sera and in the supernatants of skin cultured fibroblasts by a colorimetric sandwich enzyme linked immunosorbent assay (ELISA). MIF concentrations in culture medium samples and in serum samples were compared by Student's two tailed t test for unpaired data. Results: Anti-MIF antibody immunostained the basal and mainly suprabasal keratinocytes. Small perivascular clusters of infiltrating mononuclear cells were positive; scattered spindle fibroblast-like cells were immunostained in superficial and deep dermal layers. The serum concentrations of MIF in patients with dSSc (mean (SD) 10705.6 (9311) pg/ml) were significantly higher than in controls (2157.5 (1288.6) pg/ml; p=0.011); MIF levels from dSSc fibroblast cultures (mean (SD) 1.74 (0.16) ng/2x105 cells) were also significantly higher than in controls (0.6 (0.2) ng/2x105 cells; p=0.008). Conclusion: These results suggest that MIF may be involved in the amplifying proinflammatory loop leading to scleroderma tissue remodelling. PMID:12695161

  20. Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

    PubMed Central

    Rajasekaran, Deepa; Gröning, Sabine; Schmitz, Corinna; Zierow, Swen; Drucker, Natalie; Bakou, Maria; Kohl, Kristian; Mertens, André; Lue, Hongqi; Weber, Christian; Xiao, Annie; Luker, Gary; Kapurniotu, Aphrodite; Lolis, Elias; Bernhagen, Jürgen

    2016-01-01

    An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4. PMID:27226569

  1. Tumor necrosis factor production by human sarcoid alveolar macrophages.

    PubMed Central

    Bachwich, P. R.; Lynch, J. P.; Larrick, J.; Spengler, M.; Kunkel, S. L.

    1986-01-01

    Tumor necrosis factor (TNF) is an oncolytic peptide that may also exert many other biologic effects. Experimentally, immunologically activated mononuclear phagocytes stimulated with endotoxin (LPS) produce TNF, while resting mononuclear phagocytes stimulated with LPS produce little TNF. To date, the ability of human alveolar macrophages (AMs) to produce TNF has not been clearly delineated. As pulmonary sarcoidosis is a granulomatous inflammatory disorder characterized by immunologically activated AMs, we investigated the production of TNF by AMs obtained by bronchoalveolar lavage from 7 normal volunteers and 13 patients with pulmonary sarcoidosis. The AMs were cultured with and without LPS, and TNF production was assessed by an in vitro cytotoxicity assay. Unstimulated sarcoid and normal AMs produced little TNF, but LPS stimulation enhanced TNF production by both normal and sarcoid AMs. Furthermore, LPS-stimulated sarcoid AMs produced more TNF than normal AMs (84.9 +/- 16.7 versus 32.5 +/- 10.2 units/million cells, P less than 0.05). It is concluded that human AMs can produce TNF and that sarcoid AMs are primed and can produce significantly more TNF, compared with normal AMs. PMID:3799813

  2. Platelet activating factor raises intracellular calcium ion concentration in macrophages

    PubMed Central

    1986-01-01

    Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in

  3. 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

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

    PubMed

    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.

  5. RNY (YRNA)-derived small RNAs regulate cell death and inflammation in monocytes/macrophages.

    PubMed

    Hizir, Zoheir; Bottini, Silvia; Grandjean, Valerie; Trabucchi, Michele; Repetto, Emanuela

    2017-01-05

    The recent discovery of new classes of small RNAs has opened unknown territories to explore new regulations of physiopathological events. We have recently demonstrated that RNY (or Y RNA)-derived small RNAs (referred to as s-RNYs) are an independent class of clinical biomarkers to detect coronary artery lesions and are associated with atherosclerosis burden. Here, we have studied the role of s-RNYs in human and mouse monocytes/macrophages and have shown that in lipid-laden monocytes/macrophages s-RNY expression is timely correlated to the activation of both NF-κB and caspase 3-dependent cell death pathways. Loss- or gain-of-function experiments demonstrated that s-RNYs activate caspase 3 and NF-κB signaling pathways ultimately promoting cell death and inflammatory responses. As, in atherosclerosis, Ro60-associated s-RNYs generated by apoptotic macrophages are released in the blood of patients, we have investigated the extracellular function of the s-RNY/Ro60 complex. Our data demonstrated that s-RNY/Ro60 complex induces caspase 3-dependent cell death and NF-κB-dependent inflammation, when added to the medium of cultured monocytes/macrophages. Finally, we have shown that s-RNY function is mediated by Toll-like receptor 7 (TLR7). Indeed using chloroquine, which disrupts signaling of endosome-localized TLRs 3, 7, 8 and 9 or the more specific TLR7/9 antagonist, the phosphorothioated oligonucleotide IRS954, we blocked the effect of either intracellular or extracellular s-RNYs. These results position s-RNYs as relevant novel functional molecules that impacts on macrophage physiopathology, indicating their potential role as mediators of inflammatory diseases, such as atherosclerosis.

  6. Characterization of the interaction of African swine fever virus with monocytes and derived macrophage subsets.

    PubMed

    Franzoni, Giulia; Graham, Simon P; Giudici, Silvia Dei; Bonelli, Piero; Pilo, Giovannantonio; Anfossi, Antonio G; Pittau, Marco; Nicolussi, Paola S; Laddomada, Alberto; Oggiano, Annalisa

    2017-01-01

    African swine fever (ASF) is a devastating disease for which there is no vaccine available. The ASF virus (ASFV) primarily infects cells of the myeloid lineage and this tropism is thought to be crucial for disease pathogenesis. A detailed in vitro characterization of the interactions of a virulent Sardinian isolate (22653/14) and a tissue culture adapted avirulent strain (BA71V) of ASFV with porcine monocytes, un-activated (moMΦ), classically (moM1) and alternatively (moM2) activated monocyte-derived macrophages was conducted in an attempt to better understand this relationship. Using a multiplicity-of-infection (MOI) of 1, both viruses were able to infect monocytes and macrophage subsets, but BA71V presented a reduced ability to infect moM1 compared to 22653/14, with higher expression of early compared to late proteins. Using an MOI of 0.01, only 22653/14 was able to replicate in all the macrophage subsets, with initially lowest in moM1 and moM2. No differences were observed in the expression of CD163 between ASFV infected and uninfected bystander cells. ASFV down-regulated CD16 expression but did not modulate MHC class II levels in monocytes and macrophage subsets. BA71V-infected but not 22653/14-infected moMΦ and moM2 presented with a reduced expression of MHC class I compared to the mock-infected controls. Higher levels of IL-18, IL1-β and IL-1α were released from moM1 after infection with BA71V compared to 22653/14 or mock-infected control. These results revealed differences between these ASFV strains, suggesting that virulent isolates have evolved mechanisms to counteract activated macrophages responses, promoting their survival, dissemination in the host and so ASF pathogenesis.

  7. Production of modified C-reactive protein in U937-derived macrophages.

    PubMed

    Ciubotaru, Irina; Potempa, Lawrence A; Wander, Rosemary C

    2005-11-01

    Plasma C-reactive protein (CRP) has been proposed to be a strong independent predictor for cardiovascular disease. This circulating form of CRP (native CRP or nCRP) is well described. Recently, the existence of a conformationally distinct isoform of CRP (modified CRP or mCRP) has been reported. The relevance of each CRP isoform to atherosclerotic disease is unknown. The purpose of this study was to examine the natural expression of CRP in undifferentiated, differentiated, and stimulated macrophages, cells known to contribute to atherogenesis in vivo, and to determine whether transcribed CRP was expressed as nCRP or mCRP. Macrophages were generated from U937 monocytes using phorbol 12-myristate 13-acetate. Differentiated macrophages were further stimulated with lipopolysaccharides (LPS). In undifferentiated, differentiated, and stimulated cells, CRP expression was assessed by reverse transcription-polymerase chain reaction, and CRP protein production was measured by fluorescence microscopy and flow cytometry (cellular CRP) or high-sensitivity enzyme-linked immunosorbent assay (secreted CRP). CRP transcript was minimally expressed in undifferentiated cells. Expression increased markedly in macrophages during differentiation and was not affected by LPS at 24 hrs. Cellular CRP protein increased in a time-dependent manner after LPS stimulation, and this induction was mediated via interleukin (IL)-6 and IL-1beta. A small amount of secreted CRP was detected in the media of differentiated cells, but it was not significantly increased after LPS stimulation. Using specific monoclonal antibodies, our data indicate that cellular CRP is directly translated as the mCRP rather than the nCRP isomer. These results indicate that U937-derived macrophages are a good cell model to further study the production of mCRP under conditions relevant for the atherogenic process.

  8. Hemozoin Enhances Maturation of Murine Bone Marrow Derived Macrophages and Myeloid Dendritic Cells.

    PubMed

    Waseem, Shahid; Ur-Rehman, Kashif; Kumar, Ramesh; Mahmood, Tariq

    2016-03-01

    Falciparum malaria is a severe health burden worldwide. Antigen presenting cells are reported to be affected by erythrocytic stage of the parasite. Malarial hemozoin (HZ), a metabolite of malaria parasite, has adjuvant properties and may play a role in the induction of immune response against the parasite. To determine the immunological impact of hemozoin on the capacity of innate immune cells maturation. Plasmodium falciparum (F32 strain) was cultured in O+ blood group up to 18% parasitemia. Natural hemozoin was extracted from infected red blood cells. Murine bone marrow derived macrophages and myeloid dendritic cells were stimulated with 4 μg/mL or 40 μg/mL of synthetic hemozoin (β-hematin) or natural hemozoin. We assessed the immunomodulatory role of synthetic or natural hemozoinin vitro by flowcytometric analysis. The maturation markers MHC-II, CD80 and CD86 were significantly upregulated (p<0.05) on the surface of murine bone marrow derived macrophages or myeloid dendritic cells. Data confirmed the potential of macrophages or myeloid dendritic cells, through hemozoin activation, to establish an innate immune response against malaria parasites. Both synthetic and natural hemozoin are potent inducers of cellular immunity against malaria infection. However, natural hemozoin is a stronger inducer as compared to synthetic hemozoin.

  9. Ca(2+)-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

    PubMed

    Vashi, Nimi; Andrabi, Syed Bilal Ahmad; Ghanwat, Swapnil; Suar, Mrutyunjay; Kumar, Dhiraj

    2017-03-31

    The role of Golgi apparatus during phagocytic uptake by macrophages has been ruled out in the past. Notably, all such reports were limited to Fcγ receptor-mediated phagocytosis. Here, we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos, except the IgG-coated ones. We report recruitment of mannosidase-II-positive Golgi-derived vesicles during uptake of diverse targets, including latex beads, Escherichia coli, Salmonella typhimurium, and Mycobacterium tuberculosis in human and mouse macrophages. The recruitment of mannosidase-II vesicles was an early event mediated by focal exocytosis and coincided with the recruitment of transferrin receptor, VAMP3, and dynamin-2. Brefeldin A treatment inhibited mannosidase-II recruitment and phagocytic uptake of serum-coated or -uncoated latex beads and E. coli However, consistent with previous studies, brefeldin A treatment did not affect uptake of IgG-coated latex beads. Mechanistically, recruitment of mannosidase-II vesicles during phagocytic uptake required Ca(2+) from both extra- and intracellular sources apart from PI3K, microtubules, and dynamin-2. Extracellular Ca(2+) via voltage-gated Ca(2+) channels established a Ca(2+)-dependent local phosphatidylinositol 1,4,5-trisphosphate gradient, which guides the focal movement of Golgi-derived vesicles to the site of uptake. We confirmed Golgi-derived vesicles recruited during phagocytosis were secretory vesicles as their recruitment was sensitive to depletion of VAMP2 or NCS1, whereas recruitment of the recycling endosome marker VAMP3 was unaffected. Depletion of both VAMP2 and NCS1 individually resulted in the reduced uptake by macrophages. Together, the study provides a previously unprecedented role of Golgi-derived secretory vesicles in phagocytic uptake, the key innate defense function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Macrophage Migration Inhibitory Factor Deficiency Augments Doxorubicin‐Induced Cardiomyopathy

    PubMed Central

    Xu, Xihui; Bucala, Richard; Ren, Jun

    2013-01-01

    Background Recent evidence has depicted a role of macrophage migration inhibitory factor (MIF) in cardiac homeostasis under pathological conditions. This study was designed to evaluate the role of MIF in doxorubicin‐induced cardiomyopathy and the underlying mechanism involved with a focus on autophagy. Methods and Results Wild‐type (WT) and MIF knockout (MIF−/−) mice were given saline or doxorubicin (20 mg/kg cumulative, i.p.). A cohort of WT and MIF−/− mice was given rapamycin (6 mg/kg, i.p.) with or without bafilomycin A1 (BafA1, 3 μmol/kg per day, i.p.) for 1 week prior to doxorubicin challenge. To consolidate a role for MIF in the maintenance of cardiac homeostasis following doxorubicin challenge, recombinant mouse MIF (rmMIF) was given to MIF−/− mice challenged with or without doxorubicin. Echocardiographic, cardiomyocyte function, and intracellular Ca2+ handling were evaluated. Autophagy and apoptosis were examined. Mitochondrial morphology and function were examined using transmission electron microscopy, JC‐1 staining, MitoSOX Red fluorescence, and mitochondrial respiration complex assay. DHE staining was used to evaluate reactive oxygen species (ROS) generation. MIF knockout exacerbated doxorubicin‐induced mortality and cardiomyopathy (compromised fractional shortening, cardiomyocyte and mitochondrial function, apoptosis, and ROS generation). These detrimental effects of doxorubicin were accompanied by defective autophagolysosome formation, the effect of which was exacerbated by MIF knockout. Rapamycin pretreatment rescued doxorubicin‐induced cardiomyopathy in WT and MIF−/− mice. Blocking autophagolysosome formation using BafA1 negated the cardioprotective effect of rapamycin and rmMIF. Conclusions Our data suggest that MIF serves as an indispensable cardioprotective factor against doxorubicin‐induced cardiomyopathy with an underlying mechanism through facilitating autophagolysosome formation. PMID:24334905

  11. Granulocyte macrophage colony stimulating factor ameliorates DSS induced experimental colitis

    PubMed Central

    Sainathan, Satheesh K.; Hanna, Eyad M.; Gong, Qingqing; Bishnupuri, Kumar S.; Luo, Qizhi; Colonna, Marco; White, Frances V.; Croze, Ed; Houchen, Courtney; Anant, Shrikant; Dieckgraefe, Brian K.

    2015-01-01

    Background Sargramostim, granulocyte–macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that Sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulphate sodium (DSS) induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs). Methods Acute colitis was induced in Balb/c mice by administration of DSS in drinking water. Mice were treated with daily GM-CSF or PBS. To probe the role of plasmacytoid DCs (pDCs) in the response to GM-CSF, we further examine the effects of monoclonal antibody 440c, which is specific for a sialic acid-binding immunoglobulin (Ig)-like lectin expressed on pDCs. Results GM-CSF ameliorates acute DSS-induced colitis; resulting in significantly improved clinical parameters and histology. Microarray analysis showed reduced expression of pro-inflammatory genes including TNFα and IL1β; results further confirmed by real-time RT-PCR and serum Bio-plex analysis. GM-CSF treatment significantly expands pDCs and type 1 IFN production. Administration of mAb 440c completely blocked the therapeutic effect of GM-CSF. GM-CSF is also effective in RAG1−/− mice, demonstrating activity independent effects on T and B cells. IFN-β administration mimics the therapeutic effect of GM-CSF in DSS-treated mice. GM-CSF increases systemic and mucosal type 1 IFN expression and exhibits synergy with pDC activators, such as microbial CpG DNA. Conclusions GM-CSF is effective in the treatment of DSS colitis in a mechanism involving the 440c+ plasmacytoid DC population. PMID:17932977

  12. Macrophage migration inhibitory factor involvement in breast cancer (Review)

    PubMed Central

    RICHARD, VINCENT; KINDT, NADÈGE; SAUSSEZ, SVEN

    2015-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine involved in many cellular processes and in particular carcinogenesis. Here, we review the experimental and clinical published data on MIF and its pathways in breast cancer. Experimental data show that MIF is overexpressed in breast cancer cells (BCC) due, at least partly, to its stabilization by HSP90 and upregulation by HIF-1α. MIF interacts with its main receptor CD74 and its co-receptor CXCR-4, both overexpressed, promoting cell survival by PI3K/Akt activation, a possible link with EGFR and HER2 pathways and inhibition of autophagy. Besides these auto- and paracrine effects on BCC, MIF interacts with BCC micro-environment by several mechanisms: immunomodulation by increasing the prevalence of immune suppressive cells, neo-angiogenesis by its link to HIF-1, and finally BCC transendothelial migration. Clinical studies show higher levels of MIF in breast cancer patients serum compared to healthy volunteers but without obvious clinical significance. In breast cancer tissue, MIF and CD74 are overexpressed in the cancer cells and in the stroma but correlations with classical prognostic factors or survival are elusive. However, an inverse correlation with the tumor size for stromal MIF and a positive correlation with the triple receptor negative tumor status for stromal CD74 seem to be showed. This set of experimental and clinical data shows the involvement of MIF pathways in breast carcinogenesis. Several anti-MIF targeted strategies are being explored in therapeutic goals and should merit further investigations. PMID:26412712

  13. Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC).

    PubMed

    Jackson, Megan V; Krasnodembskaya, Anna D

    2017-05-05

    Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM through tunnelling nanotubes (TNT). To model the human AM, primary monocytes were isolated from human donor blood and differentiated into macrophages (monocyte derived macrophages, MDM) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), thus allowing adaptation of an AM-like phenotype (de Almeida et al., 2000; Guilliams et al., 2013). Human bone-marrow derived MSC, were labelled with mitochondria-specific fluorescent stain, washed extensively, seeded into the tissue culture plate with MDMs at the ratio of 1:20 (MSC/MDM) and co-cultured for 24 h. TNT formation and mitochondrial transfer were visualised by confocal microscopy and semi-quantified by flow cytometry. By using the method we described here we established that MSC use TNTs as the means to transfer mitochondria to macrophages. Further studies demonstrated that mitochondrial transfer enhances macrophage oxidative phosphorylation and phagocytosis. When TNT formation was blocked by cytochalasin B, MSC effect on macrophage phagocytosis was completely abrogated. This is the first study to demonstrate TNT-mediated mitochondrial transfer from MSC to innate immune cells.

  14. [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

  15. Macrophage migration inhibitory factor is required for recruitment of scar-associated macrophages during liver fibrosis.

    PubMed

    Barnes, Mark A; McMullen, Megan R; Roychowdhury, Sanjoy; Madhun, Nabil Z; Niese, Kathryn; Olman, Mitchell A; Stavitsky, Abram B; Bucala, Richard; Nagy, Laura E

    2015-01-01

    Recruitment of peripheral monocytes to the liver is a key contributor to the response to injury. MIF can act as a chemokine and cytokine, regulating innate immune responses in many tissues and cell types. We hypothesized that MIF contributes to the progression of CCl4-induced hepatic fibrosis by regulating recruitment of SAM. SAMs dynamically regulate HSC activation and ECM degradation. To gain insight into the role of MIF in progression of liver fibrosis, we investigated markers of fibrosis and immune responses after chronic CCl4 administration to female C57BL/6 and MIF(-/-) mice. Chronic CCl4 exposure increased activation of HSC in WT mice, indicated by increased expression of αSMA mRNA and protein, as well as mRNA for collagen 1α1; these responses were blunted in female MIF(-/-) mice. Despite lower activation of HSC in MIF(-/-) mice, accumulation of ECM was similar in WT and MIF(-/-)mice, suggesting a decreased rate of ECM degradation. Recruitment of SAMs was lower in MIF(-/-) mice compared with WT mice, both in their initial inflammatory phenotype, as well as in the later phase as proresolution macrophages. The decreased presence of resolution macrophages was associated with lower expression of MMP13 in MIF(-/-) mice. Taken together, these data indicate that MIF-dependent recruitment of SAMs contributes to degradation of ECM via MMP13, highlighting the importance of appropriate recruitment and phenotypic profile of macrophages in the resolution of fibrosis. © Society for Leukocyte Biology.

  16. CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages.

    PubMed

    Zhang, Hanrui; Shi, Jianting; Hachet, Melanie A; Xue, Chenyi; Bauer, Robert C; Jiang, Hongfeng; Li, Wenjun; Tohyama, Junichiro; Millar, John; Billheimer, Jeffrey; Phillips, Michael C; Razani, Babak; Rader, Daniel J; Reilly, Muredach P

    2017-09-07

    To gain mechanistic insights into the role of LIPA(lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats )/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA (LIPA(-/-)) had barely detectable LAL enzymatic activity. Control and LIPA(-/-) IPSDM were loaded with [(3)H]-cholesteryl oleate-labeled acetylated low-density lipoprotein followed by efflux to apolipoprotein A-I. Efflux of liberated [(3)H]-cholesterol to apolipoprotein A-I was abolished in LIPA(-/-) IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [(3)H]-cholesterol-labeled AcLDL (acetylated low-density lipoprotein), [(3)H]-cholesterol efflux was, however, not different between control and LIPA(-/-) IPSDM. ABCA1(ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA(-/-) IPSDM. In nonlipid-loaded state, LIPA(-/-) IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA(-/-) IPSDM. LIPA(-/-) did not impact lysosomal apolipoprotein-B degradation or expression of IL1B, IL6, and CCL5. CONCLUSIONS: LIPA(-/-) IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease

  17. Differential induction of macrophage-derived cytokines by live and dead intracellular bacteria in vitro.

    PubMed

    Zhan, Y; Cheers, C

    1995-02-01

    Marked differences in the abilities of living and heat-killed Brucella abortus and Listeria monocytogenes organisms to induce production of tumor necrosis factor alpha by in vitro-cultured macrophages were observed. Interleukin-1 and interleukin-6 appeared to be under different control. The results are discussed in relation to the induction of gamma interferon-producing Th1 cells and acquired cellular resistance to infection by living vaccines but not killed vaccines.

  18. Physiology of Continuous Bone Marrow Culture Derived Permanent Granulocyte-Macrophage Progenitor Cells

    DTIC Science & Technology

    1983-08-01

    capable of differentiating to mature neutrophillic granulocytes and granulocyte-macrophage progenitor cells . Several T- cell lines including K45, JURKAT...CEM, K230 have been screened for produc- tion of Interleukin-3 by assay of supernatant from cell lines for proliferation of mouse IL-3 dependent...hematopoietic progeni- tor cell lines Lines 45 and 230 produce low levels of activity. "In contrast, IL-2 (T- cell growth factor) dependent human T- cell

  19. 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

  20. Macrophage migration inhibitory factor (MIF) enzymatic activity and lung cancer.

    PubMed

    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

    2015-04-16

    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.

  1. 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

  2. Cross talk between inflammatory cytokines and granulocyte-macrophage colony-stimulating factor in transplant vasculopathy.

    PubMed

    Sterpetti, Antonio V; Borrelli, Valeria; Ventura, Marco; Cucina, Alessandra

    2017-05-15

    Transplant vasculopathy limits the clinical results of solid organ transplantation. Thirty-three arterial grafts were implanted in the abdominal aorta of Lewis rats. The animals were humanely sacrificed 4 wk after surgery. The study groups had 15 arterial isografts and 18 arterial allografts. Growth factors and inflammatory cytokines, released by the removed grafts, were studied in organ culture. The released growth factors were analyzed in vitro to assess their effect on the proliferation of endothelial, smooth muscle cells and fibroblasts. In arterial isogenic and allogenic grafts, platelet-derived growth factor and basic fibroblastic growth factor release was minimal (P < 0.01). There was a significant release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α (TNF-α; P < 0.001) in allografts. GM-CSF and TNF-α, at concentrations in the allograft organ cultures, stimulated significantly the growth of smooth muscle cells. The simultaneous action of TNF-α and GM-CSF had an exponential growth effect on endothelial cells and smooth muscle cells. Interleukin (IL)-1, IL-2, and IL-9 were released in high quantities by allografts. In vitro, IL-1, IL-2, and IL-9 facilitated the growth effect of GM-CSF and TNF-α. Transplant vasculopathy depends on the simultaneous and complementary additive effects of several growth factors and cytokines, which have a continuous "cross talk." Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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

  4. Measurement of reactive oxygen metabolites produced by human monocyte-derived macrophages exposed to mineral dusts.

    PubMed Central

    Nyberg, P.; Klockars, M.

    1990-01-01

    The aim of the present work was to develop an in-vitro model for studying mineral dust-induced production of reactive oxygen metabolites by human macrophages. Monocytes isolated from human buffy coats were cultured in vitro for 1-6 days. Quartz particles induced both luminol- and lucigenin-dependent chemiluminescence (CL) by the adherent cells. However, the luminol response decreased form day to day, obviously due to a decrease in the myeloperoxidase (MPO) activity of the cells, whereas the lucigenin response showed no such MPO dependence. The luminol response was inhibited by superoxide dismutase (SOD), catalase, and the MPO-inhibitor azide, while the lucigenin response was inhibited by SOD and catalase but stimulated by azide. There was a positive correlation between the lucigenin responses and the results obtained with the established cytochrome c assay for superoxide, when opsonized zymosan was used as a stimulant. The effects of quartz, titanium dioxide, chrysotile asbestos, and wollastonite particles were investigated with the lucigenin assay. Quartz and chrysotile caused prominent light emission by 6-day-old macrophages, whereas titanium dioxide and wollastonite caused weak responses. We conclude that mineral dusts induce production of reactive oxygen metabolites by human monocyte-derived macrophages, and that the quantitative responses depend on both physical and physicochemical dust properties, the nature of which are still to be defined. PMID:2169299

  5. Autocrine Human Urotensin II Enhances Macrophage-Derived Foam Cell Formation in Transgenic Rabbits.

    PubMed

    Zhao, Sihai; Li, Yafeng; Gao, Shoucui; Wang, Xiaojing; Sun, Lijing; Cheng, Daxing; Bai, Liang; Guan, Hua; Wang, Rong; Fan, Jianglin; Liu, Enqi

    2015-01-01

    Circulating urotensin II (UII) is involved in the development of atherosclerosis. However, the role of autocrine UII in the development of atherosclerosis remains unclear. Here, we tested the hypothesis that autocrine UII would promote atherosclerosis. Transgenic rabbits were created as a model to study macrophage-specific expressing human UII (hUII) and used to investigate the role of autocrine UII in the development of atherosclerosis. Transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet to induce atherosclerosis. Comparing the transgenic rabbits with their nontransgenic littermates, it was observed that hUII expression increased the macrophage-positive area in the atherosclerotic lesions by 45% and the positive area ratio by 56% in the transgenic rabbits. Autocrine hUII significantly decreased the smooth muscle cell-positive area ratio in transgenic rabbits (by 54%), without affecting the plasma levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and glucose and adipose tissue contents. These results elucidated for the first time that autocrine UII plays an important role in the development of atherosclerosis by increasing the accumulation of macrophage-derived foam cell.

  6. Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.

    PubMed

    Quiros, Miguel; Nishio, Hikaru; Neumann, Philipp A; Siuda, Dorothee; Brazil, Jennifer C; Azcutia, Veronica; Hilgarth, Roland; O'Leary, Monique N; Garcia-Hernandez, Vicky; Leoni, Giovanna; Feng, Mingli; Bernal, Gabriela; Williams, Holly; Dedhia, Priya H; Gerner-Smidt, Christian; Spence, Jason; Parkos, Charles A; Denning, Timothy L; Nusrat, Asma

    2017-09-01

    In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

  7. Autocrine Human Urotensin II Enhances Macrophage-Derived Foam Cell Formation in Transgenic Rabbits

    PubMed Central

    Zhao, Sihai; Li, Yafeng; Gao, Shoucui; Wang, Xiaojing; Sun, Lijing; Cheng, Daxing; Bai, Liang; Guan, Hua; Wang, Rong; Fan, Jianglin; Liu, Enqi

    2015-01-01

    Circulating urotensin II (UII) is involved in the development of atherosclerosis. However, the role of autocrine UII in the development of atherosclerosis remains unclear. Here, we tested the hypothesis that autocrine UII would promote atherosclerosis. Transgenic rabbits were created as a model to study macrophage-specific expressing human UII (hUII) and used to investigate the role of autocrine UII in the development of atherosclerosis. Transgenic rabbits and their nontransgenic littermates were fed a high cholesterol diet to induce atherosclerosis. Comparing the transgenic rabbits with their nontransgenic littermates, it was observed that hUII expression increased the macrophage-positive area in the atherosclerotic lesions by 45% and the positive area ratio by 56% in the transgenic rabbits. Autocrine hUII significantly decreased the smooth muscle cell-positive area ratio in transgenic rabbits (by 54%), without affecting the plasma levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and glucose and adipose tissue contents. These results elucidated for the first time that autocrine UII plays an important role in the development of atherosclerosis by increasing the accumulation of macrophage-derived foam cell. PMID:26640798

  8. 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

  9. Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulate macrophages

    SciTech Connect

    Jue, Dae-Myung; Sherry, B.; Luedke, C.; Manogue, K.R.; Cerami, A. )

    1990-09-11

    The biosynthesis and processing of cachetin/tumor necrosis factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipipolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa prosequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.

  10. Macrophage Migration Inhibitory Factor as an Emerging Drug Target to Regulate Antioxidant Response Element System

    PubMed Central

    Yukitake, Hiroshi; Takizawa, Masayuki

    2017-01-01

    Oxidative stress is involved in pathophysiology and pathological conditions of numerous human diseases. Thus, understanding the mechanisms underlying the redox homeostasis in cells and organs is valuable for discovery of therapeutic drugs for oxidative stress-related diseases. Recently, by applying chemical biology approach with an ARE activator, BTZO-1, we found macrophage migration inhibitory factor (MIF) as a new regulator of antioxidant response element- (ARE-) mediated gene transcription. BTZO-1 and its active derivatives bound to MIF and protected cells and organs from oxidative insults via ARE activation in animal models with oxidative stress such as ischemia/reperfusion injury, inflammatory bowel diseases, and septic shock. In this review, we briefly highlight key findings in understanding the MIF-ARE system. PMID:28191280

  11. 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

  12. Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages

    PubMed Central

    Shatalin, Konstantin; Gusarov, Ivan; Avetissova, Ekaterina; Shatalina, Yelena; McQuade, Lindsey E.; Lippard, Stephen J.; Nudler, Evgeny

    2008-01-01

    Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen—Bacillus anthracis—critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases. PMID:18215992

  13. 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

  14. 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

    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

    2015-06-20

    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. 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. 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. 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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Changes of peripheral TGF-β1 depend on monocytes-derived macrophages in Huntington disease

    PubMed Central

    2013-01-01

    Background Huntington Disease (HD) is a neurodegenerative disorder resulting from the expansion of polyglutamine stretch in the huntingtin protein (Htt). Mutant HTT (mHtt) leads to progressive impairment of several molecular pathways that have been linked to disease pathogenesis. Defects in the production of a number of neurotrophic factors have been described as important determinants contributing to the development of HD. We have previously demonstrated that production of transforming growth factor-β1 (TGF-β1) is also deregulated in HD. Peripheral levels of TGF-β1 were markedly reduced early in the disease and returned to normal levels with disease severity. However, the cause and the biochemical origin of such abnormalities are still unclear. Results We report here that the abnormal production of peripheral TGF-β1 depends on the changes in the percentage of TGF-β1-producing macrophages along disease course. Variation in the number of TGF-β1-producing macrophages resulted from differential activation state of the same cells, which displayed phenotypic and functional heterogeneity throughout the clinical course of HD. We further demonstrated that, similar to the periphery, the number of TGF-β1-immunoreactive cells in human post-mortem brain with HD, varied with neuropathological changes. Conclusions Our data indicate that reduced bioavailability of TGF-β1 in the serum of HD subjects is attributable to the variation of the number of TGF-β1-producing macrophages. Macrophages display a differential ability to produce TGF-β1, which reflects diversity in cells polarization throughout the disease course. Besides elucidating the biochemical origin of TGF-β1 fluctuations in HD, our study highlights an interesting parallelism between periphery and central compartment and underlines the potential of TGF-β1 as a possible indicator suitable for prediction of disease onset in HD. PMID:24330808

  16. Changes of peripheral TGF-β1 depend on monocytes-derived macrophages in Huntington disease.

    PubMed

    Di Pardo, Alba; Alberti, Silvia; Maglione, Vittorio; Amico, Enrico; Cortes, Etty P; Elifani, Francesca; Battaglia, Giuseppe; Busceti, Carla L; Nicoletti, Ferdinando; Vonsattel, Jean Paul G; Squitieri, Ferdinando

    2013-12-13

    Huntington Disease (HD) is a neurodegenerative disorder resulting from the expansion of polyglutamine stretch in the huntingtin protein (Htt). Mutant HTT (mHtt) leads to progressive impairment of several molecular pathways that have been linked to disease pathogenesis. Defects in the production of a number of neurotrophic factors have been described as important determinants contributing to the development of HD. We have previously demonstrated that production of transforming growth factor-β1 (TGF-β1) is also deregulated in HD. Peripheral levels of TGF-β1 were markedly reduced early in the disease and returned to normal levels with disease severity. However, the cause and the biochemical origin of such abnormalities are still unclear. We report here that the abnormal production of peripheral TGF-β1 depends on the changes in the percentage of TGF-β1-producing macrophages along disease course. Variation in the number of TGF-β1-producing macrophages resulted from differential activation state of the same cells, which displayed phenotypic and functional heterogeneity throughout the clinical course of HD. We further demonstrated that, similar to the periphery, the number of TGF-β1-immunoreactive cells in human post-mortem brain with HD, varied with neuropathological changes. Our data indicate that reduced bioavailability of TGF-β1 in the serum of HD subjects is attributable to the variation of the number of TGF-β1-producing macrophages. Macrophages display a differential ability to produce TGF-β1, which reflects diversity in cells polarization throughout the disease course. Besides elucidating the biochemical origin of TGF-β1 fluctuations in HD, our study highlights an interesting parallelism between periphery and central compartment and underlines the potential of TGF-β1 as a possible indicator suitable for prediction of disease onset in HD.

  17. 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.

  18. CXCL17 is a major chemotactic factor for lung macrophages

    PubMed Central

    Burkhardt, Amanda M.; Maravillas-Montero, José L.; Carnevale, Christina D.; Vilches-Cisneros, Natalia; Flores, Juan P.; Hevezi, Peter A.; Zlotnik, Albert

    2014-01-01

    Chemokines are a superfamily of chemotactic cytokines that direct the movement of cells throughout the body under homeostatic and inflammatory conditions. The mucosal chemokine CXCL17 was the last ligand of this superfamily to be characterized. Several recent studies have provided greater insight into the basic biology of this chemokine and have implicated CXCL17 in several human diseases. We sought to better characterize CXCL17's activity in vivo. To this end, we analyzed its chemoattractant properties in vivo and characterized a Cxcl17-/- mouse. This mouse has a significantly reduced number of macrophages in their lungs compared to WT mice. Additionally, we observed a concurrent increase in a new population of macrophage-like cells that are F4/80+CDllcmid. These results indicate that CXCL17 is a novel macrophage chemoattractant that operates in mucosal tissues. Given the importance of macrophages in inflammation, these observations strongly suggest that CXCL17 is a major regulator of mucosal inflammatory responses. PMID:24973458

  19. TGF-β/Smad3 signalling regulates the transition of bone marrow-derived macrophages into myofibroblasts during tissue fibrosis.

    PubMed

    Wang, Shuang; Meng, Xiao-Ming; Ng, Yee-Yung; Ma, Frank Y; Zhou, Shuang; Zhang, Yang; Yang, Chen; Huang, Xiao-Ru; Xiao, Jun; Wang, Ying-Ying; Ka, Shuk-Man; Tang, Yong-Jiang; Chung, Arthur C K; To, Ka-Fai; Nikolic-Paterson, David J; Lan, Hui-Yao

    2016-02-23

    Myofibroblasts are a main cell-type of collagen-producing cells during tissue fibrosis, but their origins remains controversial. While bone marrow-derived myofibroblasts in renal fibrosis has been reported, the cell origin and mechanisms regulating their transition into myofibroblasts remain undefined. In the present study, cell lineage tracing studies by adoptive transfer of GFP+ or dye-labelled macrophages identified that monocyte/macrophages from bone marrow can give rise to myofibroblasts via the process of macrophage-myofibroblast transition (MMT) in a mouse model of unilateral ureteric obstruction. The MMT cells were a major source of collagen-producing fibroblasts in the fibrosing kidney, accounting for more than 60% of α-SMA+ myofibroblasts. The MMT process occurred predominantly within M2-type macrophages and was regulated by TGF-β/Smad3 signalling as deletion of Smad3 in the bone marrow compartment of GFP+ chimeric mice prevented the M2 macrophage transition into the MMT cells and progressive renal fibrosis. In vitro studies in Smad3 null bone marrow macrophages also showed that Smad3 was required for TGF-β1-induced MMT and collagen production. In conclusion, we have demonstrated that bone marrow-derived fibroblasts originate from the monocyte/macrophage population via a process of MMT. This process contributes to progressive renal tissue fibrosis and is regulated by TGF-β/Smad3 signalling.

  20. Echinacea purpurea Extract Polarizes M1 Macrophages in Murine Bone Marrow-Derived Macrophages Through the Activation of JNK.

    PubMed

    Fu, Aikun; Wang, Yang; Wu, Yanping; Chen, Hongliang; Zheng, Shasha; Li, Yali; Xu, Xin; Li, Weifen

    2017-09-01

    Echinacea purpurea is an indigenous North American purple cone flower used by North Americans for treatment of various infectious diseases and wounds. This study investigated the effect of polysaccharide enriched extract of Echinacea purpurea (EE) on the polarization of macrophages. The results showed that 100 µg/mL of EE could markedly activate the macrophage by increasing the expression of CD80, CD86, and MHCII molecules. Meanwhile, EE upregulated the markers of classically activated macrophages (M1) such as CCR7 and the production of IL-1β, IL-6, IL-12p70, TNF-αand NO. The functional tests showed that EE enhanced the phagocytic and intracellular bactericidal activity of macrophage against ST. Furthermore, we demonstrated that JNK are required for EE-induced NO and M1-related cytokines production. Together, these results demonstrated that EE can polarize macrophages towards M1 phenotype, which is dependent on the JNK signaling pathways. J. Cell. Biochem. 118: 2664-2671, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Foxm1 Transcription Factor is required for Macrophage Migration during Lung Inflammation and Tumor Formation

    PubMed Central

    Balli, David; Ren, Xiaomeng; Chou, Fu-Sheng; Cross, Emily; Zhang, Yufang; Kalinichenko, Vladimir V.; Kalin, Tanya V.

    2011-01-01

    Macrophages play a key role in tumor-associated pulmonary inflammation that supports proliferation of tumor cells and promotes lung tumor growth. Although increased numbers of tumor-associated macrophages (TAM) are linked to poor prognosis in lung cancer patients, little is known regarding the transcriptional mechanisms controlling recruitment of macrophages during lung tumorigenesis. Forkhead Box m1 (Foxm1) transcription factor is induced in multiple cell types within tumor lesions and its increased expression is associated with poor prognosis in patients with lung adenocarcinomas. To determine the role of Foxm1 in recruitment of TAM, a mouse line with macrophage-specific Foxm1 deletion was generated (macFoxm1−/−). Lung tumorigenesis was induced using a MCA/BHT tumor initiation/promotion protocol. Ablation of Foxm1 in macrophages reduced the number and size of lung tumors in macFoxm1−/− mice. Decreased tumorigenesis was associated with diminished proliferation of tumor cells and decreased recruitment of macrophages during the early stages of tumor formation. Expression levels of pro-inflammatory genes iNOS, Cox-2, IL-1b and IL-6, as well as migration related genes MIP-1α, MIP-2 and MMP-12, were decreased in macrophages isolated from macFoxm1−/− mice. Migration of Foxm1-deficient macrophages was reduced in vitro. The chemokine receptors responsible for monocyte recruitment to the lung, CX3CR1 and CXCR4, were decreased in Foxm1-deficient monocytes. In co-transfection experiments, Foxm1 directly bound to and transcriptionally activated CX3CR1 promoter. Adoptive transfer of wild type monocytes to macFoxm1−/− mice restored BHT-induced pulmonary inflammation to the levels observed in control mice. Expression of Foxm1 in macrophages is required for pulmonary inflammation, recruitment of macrophages into tumor sites and lung tumor growth. PMID:22139074

  2. Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

    PubMed Central

    Tan, T H; Edgerton, S A; Kumari, R; McAlister, M S; Roe, S M; Nagl, S; Pearl, L H; Selkirk, M E; Bianco, A E; Totty, N F; Engwerda, C; Gray, C A; Meyer, D J; Rowe, S M

    2001-01-01

    cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is

  3. 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

  4. A modulatory effect of novel kojic acid derivatives on cancer cell proliferation and macrophage activation.

    PubMed

    Yoo, Dae Sung; Lee, Jaehwi; Choi, Sun Shim; Rho, Ho Sik; Cho, Dong Ha; Shin, Won Cheol; Cho, Jae Youl

    2010-04-01

    We examined whether several newly synthesized derivatives of kojic acid, a compound with known antiinflammatory, anti-proliferative, and anti-oxidative properties, were able to modulate glioma cell proliferation and Toll-like receptor (TLR) 4-mediated functional activation of macrophage-managed tumor microenvironments. Anti-cancer effects on C6 glioma and SYF cells were examined by cell proliferation assays, DNA laddering assays, nuclear staining experiments, and Western blot analysis. The anti-inflammatory activities of the derivatives were assessed by measuring the production of nitric oxide (NO) and cytokine expression in macrophages (RAW264.7 cells) stimulated with the TLR 4 ligand lipopolysacchride (LPS). Among the various derivatives tested, RHS-0110 exhibited the strongest inhibitory activity on the proliferation of C6 glioma cells, with an IC50 value of 4.7 microM. However, the inhibitory effect of this compound was abrogated with respect to the proliferation of SYF cells, a cell line lacking Src, Yes, and Fyn kinases, similar to effects observed with the Src kinase inhibitor PP2. In agreement with these findings, RHS-0110 decreased the expression of Src but not the activation of Yes and Fyn. Based on DNA laddering tests and nucleus staining experiments, the anti-proliferative effects of RHS-0110 appeared to be due to a necrotic pathway. Kojic acid derivatives also suppressed LPS-induced NO production and interleukin (IL)-6 expression in RAW264.7 cells under lowered or non-cytotoxic concentrations of compounds. Because of their anti-proliferative and anti-TLR4-mediated microenvironmental formation features, our results suggest that kojic acid derivatives, including RHS-0110, may be useful as novel anti-cancer drugs.

  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. Macrophage depletion impairs skeletal muscle regeneration: The roles of regulatory factors for muscle regeneration.

    PubMed

    Liu, Xiaoguang; Liu, Yu; Zhao, Linlin; Zeng, Zhigang; Xiao, Weihua; Chen, Peijie

    2017-03-01

    Though macrophages are essential for skeletal muscle regeneration, which is a complex process, the roles and mechanisms of the macrophages in the process of muscle regeneration are still not fully understood. The objective of this study is to explore the roles of macrophages and the mechanisms involved in the regeneration of injured skeletal muscle. One hundred and twelve C57BL/6 mice were randomly divided into muscle contusion and macrophages depleted groups. Their gastrocnemius muscles were harvested at the time points of 12 h, 1, 3, 5, 7, 14 d post-injury. The changes in skeletal muscle morphology were assessed by hematoxylin and eosin (HE) stain. The gene expression was analyzed by real-time polymerase chain reaction. The data showed that CL-liposomes treatment did affect the expression of myogenic regulatory factors (MyoD, myogenin) after injury. In addition, CL-liposomes treatment decreased the expression of regulatory factors of muscle regeneration (HGF, uPA, COX-2, IGF-1, MGF, FGF6) and increased the expression of inflammatory cytokines (TGF-β1, TNF-α, IL-1β, RANTES) in the late stage of regeneration. Moreover, there were significant correlations between macrophages and some regulatory factors (such as HGF, uPA) for muscle regeneration. These results suggested that macrophages depletion impairs skeletal muscle regeneration and that the regulatory factors for muscle regeneration may play important roles in this process.

  7. Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells.

    PubMed Central

    Corey, S; Eguinoa, A; Puyana-Theall, K; Bolen, J B; Cantley, L; Mollinedo, F; Jackson, T R; Hawkins, P T; Stephens, L R

    1993-01-01

    The signalling pathways used by the GM-CSF receptor are currently unknown. Here we show that in human myeloid derived cells GM-CSF can stimulate; (i) the accumulation of PtdIns(3,4,5)P3; (ii) increases in p53/p56lyn and p62c-yes directed protein tyrosine kinase activities in anti-lyn and anti-c-yes antibody directed immunoprecipitates, respectively and; (iii) increases in phosphoinositide 3OH-kinase activity in antiphosphotyrosine, anti-p53/p56lyn and anti-p62c-yes antibody directed immunoprecipitates. These results suggest that GM-CSF can stimulate formation of protein tyrosine kinase co-ordinated signalling complexes, that contain p53/p56lyn, p62c-yes and an activated PtdInsP2 directed phosphoinositide 3OH-kinase, which can drive the accumulation of the putative second-messenger PtdIns(3,4,5)P3. Images PMID:8392933

  8. 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.

  9. 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.

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

    PubMed

    Ohyama, M; Otake, T; Morinaga, K

    2001-10-01

    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.

  11. 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

  12. The combination of CRP isoforms with oxLDL decreases TNF-α and IL-6 release by U937-derived macrophages.

    PubMed

    Krayem, Imtissal; Bazzi, Samer; Karam, Marc

    2017-09-01

    C-reactive protein (CRP) and oxidized low density lipoprotein (oxLDL) serve major roles at both early and advanced stages of atherosclerosis. CRP exists in two isoforms, monomeric (m) and pentameric (p), that bring about pro- or anti-inflammatory effects in macrophages. In addition, CRP may form a complex with oxidized low-density lipoprotein (oxLDL) via phosphatidylcholine, thus decreasing its pro-inflammatory effects within macrophages. The aim of the present study was to investigate the single and the combined effects of mCRP, pCRP and oxLDL on U937-derived macrophages. In the current study, U937-derived macrophages were treated in vitro with different combinations of CRP isoforms with or without oxLDL. The levels of major inflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α] along with the production of reactive oxygen species (ROS) were determined. TNF-α and IL-6 levels were significantly decreased (P<0.05) by the effect of mCRP and pCRP combined with oxLDL. No significant changes were observed in IL-1β, IL-8 or ROS levels.

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

    PubMed

    Siniscalco, Dario; Bradstreet, James Jeffrey; Cirillo, Alessandra; Antonucci, Nicola

    2014-04-17

    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. To achieve these goals, we used biomolecular, biochemical and immunocytochemical methods. 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. 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.

  14. 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. © Society for Leukocyte Biology.

  15. Mouse Mesenchymal Progenitor Cells Expressing Adipogenic and Osteogenic Transcription Factors Suppress the Macrophage Inflammatory Response.

    PubMed

    Fernandez, Natalie; Renna, Heather; McHugh, Lauren; Mazolkova, Katie; Crugnola, William; Evans, Jodi F

    2017-01-01

    Mesenchymal progenitor cell characteristics that can identify progenitor populations with specific functions in immunity are actively being investigated. Progenitors from bone marrow and adipose tissue regulate the macrophage (MΦ) inflammatory response by promoting the switch from an inflammatory to an anti-inflammatory phenotype. Conversely, mesenchymal progenitors from the mouse aorta (mAo) support and contribute to the MΦ response under inflammatory conditions. We used cell lines with purported opposing immune-regulatory function, a bone marrow derived mesenchymal progenitor cell line (D1) and a mouse aorta derived mesenchymal progenitor cell line (mAo). Their interaction and regulation of the MΦ cell response to the inflammatory mediator, lipopolysaccharide (LPS), was examined by coculture. As expected, D1 cells suppressed NO, TNF-α, and IL-12p70 production but MΦ phagocytic activity remained unchanged. The mAo cells enhanced NO and TNF-α production in coculture and enhanced MΦ phagocytic activity. Using flow cytometry and PCR array, we then sought to identify sets of MSC-associated genes and markers that are expressed by these progenitor populations. We have determined that immune-supportive mesenchymal progenitors highly express chondrogenic and tenogenic transcription factors while immunosuppressive mesenchymal progenitors highly express adipogenic and osteogenic transcription factors. These data will be useful for the isolation, purification, and modification of mesenchymal progenitors to be used in the treatment of inflammatory diseases.

  16. Mouse Mesenchymal Progenitor Cells Expressing Adipogenic and Osteogenic Transcription Factors Suppress the Macrophage Inflammatory Response

    PubMed Central

    Fernandez, Natalie; Renna, Heather; McHugh, Lauren; Mazolkova, Katie; Crugnola, William

    2017-01-01

    Mesenchymal progenitor cell characteristics that can identify progenitor populations with specific functions in immunity are actively being investigated. Progenitors from bone marrow and adipose tissue regulate the macrophage (MΦ) inflammatory response by promoting the switch from an inflammatory to an anti-inflammatory phenotype. Conversely, mesenchymal progenitors from the mouse aorta (mAo) support and contribute to the MΦ response under inflammatory conditions. We used cell lines with purported opposing immune-regulatory function, a bone marrow derived mesenchymal progenitor cell line (D1) and a mouse aorta derived mesenchymal progenitor cell line (mAo). Their interaction and regulation of the MΦ cell response to the inflammatory mediator, lipopolysaccharide (LPS), was examined by coculture. As expected, D1 cells suppressed NO, TNF-α, and IL-12p70 production but MΦ phagocytic activity remained unchanged. The mAo cells enhanced NO and TNF-α production in coculture and enhanced MΦ phagocytic activity. Using flow cytometry and PCR array, we then sought to identify sets of MSC-associated genes and markers that are expressed by these progenitor populations. We have determined that immune-supportive mesenchymal progenitors highly express chondrogenic and tenogenic transcription factors while immunosuppressive mesenchymal progenitors highly express adipogenic and osteogenic transcription factors. These data will be useful for the isolation, purification, and modification of mesenchymal progenitors to be used in the treatment of inflammatory diseases. PMID:28191017

  17. Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment

    PubMed Central

    Hasan, Al Shaimaa; Luo, Lan; Yan, Chen; Zhang, Tian-Xia; Urata, Yoshishige; Goto, Shinji; Mangoura, Safwat A.; Abdel-Raheem, Mahmoud H.; Zhang, Shouhua; Li, Tao-Sheng

    2016-01-01

    Cardiosphere-derived cells (CDCs), one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1) into a regulatory anti-inflammatory phenotype (M2). Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages), but decreased the expression of CD86 (a marker for M1 macrophages) 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair. PMID:27764217

  18. Glucocorticoid-induced tumour necrosis factor receptor family related protein (GITR) mediates inflammatory activation of macrophages that can destabilize atherosclerotic plaques

    PubMed Central

    Kim, Won-Jung; Bae, Eun-Mi; Kang, Yoon-Joong; Bae, Hyung-Uk; Hong, Su Hyung; Lee, Joo Y; Park, Jeong-Euy; Kwon, Byoung S; Suk, Kyoungho; Lee, Won-Ha

    2006-01-01

    Glucocorticoid-induced tumour necrosis factor receptor family related protein (GITR) is the 18th member of the tumour necrosis factor receptor superfamily (TNFRSF18) and is known to interact with its cognate ligand GITRL (TNFSF18). We investigated the potential role of GITR in the pro-inflammatory activation of macrophages. Immunohistochemistry and in situ hybridization analyses of human atherosclerotic plaques demonstrated that GITR and its ligand are expressed mainly in lipid-rich macrophages. We then investigated the role of GITR in human and mouse monocyte/macrophage functions. Stimulation of GITR caused nuclear factor (NF)-κB-dependent activation of matrix metalloproteinase-9 (MMP-9) and pro-inflammatory cytokine expression in both the human and mouse monocytic/macrophage cell lines, THP-1 and RAW264.7, respectively. These cellular responses were also observed when the THP-1 cells were treated with phorbol-12 myristate-13 acetate (PMA), which is known to induce macrophage differentiation. To demonstrate that these responses are not restricted to cultured cell lines, we tested primary macrophages. Both peritoneal and bone marrow-derived macrophages responded to GITR stimulation with induction of MMP-9 and tumour necrosis factor-α (TNF-α). Furthermore, the GITR staining pattern overlapped with those of MMP-9 and TNF-α in atherosclerotic plaques. These data indicate that GITR-mediated macrophage activation may promote atherogenesis via the induction of pro-atherogenic cytokines/chemokines, and destabilize the atherosclerotic plaques via the induction of the matrix-degrading enzyme, MMP-9. PMID:17067317

  19. Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury

    PubMed Central

    Saha, Subhrajit; Aranda, Evelyn; Hayakawa, Yoku; Bhanja, Payel; Atay, Safinur; Brodin, N Patrik; Li, Jiufeng; Asfaha, Samuel; Liu, Laibin; Tailor, Yagnesh; Zhang, Jinghang; Godwin, Andrew K.; Tome, Wolfgang A.; Wang, Timothy C.; Guha, Chandan; Pollard, Jeffrey W.

    2016-01-01

    WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation. PMID:27734833

  20. 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.

  1. Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro

    PubMed Central

    Heinrich, Franziska; Lehmbecker, Annika; Raddatz, Barbara B.; Kegler, Kristel; Tipold, Andrea; Stein, Veronika M.; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner

    2017-01-01

    Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as “respiratory burst”, whereas M2-polarization was associated with processes such as “mitosis”. Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for

  2. 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.

  3. A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens

    PubMed Central

    Laubier, Aurélie; Bleves, Sophie; Blanc-Potard, Anne-Béatrice

    2015-01-01

    Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus

  4. A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens.

    PubMed

    Belon, Claudine; Soscia, Chantal; Bernut, Audrey; Laubier, Aurélie; Bleves, Sophie; Blanc-Potard, Anne-Béatrice

    2015-06-01

    Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus

  5. c-Maf-dependent growth of Mycobacterium tuberculosis in a CD14(hi) subpopulation of monocyte-derived macrophages.

    PubMed

    Dhiman, Rohan; Bandaru, Anuradha; Barnes, Peter F; Saha, Sudipto; Tvinnereim, Amy; Nayak, Ramesh C; Paidipally, Padmaja; Valluri, Vijaya Lakshmi; Rao, L Vijaya Mohan; Vankayalapati, Ramakrishna

    2011-02-01

    Macrophages are a major component of the innate immune response, comprising the first line of defense against various intracellular pathogens, including Mycobacterium tuberculosis. In this report, we studied the factors that regulate growth of M. tuberculosis H37Rv in subpopulations of human monocyte-derived macrophages (MDMs). In healthy donors, M. tuberculosis H37Rv grew 5.6-fold more rapidly in CD14(hi) MDMs compared with that in CD14(lo)CD16(+) MDMs. Compared with CD14(lo)CD16(+) cells, M. tuberculosis H37Rv-stimulated CD14(hi) monocytes produced more IL-10 and had increased mRNA expression for c-Maf, a transcription factor that upregulates IL-10 gene expression. c-Maf small interfering RNA (siRNA) inhibited IL-10 production and growth of M. tuberculosis in CD14(hi) cells. Compared with CD14(lo)CD16(+) monocytes, M. tuberculosis H37Rv-stimulated CD14(hi) cells had increased expression of 22 genes whose promoters contained a c-Maf binding site, including hyaluronan synthase 1 (HAS1). c-Maf siRNA inhibited HAS1 expression in M. tuberculosis-stimulated CD14(hi) monocytes, and HAS1 siRNA inhibited growth of M. tuberculosis in CD14(hi) MDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product, hyaluronan, in CD14(hi) MDMs. We conclude that M. tuberculosis grows more rapidly in CD14(hi) than in CD14(lo)CD16(+) MDMs because CD14(hi) cells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10) that promote growth of M. tuberculosis.

  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.

  7. Coxsackievirus B4 Can Infect Human Peripheral Blood-Derived Macrophages.

    PubMed

    Alidjinou, Enagnon Kazali; Sané, Famara; Trauet, Jacques; Copin, Marie-Christine; Hober, Didier

    2015-11-24

    Beyond acute infections, group B coxsackieviruses (CVB) are also reported to play a role in the development of chronic diseases, like type 1 diabetes. The viral pathogenesis mainly relies on the interplay between the viruses and innate immune response in genetically-susceptible individuals. We investigated the interaction between CVB4 and macrophages considered as major players in immune response. Monocyte-derived macrophages (MDM) generated with either M-CSF or GM-CSF were inoculated with CVB4, and infection, inflammation, viral replication and persistence were assessed. M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. In addition, enhancing serum was not needed to infect MDM in contrast with parental monocytes. The expression of viral receptor (CAR) mRNA was similar in both M-CSF and GM-CSF MDM. CVB4 induced high levels of pro-inflammatory cytokines (IL-6 and TNFα) in both MDM populations. CVB4 effectively replicated and persisted in M-CSF MDM, but IFNα was produced in the early phase of infection only. Our results demonstrate that CVB4 can replicate and persist in MDM. Further investigations are required to determine whether the interaction between the virus and MDM plays a role in the pathogenesis of CVB-induced chronic diseases.

  8. Coxsackievirus B4 Can Infect Human Peripheral Blood-Derived Macrophages

    PubMed Central

    Alidjinou, Enagnon Kazali; Sané, Famara; Trauet, Jacques; Copin, Marie-Christine; Hober, Didier

    2015-01-01

    Beyond acute infections, group B coxsackieviruses (CVB) are also reported to play a role in the development of chronic diseases, like type 1 diabetes. The viral pathogenesis mainly relies on the interplay between the viruses and innate immune response in genetically-susceptible individuals. We investigated the interaction between CVB4 and macrophages considered as major players in immune response. Monocyte-derived macrophages (MDM) generated with either M-CSF or GM-CSF were inoculated with CVB4, and infection, inflammation, viral replication and persistence were assessed. M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. In addition, enhancing serum was not needed to infect MDM in contrast with parental monocytes. The expression of viral receptor (CAR) mRNA was similar in both M-CSF and GM-CSF MDM. CVB4 induced high levels of pro-inflammatory cytokines (IL-6 and TNFα) in both MDM populations. CVB4 effectively replicated and persisted in M-CSF MDM, but IFNα was produced in the early phase of infection only. Our results demonstrate that CVB4 can replicate and persist in MDM. Further investigations are required to determine whether the interaction between the virus and MDM plays a role in the pathogenesis of CVB-induced chronic diseases. PMID:26610550

  9. Myeloid-derived suppressor cells impair alveolar macrophages through PD-1 receptor ligation during Pneumocystis pneumonia.

    PubMed

    Lei, Guang-Sheng; Zhang, Chen; Lee, Chao-Hung

    2015-02-01

    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.

  10. Transcriptional analysis of diverse strains Mycobacterium avium subspecies paratuberculosis in primary bovine monocyte derived macrophages.

    PubMed

    Zhu, Xiaochun; Tu, Zheng J; Coussens, Paul M; Kapur, Vivek; Janagama, Harish; Naser, Saleh; Sreevatsan, Srinand

    2008-10-01

    In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. paratuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequence annotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have any annotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, 10-12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation, and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modus operandi for survival in the host.

  11. Induced Pluripotent Stem Cell Derived Macrophages as a Cellular System to Study Salmonella and Other Pathogens

    PubMed Central

    Hale, Christine; Yeung, Amy; Goulding, David; Pickard, Derek; Alasoo, Kaur; Powrie, Fiona; Dougan, Gordon; Mukhopadhyay, Subhankar

    2015-01-01

    A number of pathogens, including several human-restricted organisms, persist and replicate within macrophages (Mφs) as a key step in pathogenesis. The mechanisms underpinning such host-restricted intracellular adaptations are poorly understood, in part, due to a lack of appropriate model systems. Here we explore the potential of human induced pluripotent stem cell derived macrophages (iPSDMs) to study such pathogen interactions. We show iPSDMs express a panel of established Mφ-specific markers, produce cytokines, and polarise into classical and alternative activation states in response to IFN-γ and IL-4 stimulation, respectively. iPSDMs also efficiently phagocytosed inactivated bacterial particles as well as live Salmonella Typhi and S. Typhimurium and were able to kill these pathogens. We conclude that iPSDMs can support productive Salmonella infection and propose this as a flexible system to study host/pathogen interactions. Furthermore, iPSDMs can provide a flexible and practical cellular platform for assessing host responses in multiple genetic backgrounds. PMID:25946027

  12. 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.

  13. Nerve growth factor is an autocrine factor essential for the survival of macrophages infected with HIV.

    PubMed

    Garaci, E; Caroleo, M C; Aloe, L; Aquaro, S; Piacentini, M; Costa, N; Amendola, A; Micera, A; Caliò, R; Perno, C F; Levi-Montalcini, R

    1999-11-23

    Nerve growth factor (NGF) is a neurotrophin with the ability to exert specific effects on cells of the immune system. Human monocytes/macrophages (M/M) infected in vitro with HIV type 1 (HIV-1) are able to produce substantial levels of NGF that are associated with enhanced expression of the high-affinity NGF receptor (p140 trkA) on the M/M surface. Treatment of HIV-infected human M/M with anti-NGF Ab blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high-affinity receptor, a concomitant increased expression of p75(NTR) low-affinity receptor for NGF, and the occurrence of apoptotic death of M/M. Taken together, these findings suggest a role for NGF as an autocrine survival factor that rescues human M/M from the cytopathic effect caused by HIV infection.

  14. Macrophage-derived apoptotic bodies promote the proliferation of the recipient cells via shuttling microRNA-221/222.

    PubMed

    Zhu, Ziwen; Zhang, Duo; Lee, Heedoo; Menon, Aravind Ajakumar; Wu, Jingxuan; Hu, Kebin; Jin, Yang

    2017-03-08

    Bacterial pneumonia is a common and serious clinical entity. Alveolar epithelial cells and alveolar macrophages are the first line of defense in the innate immunity against bacterial pathogens. Epithelial cells are known to release chemokines/cytokines that recruit and activate phagocytic cells. However, the signals sent from alveolar macrophages back to the lung epithelial cells remain largely unexplored. We found that LPS, a well-recognized stimulator derived from gram-negative (G(-)) bacteria, rapidly and robustly induces the secretion of macrophage-derived extracellular vesicles (EVs). The main type of EVs found in the early stages after LPS stimulation are apoptotic bodies (ABs) and not microvesicles (MVs) or exosomes (Exos). Furthermore, LPS markedly up-regulate the levels of a repertoire of microRNAs (miRNAs) in the macrophage-derived ABs, including miR-221 and miR-222. Functionally, the LPS-induced, macrophage-derived ABs promote the proliferation of malignant and/or normal lung epithelial cells. We next directly transfected miR-221 and/or miR-222 inhibitors into the LPS-induced ABs. Deletion of miR-221/222 in ABs significantly reduces the AB-mediated proliferation of lung epithelial cells. Mechanistically, AB-shuttling miR-221/222 promote cell growth by modulating cyclin-dependent kinase inhibitor 1B (CDKN1B) pathways. Collectively, LPS-induced, macrophage-derived ABs promote the proliferation of their recipient epithelial cells, partially via AB-shuttling miRNAs.

  15. Macrophage Responses to Epithelial Dysfunction Promote Lung Fibrosis in Aging

    DTIC Science & Technology

    2016-10-01

    tissue-resident macrophages or bone marrow -derived macrophages that prevent or promote fibrosis, respectively so they can be targeted for prevention or...guide therapy and factors released from tissue-resident macrophages or bone marrow -derived macrophages that prevent or promote fibrosis...aging? Subtask 1: Generate cohorts of shielded bone marrow chimeric mice. Dr. Misharin will perform the procedure, Drs. Chen and Soberanes will

  16. Acute myeloid leukemia cells polarize macrophages towards a leukemia supporting state in a Growth factor independence 1 dependent manner

    PubMed Central

    Al-Matary, Yahya S.; Botezatu, Lacramioara; Opalka, Bertram; Hönes, Judith M.; Lams, Robert F.; Thivakaran, Aniththa; Schütte, Judith; Köster, Renata; Lennartz, Klaus; Schroeder, Thomas; Haas, Rainer; Dührsen, Ulrich; Khandanpour, Cyrus

    2016-01-01

    The growth of malignant cells is not only driven by cell-intrinsic factors, but also by the surrounding stroma. Monocytes/Macrophages play an important role in the onset and progression of solid cancers. However, little is known about their role in the development of acute myeloid leukemia, a malignant disease characterized by an aberrant development of the myeloid compartment of the hematopoietic system. It is also unclear which factors are responsible for changing the status of macrophage polarization, thus supporting the growth of malignant cells instead of inhibiting it. We report herein that acute myeloid leukemia leads to the invasion of acute myeloid leukemia-associated macrophages into the bone marrow and spleen of leukemic patients and mice. In different leukemic mouse models, these macrophages support the in vitro expansion of acute myeloid leukemia cell lines better than macrophages from non-leukemic mice. The grade of macrophage infiltration correlates in vivo with the survival of the mice. We found that the transcriptional repressor Growth factor independence 1 is crucial in the process of macrophage polarization, since its absence impedes macrophage polarization towards a leukemia supporting state and favors an anti-tumor state both in vitro and in vivo. These results not only suggest that acute myeloid leukemia-associated macrophages play an important role in the progression of acute myeloid leukemia, but also implicate Growth factor independence 1 as a pivotal factor in macrophage polarization. These data may provide new insights and opportunities for novel therapies for acute myeloid leukemia. PMID:27390361

  17. Monocyte/macrophage-derived soluble CD163: a novel biomarker in multiple myeloma.

    PubMed

    Andersen, Morten N; Abildgaard, Niels; Maniecki, Maciej B; Møller, Holger J; Andersen, Niels F

    2014-07-01

    Macrophages play an important role in cancer by suppression of adaptive immunity and promotion of angiogenesis and metastasis. Tumor-associated macrophages strongly express the hemoglobin scavenger receptor CD163, which can also be found as a soluble protein in serum and other body fluids (soluble CD163, sCD163). In this study, we examined serum sCD163 as a biomarker in patients with newly diagnosed multiple myeloma. Peripheral blood (n = 104) and bone marrow (n = 17) levels of sCD163 were measured using an enzyme-linked immunosorbent assay. At diagnosis, high sCD163 was associated with higher stage according to the International Staging System (ISS) and with other known prognostic factors in multiple myeloma (creatinine, C-reactive protein, and beta-2 microglobulin). Soluble CD163 decreased upon high-dose treatment, and in a multivariate survival analysis including the covariates treatment modality and age at diagnosis, higher levels of sCD163 were associated with poor outcome (HR = 1.82; P = 0.010). The prognostic significance of sCD163 was lost when including ISS stage in the model (HR = 1.51; P = 0.085). Soluble CD163 values were significantly higher in bone marrow samples than in the matched blood samples, which indicate a localized production of sCD163 within the bone marrow microenvironment. Soluble CD163 was found to be a prognostic marker in patients with multiple myeloma. This may indicate that macrophages and/or monocytes have an important role in the bone marrow microenvironment of myeloma patients, supporting myeloma cell proliferation and survival. We propose the serum sCD163 value 1.8 mg/L as a cutoff concentration for survival analysis in patients with multiple myeloma, which should be validated in future studies. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Nuclear factor kappa-B- and activator protein-1-mediated immunostimulatory activity of compound K in monocytes and macrophages.

    PubMed

    Yang, Woo Seok; Yi, Young-Su; Kim, Donghyun; Kim, Min Ho; Park, Jae Gwang; Kim, Eunji; Lee, Sang Yeol; Yoon, Keejung; Kim, Jong-Hoon; Park, Junseong; Cho, Jae Youl

    2017-07-01

    Compound K (CK) is a bioactive derivative of ginsenoside Rb1 in Panax ginseng (Korean ginseng). Its biological and pharmacological activities have been studied in various disease conditions, although its immunomodulatory role in innate immunity mediated by monocytes/macrophages has been poorly understood. In this study, we aimed to elucidate the regulatory role of CK on cellular events mediated by monocytes and macrophages in innate immune responses. The immunomodulatory role of CK was explored by various immunoassays including cell-cell adhesion, fibronectin adhesion, cell migration, phagocytic uptake, costimulatory molecules, reactive oxygen species production, luciferase activity, and by the measurement of mRNA levels of proinflammatory genes. Compound K induced cell cluster formation through cell-cell adhesion, cell migration, and phagocytic activity, but it suppressed cell-tissue interactions in U937 and RAW264.7 cells. Compound K also upregulated the surface expression of the cell adhesion molecule cluster of differentiation (CD) 43 (CD43) and costimulatory molecules CD69, CD80, and CD86, but it downregulated the expression of monocyte differentiation marker CD82 in RAW264.7 cells. Moreover, CK induced the release of reactive oxygen species and induced messenger RNA expression of proinflammatory genes, inducible nitric oxide synthase, and tumor necrosis factor-alpha by enhancing the nuclear translocation and transcriptional activities of nuclear factor kappa-B and activator protein-1. Our results suggest that CK has an immunomodulatory role in innate immune responses through regulating various cellular events mediated by monocytes and macrophages.

  19. Modulation of Intracellular Restriction Factors Contributes to Methamphetamine-Mediated Enhancement of Acquired Immune Deficiency Syndrome Virus Infection of Macrophages

    PubMed Central

    Wang, Xu; Wang, Yizhong; Ye, Li; Li, Jieliang; Zhou, Yu; Sakarcan, Sinem; Ho, Wenzhe

    2014-01-01

    Epidemiological studies have demonstrated that the use of methamphetamine (METH), a sympathomimetic stimulant, is particularly common among patients infected with HIV. In vitro studies have determined that METH enhances HIV infection of CD4+ T cells, monocyte-derived dendritic cells, and macrophages. In addition, animal studies have also showed that METH treatment increases brain viral load of SIV-infected monkeys and promotes HIV replication and viremia in HIV/hu-CycT1 transgenic mice. However, the mechanisms (s) of METH actions on HIV remain to be determined. In this study, we investigated the impact of METH on intracellular restriction factors against HIV and SIV. We demonstrated that METH treatment of human blood mononuclear phagocytes significantly affected the expression of anti-HIV microRNAs and several key elements (RIG-I, IRF-3/5, SOCS-2, 3 and PIAS-1, 3, X, Y) in the type I IFN pathway. The suppression of these innate restriction factors was associated with a reduced production of type I IFNs and the enhancement of HIV or SIV infection of macrophages. These findings indicate that METH use impairs intracellular innate antiviral mechanism(s) in macrophages, contributing to cell susceptibility to the acquired immune deficiency syndrome (AIDS) virus infection. PMID:22591364

  20. Colony-stimulating factor-1 mediates macrophage-related neural damage in a model for Charcot-Marie-Tooth disease type 1X.

    PubMed

    Groh, Janos; Weis, Joachim; Zieger, Hanna; Stanley, E Richard; Heuer, Heike; Martini, Rudolf

    2012-01-01

    Previous studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot-Marie-Tooth neuropathy, low-grade inflammation implicating phagocytosing macrophages mediates demyelination and perturbation of axons. In the present study, we focus on colony-stimulating factor-1, a cytokine implicated in macrophage differentiation, activation and proliferation and fostering neural damage in a model for Charcot-Marie-Tooth neuropathy 1B. By crossbreeding a model for the X-linked form of Charcot-Marie-Tooth neuropathy with osteopetrotic mice, a spontaneous null mutant for colony-stimulating factor-1, we demonstrate a robust and persistent amelioration of demyelination and axon perturbation. Furthermore, functionally important domains of the peripheral nervous system, such as juxtaparanodes and presynaptic terminals, were preserved in the absence of colony-stimulating factor-1-dependent macrophage activation. As opposed to other Schwann cell-derived cytokines, colony-stimulating factor-1 is expressed by endoneurial fibroblasts, as revealed by in situ hybridization, immunocytochemistry and detection of β-galactosidase expression driven by the colony-stimulating factor-1 promoter. By both light and electron microscopic studies, we detected extended cell-cell contacts between the colony-stimulating factor-1-expressing fibroblasts and endoneurial macrophages as a putative prerequisite for the effective and constant activation of macrophages by fibroblasts in the chronically diseased nerve. Interestingly, in human biopsies from patients with Charcot-Marie-Tooth type 1, we also found frequent cell-cell contacts between macrophages and endoneurial fibroblasts and identified the latter as main source for colony-stimulating factor-1. Therefore, our study provides strong evidence for a similarly pathogenic role of colony-stimulating factor-1 in genetically mediated demyelination in mice and Charcot-Marie-Tooth type 1

  1. Colony-stimulating factor-1 mediates macrophage-related neural damage in a model for Charcot–Marie–Tooth disease type 1X

    PubMed Central

    Groh, Janos; Weis, Joachim; Zieger, Hanna; Stanley, E. Richard; Heuer, Heike

    2012-01-01

    Previous studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot–Marie–Tooth neuropathy, low-grade inflammation implicating phagocytosing macrophages mediates demyelination and perturbation of axons. In the present study, we focus on colony-stimulating factor-1, a cytokine implicated in macrophage differentiation, activation and proliferation and fostering neural damage in a model for Charcot–Marie–Tooth neuropathy 1B. By crossbreeding a model for the X-linked form of Charcot–Marie–Tooth neuropathy with osteopetrotic mice, a spontaneous null mutant for colony-stimulating factor-1, we demonstrate a robust and persistent amelioration of demyelination and axon perturbation. Furthermore, functionally important domains of the peripheral nervous system, such as juxtaparanodes and presynaptic terminals, were preserved in the absence of colony-stimulating factor-1-dependent macrophage activation. As opposed to other Schwann cell-derived cytokines, colony-stimulating factor-1 is expressed by endoneurial fibroblasts, as revealed by in situ hybridization, immunocytochemistry and detection of β-galactosidase expression driven by the colony-stimulating factor-1 promoter. By both light and electron microscopic studies, we detected extended cell–cell contacts between the colony-stimulating factor-1-expressing fibroblasts and endoneurial macrophages as a putative prerequisite for the effective and constant activation of macrophages by fibroblasts in the chronically diseased nerve. Interestingly, in human biopsies from patients with Charcot–Marie–Tooth type 1, we also found frequent cell–cell contacts between macrophages and endoneurial fibroblasts and identified the latter as main source for colony-stimulating factor-1. Therefore, our study provides strong evidence for a similarly pathogenic role of colony-stimulating factor-1 in genetically mediated demyelination in mice and Charcot

  2. Macrophage colony stimulating factor regulation by nuclear factor kappa B: a relevant pathway in human immunodeficiency virus type 1 infected macrophages.

    PubMed

    Kogan, Michael; Haine, Valerie; Ke, Yuxong; Wigdahl, Brian; Fischer-Smith, Tracy; Rappaport, Jay

    2012-03-01

    Macrophage colony stimulating factor (M-CSF) is a cytokine that promotes monocyte differentiation and survival. When overexpressed, M-CSF contributes to pathology in a wide variety of diseases, including osteoporosis, obesity, certain human cancers, and in human immunodeficiency virus type 1 (HIV-1) infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1 associated central nervous system disorders. In this study, our aim was to expand the current knowledge of M-CSF regulation, focusing on nuclear factor kappa B (NF-κB), a transcription factor playing a prominent role during inflammation and HIV-1 infection. Our results suggest that tumor necrosis factor alpha (TNF-α) promotes M-CSF secretion in primary macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-κB pathway diminish this response. We identified four putative NF-κB and four CCAAT-enhancer-binding protein beta binding sites within the M-CSF promoter. Our findings, using promoter constructs mutated at individual NF-κB sites within the M-CSF promoter region, suggest that these sites are redundant with respect to NF-κB regulation. TNF-α treatment promoted NF-κB p65 binding to the M-CSF promoter in phorbol 12-myristate 13-acetate (PMA) treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-κB response. In conclusion, our findings demonstrate that NF-κB induces M-CSF expression on a promoter level via multiple functional NF-κB binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages.

  3. 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.

  4. Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures.

    PubMed

    Kwon, Tae-Ho; Kim, Young-Sook; Lee, Jae-Hwa; Yang, Moon-Sik

    2003-09-01

    A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 microg l(-1) after 10 d' cultivation.

  5. 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.

  6. Lipophilic derivative of muramyl dipeptide is more active than muramyl dipeptide in priming macrophages to release superoxide anion.

    PubMed Central

    Pabst, M J; Cummings, N P; Shiba, T; Kusumoto, S; Kotani, S

    1980-01-01

    Mouse peritoneal macrophages, when treated with a lipophilic derivative of muramyl dipeptide either in vitro or in vivo by intraperitoneal injection, showed a more than fivefold increase in their ability to generate superoxide anion after stimulation of the macrophages with phorbol myristate acetate. This response was more than twice that observed with the parent molecule, muramyl dipeptide (MDP). Unlike MDP, which has a systemic effect, the lipophilic derivative, [B30]-MDP, did not alter the response of peritoneal macrophages when given subcutaneously in the flank, suggesting that [B30]-MDP remains localized at the site of injection. The enhanced effect of [B30]-MDP over MDP appeared to be due to the inherent lipophilicity of the molecule, and was probably not due to either stimulation of T lymphocytes or activation of the alternative pathway of complement. PMID:6260655

  7. Gut-derived lipopolysaccharide augments adipose macrophage accumulation but is not essential for impaired glucose or insulin tolerance in mice.

    PubMed

    Caesar, Robert; Reigstad, Christopher S; Bäckhed, Helene Kling; Reinhardt, Christoph; Ketonen, Maria; Lundén, Gunnel Östergren; Cani, Patrice D; Bäckhed, Fredrik

    2012-12-01

    Obesity is associated with accumulation of macrophages in white adipose tissue (WAT), which contribute to the development of insulin resistance. Germ-free (GF) mice have reduced adiposity and are protected against diet-induced obesity, To investigate whether the gut microbiota and, specifically, gut-derived lipopolysaccharide (LPS) promote WAT inflammation and contribute to impaired glucose metabolism. Macrophage composition and expression of proinflammatory and anti-inflammatory markers were compared in WAT of GF, conventionally raised and Escherichia coli-monocolonised mice. Additionally, glucose and insulin tolerance in these mice was determined. The presence of a gut microbiota resulted in impaired glucose metabolism and increased macrophage accumulation and polarisation towards the proinflammatory M1 phenotype in WAT. Monocolonisation of GF mice for 4 weeks with E. coli W3110 or the isogenic strain MLK1067 (which expresses LPS with reduced immunogenicity) resulted in impaired glucose and insulin tolerance and promoted M1 polarisation of CD11b cells in WAT. However, colonisation with E. coli W3110 but not MLK1067 promoted macrophage accumulation and upregulation of proinflammatory and anti-inflammatory gene expression as well as JNK phosphorylation. Gut microbiota induced LPS-dependent macrophage accumulation in WAT, whereas impairment of systemic glucose metabolism was not dependent on LPS. These results indicate that macrophage accumulation in WAT does not always correlate with impaired glucose metabolism.

  8. THP-1-derived macrophages render lung epithelial cells hypo-responsive to Legionella pneumophila - a systems biology study.

    PubMed

    Schulz, Christine; Lai, Xin; Bertrams, Wilhelm; Jung, Anna Lena; Sittka-Stark, Alexandra; Herkt, Christina Elena; Janga, Harshavadhan; Zscheppang, Katja; Stielow, Christina; Schulte, Leon; Hippenstiel, Stefan; Vera, Julio; Schmeck, Bernd

    2017-09-20

    Immune response in the lung has to protect the huge alveolar surface against pathogens while securing the delicate lung structure. Macrophages and alveolar epithelial cells constitute the first line of defense and together orchestrate the initial steps of host defense. In this study, we analysed the influence of macrophages on type II alveolar epithelial cells during Legionella pneumophila-infection by a systems biology approach combining experimental work and mathematical modelling. We found that L. pneumophila-infected THP-1-derived macrophages provoke a pro-inflammatory activation of neighboring lung epithelial cells, but in addition render them hypo-responsive to direct infection with the same pathogen. We generated a kinetic mathematical model of macrophage activation and identified a paracrine mechanism of macrophage-secreted IL-1β inducing a prolonged IRAK-1 degradation in lung epithelial cells. This intercellular crosstalk may help to avoid an overwhelming inflammatory response by preventing excessive local secretion of pro-inflammatory cytokines and thereby negatively regulating the recruitment of immune cells to the site of infection. This suggests an important but ambivalent immunomodulatory role of macrophages in lung infection.

  9. 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.

  10. Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries.

    PubMed

    Sacco, R E; Waters, W R; Rudolph, K M; Drew, M L

    2006-01-01

    Bighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (INOS) inhibitor, N(G)-monomethyl-L-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.

  11. 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.

  12. Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells.

    PubMed

    Wang, Chongzhen; Luo, Haiying; Zhu, Linnan; Yang, Fan; Chu, Zhulang; Tian, Hongling; Feng, Meifu; Zhao, Yong; Shang, Peng

    2014-01-01

    Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear. RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control. Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals. LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1β expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity. Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.

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

    PubMed Central

    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, Alicia E.; Jewett, Ethan M.; Germiller, John A.; Barald, Kate F.

    2012-01-01

    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

  14. Effect of Corynebacterium parvum on colony-stimulating factor and granulocyte-macrophage colony formation.

    PubMed

    Foster, R S; MacPherson, B R; Browdie, D A

    1977-05-01

    Because Corynebacterium parvum has tumor-inhibitory properties and stimulates granulocyte-macrophage production, it may have clinical value in combination with chemotherapy. The leukopoietic effect of killed suspensions of C. parvum was studied in mice using the technique of in vitro clonal culture of hematopoietic cells. After C. parvum injection, there was a prompt, sustained elevation of serum colony-stimulating factor followed by an increase in granulocyte-macrophage precursor cells in the spleen and increases in blood mononuclear and granulocyte cells. Colony-stimulating factor production is suggested as a major mechanism of stimulation of granulocyte-macrophage proliferation by C. parvum. Since rapidly proliferating hematopoietic cells may have increased sensititity to cytotoxic agents, the details of hematopoietic stimulation by C. parvum may be critical in the sequential timing of combined C. parvum and chemotherapy treatment to obtain maximal tumor inhibition and minimal hematopoietic toxicity.

  15. IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation.

    PubMed

    Dai, Lue; Lidie, Kristy B; Chen, Qian; Adelsberger, Joseph W; Zheng, Xin; Huang, DaWei; Yang, Jun; Lempicki, Richard A; Rehman, Tauseef; Dewar, Robin L; Wang, Yanmei; Hornung, Ronald L; Canizales, Kelsey A; Lockett, Stephen J; Lane, H Clifford; Imamichi, Tomozumi

    2013-03-11

    The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1-mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27-treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.

  16. Macrophage migration inhibitory factor deficiency is associated with impaired killing of gram-negative bacteria by macrophages and increased susceptibility to Klebsiella pneumoniae sepsis.

    PubMed

    Roger, Thierry; Delaloye, Julie; Chanson, Anne-Laure; Giddey, Marlyse; Le Roy, Didier; Calandra, Thierry

    2013-01-15

    The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

  17. Increased formation of autophagosomes in ectromelia virus-infected primary culture of murine bone marrow-derived macrophages.

    PubMed

    Martyniszyn, L; Szulc-Dąbrowska, L; Boratyńska-Jasińska, A; Niemiałtowski, M

    2013-01-01

    Induction of autophagy by ectromelia virus (ECTV) in primary cultures of bone marrow-derived macrophages (BMDMs) was investigated. The results showed that ECTV infection of BMDMs resulted in increased formation of autophagosomes, increased level of LC3-II protein present in aggregates and extensive cytoplasmic vacuolization. These data indicate an increased autophagic activity in BMDMs during ECTV infection.

  18. Colony-stimulating factor-1 promotes kidney growth and repair via alteration of macrophage responses.

    PubMed

    Alikhan, Maliha A; Jones, Christina V; Williams, Timothy M; Beckhouse, Anthony G; Fletcher, Anne L; Kett, Michelle M; Sakkal, Samy; Samuel, Chrishan S; Ramsay, Robert G; Deane, James A; Wells, Christine A; Little, Melissa H; Hume, David A; Ricardo, Sharon D

    2011-09-01

    Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.

  19. Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

    PubMed Central

    Alikhan, Maliha A.; Jones, Christina V.; Williams, Timothy M.; Beckhouse, Anthony G.; Fletcher, Anne L.; Kett, Michelle M.; Sakkal, Samy; Samuel, Chrishan S.; Ramsay, Robert G.; Deane, James A.; Wells, Christine A.; Little, Melissa H.; Hume, David A.; Ricardo, Sharon D.

    2011-01-01

    Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury. PMID:21762674

  20. uPAR induces expression of transforming growth factor β and interleukin-4 in cancer cells to promote tumor-permissive conditioning of macrophages.

    PubMed

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

    2014-12-01

    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.

  1. 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

  2. Chicken tumor necrosis-like factor. I. In vitro production by macrophages stimulated with Eimeria tenella or bacterial lipopolysaccharide.

    PubMed

    Zhang, S; Lillehoj, H S; Ruff, M D

    1995-08-01

    HD11, a transformed avian macrophage cell line, and chicken peripheral blood leukocyte-derived macrophages (PBL-M phi) were stimulated with bacterial endotoxin lipopolysaccharide (LPS) or Eimeria tenella sporozoites and merozoites. The specific cytotoxicities of the culture supernatants against different target cell lines were measured, and the kinetics of tumor necrosis-like factor (TNF) production by HD11 and PBL-M phi were also measured. The results showed that HD11 and PBL-M phi secreted a TNF-like factor when stimulated with Eimeria parasites or LPS. A time- and dose-dependent TNF-like factors production by PBL-M phi was observed poststimulation with Eimeria parasites. Chicken TNF-like factor preferentially kills CHCC OU-2 cells, a fibroblast cell line of chicken origin, when compared to LM cells, a murine cell line used for mammalian TNF. This study indicates that chicken M phi produce a significant level of TNF-like factor following coccidial infection.

  3. 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

  4. Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.

    PubMed

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M; Ochoa, Sara A; González-Pedrajo, Bertha; 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.

  5. Macrophage colony-stimulating factor mRNA and protein in atherosclerotic lesions of rabbits and humans.

    PubMed Central

    Rosenfeld, M. E.; Ylä-Herttuala, S.; Lipton, B. A.; Ord, V. A.; Witztum, J. L.; Steinberg, D.

    1992-01-01

    In this study, the authors demonstrate the expression of mRNA and the presence of protein for macrophage colony-stimulating factor (MCSF) in atherosclerotic lesions from humans and rabbits. In situ hybridization of serial sections of human fatty streaks demonstrated expression of MCSF mRNA by cells dispersed throughout the lesions. Immunocytochemical staining with a panel of MCSF-specific antibodies showed extensive cell-associated staining of all of the cell types in the lesions. Immunocytochemical studies of atherosclerotic lesions from Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed rabbits demonstrated a similar cell-associated pattern of staining. There was no MCSF-specific staining of aortas from normal rabbits or of cultured aortic smooth muscle cells from either humans or rabbits. Macrophage-derived foam cells (MFC) were isolated from the aortas of ballooned, cholesterol-fed rabbits. A Northern blot demonstrated that RNA isolated from the MFC hybridized with a human cDNA probe for MCSF. RNA from alveolar macrophages isolated simultaneously from the same rabbits did not hybridize with the MCSF probe. Conditioned media from an 18- to 24-hour incubation of the MFC contained colony-stimulating activity as demonstrated in a mouse bone marrow culture assay. Most of this colony-stimulating activity was neutralized by preincubating the conditioned media with an MCSF-specific antibody. Images Figure 2 Figure 1 Figure 1 Figure 3 PMID:1739123

  6. Differential Regulation of Macrophage Glucose Metabolism by Macrophage Colony-stimulating Factor and Granulocyte-Macrophage Colony-stimulating Factor: Implications for (18)F FDG PET Imaging of Vessel Wall Inflammation.

    PubMed

    Tavakoli, Sina; Short, John D; Downs, Kevin; Nguyen, Huynh Nga; Lai, Yanlai; Zhang, Wei; Jerabek, Paul; Goins, Beth; Sadeghi, Mehran M; Asmis, Reto

    2017-04-01

    Purpose To determine the divergence of immunometabolic phenotypes of macrophages stimulated with macrophage colony-stimulating factor (M-CSF) and granulocyte-M-CSF (GM-CSF) and its implications for fluorine 18 ((18)F) fluorodeoxyglucose (FDG) imaging of atherosclerosis. Materials and Methods This study was approved by the animal care committee. Uptake of 2-deoxyglucose and various indexes of oxidative and glycolytic metabolism were evaluated in nonactivated murine peritoneal macrophages (MΦ0) and macrophages stimulated with M-CSF (MΦM-CSF) or GM-CSF (MΦGM-CSF). Intracellular glucose flux was measured by using stable isotope tracing of glycolytic and tricyclic acid intermediary metabolites. (18)F-FDG uptake was evaluated in murine atherosclerotic aortas after stimulation with M-CSF or GM-CSF by using quantitative autoradiography. Results Despite inducing distinct activation states, GM-CSF and M-CSF stimulated progressive but similar levels of increased 2-deoxyglucose uptake in macrophages that reached up to sixfold compared with MΦ0. The expression of glucose transporters, oxidative metabolism, and mitochondrial biogenesis were induced to similar levels in MΦM-CSF and MΦGM-CSF. Unexpectedly, there was a 1.7-fold increase in extracellular acidification rate, a 1.4-fold increase in lactate production, and overexpression of several critical glycolytic enzymes in MΦM-CSF compared with MΦGM-CSF with associated increased glucose flux through glycolytic pathway. Quantitative autoradiography demonstrated a 1.6-fold induction of (18)F-FDG uptake in murine atherosclerotic plaques by both M-CSF and GM-CSF. Conclusion The proinflammatory and inflammation-resolving activation states of macrophages induced by GM-CSF and M-CSF in either cell culture or atherosclerotic plaques may not be distinguishable by the assessment of glucose uptake. (©) RSNA, 2016 Online supplemental material is available for this article.

  7. Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech.

    PubMed

    Schorn, Tilo; Drago, Francesco; Tettamanti, Gianluca; Valvassori, Roberto; de Eguileor, Magda; Vizioli, Jacopo; Grimaldi, Annalisa

    2015-03-01

    Allograft inflammatory factor-1 (AIF-1) is a 17-kDa cytokine-inducible calcium-binding protein that, in vertebrates, plays an important role in the allograft immune response. Its expression is mostly limited to the monocyte/macrophage lineage. Until recently, AIF-1 was assumed to be a novel molecule involved in inflammatory responses. To clarify this aspect, we have investigated the expression of AIF-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). Analysis of an expressed sequence tag library from the central nervous system of Hirudo revealed the presence of the gene Hmaif-1/alias Hmiba1, showing high homology with vertebrate aif-1. Immunohistochemistry with an anti-HmAIF-1 polyclonal antibody revealed the constitutive presence of this protein in spread CD68(+) macrophage-like cells. A few hours after pathogen (bacterial) injection into the body wall, the amount of these immunopositive cells co-expressing HmAIF-1 and the common leucocyte marker CD45 increased at the injected site. Moreover, the recombinant protein HmAIF-1 induced massive angiogenesis and was a potent chemoattractant for macrophages. Following rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which, in vertebrates, seems to have a role in the integrin-mediated adhesion of macrophages and in the regulation of the functional responsiveness of cells to chemoattractants. CD45 is therefore probably involved in leech macrophage-like cell activation and migration towards an inflammation site. We have also examined its potential effect on HmAIF-1-induced signalling.

  8. Identification of blue staining vaccine-derived material in inflammatory lesions using cultured canine macrophages.

    PubMed

    Scruggs, Jennifer L; LeBlanc, Casey J

    2015-03-01

    Vaccine reactions are described in cytology textbooks as having eosinophilic to magenta colored globules within and admixed with inflammatory cells. Recently, we have seen increased numbers of inflammatory lesions containing blue to blue-gray globular material, with historical information suggesting an association with rabies vaccination. The purpose of the study was to confirm the blue-gray and the eosinophilic material observed microscopically in some inflammatory lesions as being vaccine-derived. Three different vaccines were cytocentrifuged and Wright stained. Vaccine aliquots were also added to the culture media of canine-derived macrophages for 24 hours and the cells subsequently harvested, cytocentrifuged, and Wright stained. The globular material present in both preparations was compared to that observed in vaccine-induced inflammatory lesions. Morin staining was used to identify metal within vaccine material in both in vitro- and in vivo-derived cytology samples. Vaccine-derived material has a characteristic color and appearance. Appearance of the material was consistent in cytologic samples, in cells incubated with the vaccine, and in cytocentrifuged preparations of the vaccine vial contents. The blue-gray globules stained positively for Morin stain, while the eosinophilic material did not stain. Vaccine-induced inflammatory lesions may contain blue to blue-gray or magenta stained globular material. Blue-gray material was associated with administration of rabies vaccine Imrab 3 TF and the observed material may be metal-containing adjuvant. Magenta material was associated with other vaccines and negative for Morin stain, suggesting a metal-free adjuvant. © 2014 American Society for Veterinary Clinical Pathology.

  9. Acid fibroblast growth factor and peripheral nerve grafts regulate Th2 cytokine expression, macrophage activation, polyamine synthesis, and neurotrophin expression in transected rat spinal cords.

    PubMed

    Kuo, Huai-Sheng; Tsai, May-Jywan; Huang, Ming-Chao; Chiu, Chuan-Wen; Tsai, Ching-Yi; Lee, Meng-Jen; Huang, Wen-Cheng; Lin, Yi-Lo; Kuo, Wen-Chun; Cheng, Henrich

    2011-03-16

    Spinal cord injury elicits an inflammatory response that recruits macrophages to the injured spinal cord. Quantitative real-time PCR results have shown that a repair strategy combining peripheral nerve grafts with acidic fibroblast growth factor (aFGF) induced higher interleukin-4 (IL-4), IL-10, and IL-13 levels in the graft areas of rat spinal cords compared with transected spinal cords at 10 and 14 d. This led to higher arginase I-positive alternatively activated macrophage (M2 macrophage) responses. The gene expression of several enzymes involved in polyamine biosynthesis pathways was also upregulated in the graft areas of repaired spinal cords. The treatment induced a twofold upregulation of polyamine levels at 14 d, as confirmed by HPLC. Polyamines are important for the repair process, as demonstrated by the observation that treatment with inhibitors of arginase I and ornithine decarboxylase attenuates the functional recoveries of repaired rats. After 14 d, the treatment also induced the expression of neurotrophin nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as M2 macrophages within grafted nerves expressing BDNF. IL-4 was upregulated in the injury sites of transected rats that received aFGF alone compared with those that received nerve grafts alone at 10 d. Conversely, nerve graft treatment induced NGF and BDNF expression at 14 d. Macrophages expressing polyamines and BDNF may benefit axonal regeneration at 14 d. These results indicate that aFGF and nerve grafts regulate different macrophage responses, and M2 macrophages may play an important role in axonal regeneration after spinal cord injury in rats.

  10. Differential expression of distinct surface markers in early endothelial progenitor cells and monocyte-derived macrophages.

    PubMed

    Cheng, Shu-Meng; Chang, Shing-Jyh; Tsai, Tsung-Neng; Wu, Chun-Hsien; Lin, Wei-Shing; Lin, Wen-Yu; Cheng, Cheng-Chung

    2013-01-01

    Bone marrow-derived endothelial progenitor cells (EPCs) play a fundamental role in postnatal angiogenesis. Currently, EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Reports have shown that early EPCs share common properties and surface markers with adherent blood cells, especially CD14+ monocytes. Distinguishing early EPCs from circulating monocytes or monocyte-derived macrophages (MDMs) is therefore crucial to obtaining pure endothelial populations before they can be applied as part of clinical therapies. We compared the gene expression profiles of early EPCs, blood cells (including peripheral blood mononuclear cells, monocytes, and MDMs), and various endothelial lineage cells (including mature endothelial cells, late EPCs, and CD133+ stem cells). We found that early EPCs expressed an mRNA profile that showed the greatest similarity to MDMs than any other cell type tested. The functional significance of this molecular profiling data was explored by Gene Ontology database search. Novel plasma membrane genes that might potentially be novel isolation biomarkers were also pinpointed. Specifically, expression of CLEC5A was high in MDMs, whereas early EPCs expressed abundant SIGLEC8 and KCNE1. These detailed mRNA expression profiles and the identified functional modules will help to develop novel cell isolation approaches that will allow EPCs to be purified; these can then be used to target cardiovascular disease, tumor angiogenesis, and various ischemia-related diseases.

  11. Role of spleen-derived monocytes/macrophages in acute ischemic brain injury

    PubMed Central

    Kim, Eunhee; Yang, Jiwon; D Beltran, Cesar; Cho, Sunghee

    2014-01-01

    Monocytes/macrophages (MMs), mononuclear phagocytes, have been implicated in stroke-induced inflammation and injury. However, the presence of pro-inflammatory Ly-6Chigh and antiinflammatory Ly-6Clow monocyte subsets raises uncertainty regarding their role in stroke pathologic assessment. With recent identification of the spleen as an immediate reservoir of MMs, this current study addresses whether the spleen-derived MMs are required for stroke pathologic assessment. We observed that the spleen was contracted in poststroke animals and the contraction was accompanied by decreased number of Ly-6Chigh and Ly-6Clow subsets in the spleen. The deployment of these subsets from the spleen temporally coincided with respective increases in the ischemic brain. Compared to mice with the spleen, mice receiving a splenectomy just before the stroke displayed less accumulation of Ly-6Chigh and Ly-6Clow MMs in the brain. Despite the reduced accumulation of both subsets, infarct size and swelling were not reduced in the asplenic mice. The dissociative findings of infarct size and extent of MM infiltration in the postischemic brain indicate minimal involvement of spleen-derived total MMs in acute infarct development. Selective Ly-6Chigh or Ly-6Clow MM targeting is suggested to address the contribution of the individual subset to acute stroke pathologic assessment. PMID:24865998

  12. Macrophage Polarization.

    PubMed

    Murray, Peter J

    2017-02-10

    Macrophage polarization refers to how macrophages have been activated at a given point in space and time. Polarization is not fixed, as macrophages are sufficiently plastic to integrate multiple signals, such as those from microbes, damaged tissues, and the normal tissue environment. Three broad pathways control polarization: epigenetic and cell survival pathways that prolong or shorten macrophage development and viability, the tissue microenvironment, and extrinsic factors, such as microbial products and cytokines released in inflammation. A plethora of advances have provided a framework for rationally purifying, describing, and manipulating macrophage polarization. Here, I assess the current state of knowledge about macrophage polarization and enumerate the major questions about how activated macrophages regulate the physiology of normal and damaged tissues.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  14. Association of Macrophage Migration Inhibitory Factor (MIF) Polymorphisms with Risk of Meningitis from Streptococcus pneumoniae

    PubMed Central

    Doernberg, Sarah; Schaaf, Bernhard; Dalhoff, Klaus; Leng, Lin; Beitin, Anna; Quagliarello, Vincent; Bucala, Richard

    2010-01-01

    Macrophage migration inhibitory factor (MIF) is an upstream proinflammatory cytokine encoded by a functionally polymorphic locus. This study of 119 patients explored the potential relationship between MIF genotype and invasive Streptococcus pneumoniae infections. We observed an association between a high-expression MIF allele and occurrence of pneumococcal meningitis. PMID:21208809

  15. Moraxella catarrhalis stimulates the release of proinflammatory cytokines and prostaglandin E from human respiratory epithelial cells and monocyte-derived macrophages.

    PubMed

    Fink, Joshua; Mathaba, Leslie T; Stewart, Geoffrey A; Graham, Peter T; Steer, James H; Joyce, David A; McWilliam, Andrew S

    2006-03-01

    The outer membrane proteins of Moraxella catarrhalis, a bacterial pathogen which causes disease in both children and adults, play an important role in its phenotypic properties. However, their proinflammatory potential with regard to respiratory epithelium and macrophages is unclear. To this end, we examined the cytokine- and mediator-inducing capacity of a heat-killed wild-type M. catarrhalis strain and a nonautoagglutinating mutant as well as their outer membrane proteins and secretory/excretory products using the A549 respiratory epithelial cell line. The outer membrane proteins and secretory/excretory products from both isolates as well as the heat-killed bacteria all induced interleukin (IL)-6, IL-8 and prostaglandin E2, but not IL-1beta, from the A549 cell line in a dose- and time-dependent manner. Heat-killed bacteria and secretory/excretory products stimulated the release of IL-1beta, IL-6, IL-8 and prostaglandin E2 from human monocyte-derived macrophages. Both heat-killed isolates also stimulated nuclear translocation and transactivation of nuclear factor-kappaB. The heat-killed wild-type autoagglutinating isolate induced significantly greater amounts of IL-6 and IL-8 from A549 cells than the nonautoagglutinating mutant compared with the monocyte-derived macrophages but no significant differences in the amounts induced by the two strains were observed. These differences were also evident when the respiratory cell line was stimulated with outer membrane proteins as well as in the degree of nuclear factor-kappaB transactivation. There was little difference in the stimulatory activity of the secretory/excretory products. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses revealed some differences in the outer membrane proteins and secretory excretory products between the two isolates. Combined, these data show that M. catarrhalis secretory excretory products and outer membrane proteins are associated with the induction of inflammatory

  16. D4F alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and ER stress-CHOP pathway[S

    PubMed Central

    Yao, Shutong; Tian, Hua; Miao, Cheng; Zhang, Da-Wei; Zhao, Li; Li, Yanyan; Yang, Nana; Jiao, Peng; Sang, Hui; Guo, Shoudong; Wang, Yiwei; Qin, Shucun

    2015-01-01

    This study was designed to explore the protective effect of D4F, an apoA-I mimetic peptide, on oxidized LDL (ox-LDL)-induced endoplasmic reticulum (ER) stress-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) pathway-mediated apoptosis in macrophages. Our results showed that treating apoE knockout mice with D4F decreased the serum ox-LDL level and apoptosis in atherosclerotic lesions with concomitant downregulation of cluster of differentiation 36 (CD36) and inhibition of ER stress. In vitro, D4F inhibited macrophage-derived foam cell formation. Furthermore, like ER stress inhibitor 4-phenylbutyric acid (PBA), D4F inhibited ox-LDL- or tunicamycin (TM, an ER stress inducer)-induced reduction in cell viability and increase in lactate dehydrogenase leakage, caspase-3 activation, and apoptosis. Additionally, like PBA, D4F inhibited ox-LDL- or TM-induced activation of ER stress response as assessed by the reduced nuclear translocation of activating transcription factor 6 and the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, as well as the downregulation of glucose-regulated protein 78 and CHOP. Moreover, D4F mitigated ox-LDL uptake by macrophages and CD36 upregulation induced by ox-LDL or TM. These data indicate that D4F can alleviate the formation and apoptosis of macrophage-derived foam cells by suppressing CD36-mediated ox-LDL uptake and subsequent activation of the ER stress-CHOP pathway. PMID:25635126

  17. 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

  18. Nerve growth factor promotes killing of Leishmania donovani by macrophages through the induction of hydrogen peroxide.

    PubMed

    Chiba, Rieko; Amagai, Yosuke; Tanaka, Akane; Katakura, Ken; Matsuda, Hiroshi

    2014-08-01

    Visceral leishmaniasis is protozoonosis that occurs worldwide and still requires effective therapies with less toxicity. In this study, we examined the antileishmanial effect of nerve growth factor (NGF) using a murine infection model. NGF blocked the infection of macrophages by Leishmania donovani, which was completely cancelled by a hydrogen peroxide inhibitor. In vivo, not only did NGF show antileishmanial effects, but combination therapy of NGF and sodium stibogluconate synergistically exhibited the activity more potently than each monotherapy. These results indicate that NGF exerts antileishmanial effect by stimulating hydrogen peroxide production in macrophages and can be a novel therapy for leishmaniasis.

  19. 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

  20. Vitamin D binding protein-macrophage activating factor inhibits HCC in SCID mice.

    PubMed

    Nonaka, Koichi; Onizuka, Shinya; Ishibashi, Hiromi; Uto, Yoshihiro; Hori, Hitoshi; Nakayama, Toshiyuki; Matsuura, Nariaki; Kanematsu, Takashi; Fujioka, Hikaru

    2012-01-01

    A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC. The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d. DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group. DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Substrate elasticity regulates the behavior of human monocyte-derived macrophages.

    PubMed

    Adlerz, Katrina M; Aranda-Espinoza, Helim; Hayenga, Heather N

    2016-05-01

    Macrophages play a key role in atherosclerosis, cancer, and in the response to implanted medical devices. In each of these situations, the mechanical environment of a macrophage can vary from soft to stiff. However, how stiffness affects macrophage behavior remains uncertain. Using substrates of varying stiffness, we show macrophage phenotype and function depends on substrate stiffness. Notably, the cell area increases slightly from a sphere after 18 h on substrates mimicking healthy arterial stiffness (1-5 kPa), whereas macrophages on stiffer substrates (280 kPa-70 GPa) increased in area by nearly eight-fold. Macrophage migration is random regardless of substrate stiffness. The total average track speed was 7.8 ± 0.5 μm/h, with macrophages traveling fastest on the 280-kPa substrate (12.0 ± 0.5 μm/h) and slowest on the 3-kPa substrate (5.0 ± 0.4 μm/h). In addition F-actin organization in macrophages depends on substrate stiffness. On soft substrates, F-actin is spread uniformly throughout the cytoplasm, whereas on stiff substrates F-actin is functionalized into stress fibers. The proliferation rate of macrophages was faster on stiff substrates. Cells plated on the 280-kPa gel had a significantly shorter doubling time than those plated on the softer substrate. However, the ability of macrophages to phagocytose 1-μm particles did not depend on substrate stiffness. In conclusion, the results herein show macrophages are mechanosensitive; they respond to changes in stiffness by modifying their area, migration speed, actin organization, and proliferation rate. These results are important to understanding how macrophages respond in complex mechanical environments such as an atherosclerotic plaque.

  2. Transcription factor Fli-1 positively regulates lipopolysaccharide-induced interleukin-27 production in macrophages.

    PubMed

    Gao, Peng; Yuan, Ming; Ma, Xianwei; Jiang, Wei; Zhu, Lingxi; Wen, Mingyue; Xu, Jing; Liu, Qiuyan; An, Huazhang

    2016-03-01

    IL-27 is an important regulator of TLR4-activated innate immune. The mechanism by which IL-27 production is regulated in TLR4-activated innate immune remains largely unclear. Here we show that expression of transcription factor Fli-1 at protein level is increased in macrophages following LPS stimulation. Fli-1 overexpression increases LPS-activated IL-27 production in macrophages. Consistently, Fli-1 knockdown inhibits LPS-induced IL-27 production in macrophages. Chromatin immunoprecipitation (ChIP) assay reveals that Fli-1 binds the promoter of IL-27 p28 subunit. Further experiments manifest that Fli-1 binds the region between -250 and -150 bp upstream of the transcriptional start site of p28 gene and increases p28 gene promoter-controlled transcription. These results demonstrate that Fli-1 positively regulates IL-27 production in TLR4-activated immune response by promoting transcription of IL-27 p28 gene.

  3. A significant correlation between C - reactive protein levels in blood monocytes derived macrophages versus content in carotid atherosclerotic lesions

    PubMed Central

    2014-01-01

    Background Atherosclerosis is a complex disease involving different cell types, including macrophages that play a major role in the inflammatory events occurring in atherogenesis. C-Reactive Protein (CRP) is a sensitive systemic marker of inflammation and was identified as a biomarker of cardiovascular diseases. Histological studies demonstrate CRP presence in human atherosclerotic lesions, and we have previously shown that macrophages express CRP mRNA. CRP could be locally secreted in the atherosclerotic lesion by arterial macrophages and local regulation of CRP could affect its pro-atherogenic effects. Moreover, human blood derived macrophages (HMDM) expression of CRP could reflect atherosclerotic lesion secretion of CRP. Methods Ten type 2 diabetic patients and ten non-diabetic patients scheduled to undergo carotid endarterectomy were enrolled in this study, and their blood samples were used for serum CRP, lipid determination, and for preparation of HMDM further analyzed for their CRP mRNA expression and CRP content. Carotid lesions obtained from the patients were analyzed for their CRP and interleukin 6 (IL-6) content by immunohistochemistry. Results Lesions from diabetic patients showed substantially higher CRP levels by 62% (p = 0.05) than lesions from non diabetic patients, and CRP staining that co-localized with arterial macrophages. CRP carotid lesion levels positively correlated with CRP mRNA expression (r2 = 0.661) and with CRP content (r2 = 0.611) in the patient’s HMDM. Conclusions Diabetes up-regulated carotid plaques CRP levels and CRP measurements in HMDM could reflect atherosclerotic lesion macrophages secretion of CRP. Understanding the regulation of locally produced macrophage CRP in the arterial wall during atherogenesis could be of major importance in identifying the underlying mechanisms of inflammatory response pathways during atherogenesis. PMID:24588988

  4. Potent inhibition of macrophage migration inhibitory factor (MIF) by myeloperoxidase-dependent oxidation of epicatechins.

    PubMed

    Dickerhof, Nina; Magon, Nicholas J; Tyndall, Joel D A; Kettle, Anthony J; Hampton, Mark B

    2014-09-01

    MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenance of the inflammatory response. It is implicated in a number of inflammatory diseases including sepsis, arthritis and colitis, and in diseases with an inflammatory component, such as atherosclerosis, diabetes and cancer. MIF has an unusual N-terminal proline with catalytic activity, and targeting of this residue by small-molecule inhibitors has been shown to interfere with the biological activity of MIF. The objective of the present study was to determine if MIF was susceptible to modification by epicatechins, a group of dietary flavonoids with known anti-inflammatory properties. Epicatechins are substrates for peroxidases including neutrophil-derived MPO (myeloperoxidase). In the present study we show that oxidation of the catechol moiety of epicatechins to an ο-quinone by MPO generates potent MIF inhibitors. Near complete inhibition of MIF by the MPO/H2O2/epicatechin system was achieved at equimolar concentrations of epicatechin and MIF, even in the presence of other MPO substrates. We have characterized the modification introduced by oxidized (-)-epicatechin on MIF by LC-MS (liquid chromatography MS) and found it to occur at the N-terminal proline. We propose that MIF inhibition by oxidized epicatechins contributes to the anti-inflammatory activity of these compounds.

  5. Molecular cloning and expression of woodchuck granulocyte-macrophage colony stimulating factor.

    PubMed

    Wu, H L; Chen, P J; Lin, H K; Lee, R S; Lin, H L; Liu, C J; Lee, P J; Lee, J J; Chen, D S

    2001-11-01

    Granulocyte-macrophage colony stimulating factor (GM-CSF) has immunoregulatory and antiviral effects, and may thus be promising for the treatment of chronic hepatitis B. Using woodchuck hepatitis virus (WHV)-infected woodchuck as an animal model to test the efficacy and safety of GM-CSF on the therapy of chronic hepatitis B, woodchuck GM-CSF will be required due to the apparent species-specific activity of GM-CSF. The cDNA of woodchuck GM-CSF was cloned using reverse transcription-polymerase chain reaction (RT-PCR) with primers deriving from highly conserved regions of GM-CSF genes from other species. The deduced amino acids, including the signal peptide, is 138 in length and its identities to human, murine, canine and bovine GM-CSFs are 63, 49, 63, and 63% respectively. The genomic DNA of woodchuck GM-CSF was also cloned by PCR. Its organization is highly homologous to that of human and murine GM-CSF genes, consisting of four exons and three introns. Cloned woodchuck GM-CSF was expressed transiently in 293T cells. The recombinant protein expressed was found to stimulate the growth and differentiation of woodchuck bone marrow cells, indicating the protein expressed by the cloned gene is functional. These results pave the way for future studies on the potential role of GM-CSF for the treatment of chronic hepatitis B by using this animal model. Copyright 2001 Wiley-Liss, Inc.

  6. Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer

    PubMed Central

    Schinagl, Alexander; Thiele, Michael; Douillard, Patrice; Völkel, Dirk; Kenner, Lukas; Kazemi, Zahra; Freissmuth, Michael; Scheiflinger, Friedrich; Kerschbaumer, Randolf J.

    2016-01-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer. PMID:27636991

  7. 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.

  8. Interleukin-3/granulocyte macrophage colony-stimulating factor receptor promotes stem cell expansion, monocytosis, and atheroma macrophage burden in mice with hematopoietic ApoE deficiency.

    PubMed

    Wang, Mi; Subramanian, Manikandan; Abramowicz, Sandra; Murphy, Andrew J; Gonen, Ayelet; Witztum, Joseph; Welch, Carrie; Tabas, Ira; Westerterp, Marit; Tall, Alan R

    2014-05-01

    Coronary heart disease is associated with monocytosis. Studies using animal models of monocytosis and atherosclerosis such as ApoE(-/-) mice have shown bone marrow (BM) hematopoietic stem and multipotential progenitor cell (HSPC) expansion, associated with increased cell surface expression of the common β subunit of the granulocyte macrophage colony-stimulating factor/interleukin-3 receptor (CBS) on HSPCs. ApoE(-/-) mice also display increased granulocyte macrophage colony-stimulating factor-dependent monocyte production in the spleen. We investigated the role of the CBS in cholesterol-driven HSPC expansion, monocytosis, and atherosclerosis. Ldlr(-/-) mice were transplanted with ApoE(-/-)Cbs(-/-) or ApoE(-/-) BM followed by Western-type diet feeding. Compared with ApoE(-/-) BM-transplanted controls, ApoE(-/-)Cbs(-/-) BM-transplanted mice had reduced BM and splenic HSPC proliferation, fewer blood monocytes and neutrophils, and reduced macrophage content and area of early atherosclerotic lesions. More advanced lesions showed diminished macrophage and collagen content; however, lesion size was unchanged, reflecting an increase in necrotic core area, associated with a marked decrease in Abcg1 expression and increased macrophage apoptosis. Compared with wild-type mice, Western-type diet-fed ApoE(-/-) mice showed increased CBS expression on granulocyte macrophage colony-stimulating factor-producing innate response activator B cells and expansion of this population. ApoE(-/-)Cbs(-/-) BM-transplanted Ldlr(-/-) mice showed a marked decrease in innate response activator B cells compared with ApoE(-/-) BM-transplanted Ldlr(-/-) controls. Increased levels of CBS on HSPCs and splenic innate response activator B cells lead to expansion of these populations in ApoE(-/-) BM-transplanted Ldlr(-/-) mice, contributing to monocytosis and increased lesional macrophage content. However, in more advanced lesions, the CBS also has a role in atherosclerotic plaque stabilization.

  9. Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

    PubMed

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

    2015-06-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. 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

  11. Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise

    PubMed Central

    Moon, Hyo Youl; Kim, Se Hyun; Yang, Yong Ryoul; Song, Parkyong; Yu, Hyun Sook; Park, Hong Geun; Hwang, Onyou; Lee-Kwon, Whaseon; Seo, Jeong Kon; Hwang, Daehee; Choi, Jang Hyun; Bucala, Richard; Ryu, Sung Ho; Kim, Yong Sik; Suh, Pann-Ghill

    2012-01-01

    Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif−/− mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain. PMID:22826223

  12. Macrophage Migration Inhibitory Factor Activates Hypoxia-Inducible Factor in a p53-Dependent Manner

    PubMed Central

    Oda, Seiko; Oda, Tomoyuki; Nishi, Kenichiro; Takabuchi, Satoshi; Wakamatsu, Takuhiko; Tanaka, Tomoharu; Adachi, Takehiko; Fukuda, Kazuhiko; Semenza, Gregg L.; Hirota, Kiichi

    2008-01-01

    Background Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner. Methods/Principal Findings The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly. Conclusion/Significance MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF's potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF. PMID:18493321

  13. Leishmania eukaryotic initiation factor (LeIF) inhibits parasite growth in murine macrophages.

    PubMed

    Koutsoni, Olga; Barhoumi, Mourad; Guizani, Ikram; Dotsika, Eleni

    2014-01-01

    The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF), an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment), and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment), and resistance to infection was also observed at both time points tested (19 h and 72 h) after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s) that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO) and reactive oxygen species (ROS), within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α) as well as tumor necrosis factor alpha (TNF-α) expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably different

  14. Elevated ARG1 expression in primary monocytes-derived macrophages as a predictor of radiation-induced acute skin toxicities in early breast cancer patients

    PubMed Central

    Jung, Karen; Sabri, Siham; Hanson, John; Xu, Yaoxian; Wang, Ying Wayne; Lai, Raymond; Abdulkarim, Bassam S

    2015-01-01

    Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their “M1/M2” activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic factor of erythema (p = 0 .032), moist desquamation (p = 0 .027), and CTC grade (p = 0 .056). Interestingly, multivariate analysis of ARG1 mRNA expression in macrophages stimulated with IL-4 also revealed independent prognostic value for predicting acute RT-induced toxicity factors, erythema (p = 0 .069), moist desquamation (p = 0 .037), and CTC grade (p = 0 .046). To conclude, our findings underline for the first time the biological significance of increased

  15. Elevated ARG1 expression in primary monocytes-derived macrophages as a predictor of radiation-induced acute skin toxicities in early breast cancer patients.

    PubMed

    Jung, Karen; Sabri, Siham; Hanson, John; Xu, Yaoxian; Wang, Ying Wayne; Lai, Raymond; Abdulkarim, Bassam S

    2015-01-01

    Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their "M1/M2" activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic