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Sample records for mouse peritoneal macrophages

  1. Effect of lectins on mouse peritoneal macrophage phagocytic activity.

    PubMed

    Maldonado, G; Porras, F; Fernández, L; Vázquez, L; Zenteno, E

    1994-11-01

    We studied the in vitro ability of lectin-treated murine peritoneal macrophages to attach and phagocytize particulate antigens. Glucose and mannose specific lectins such as Con-A and lentil lectin, as well as complex lactosamine residues specific lectins, such as Phaseolus vulgaris var. cacahuate and Phaseolus coccineus var. alubia, increased the macrophage phagocytic activity towards heterologous erythrocytes, whereas peanut agglutinin, a galactose-specific lectin, diminished the macrophage phagocytic activity. These results suggest that a galactose-N-acetyl-D galactosamine-containing structure could participate as negative modulator of the phagocytic activity. PMID:7851961

  2. Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

    PubMed

    Ishida, Momoko; Ose, Saya; Nishi, Kosuke; Sugahara, Takuya

    2016-07-01

    We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages. PMID:27095137

  3. On the response of mouse peritoneal macrophages to titanium dioxide pigments in vitro

    SciTech Connect

    Nuuja, I.J.; Arstila, A.U.

    1982-10-01

    Acid phosphatase activity and cell morphology were followed using mouse peritoneal macrophages as a toxicity test model in vitro. The cells were given titanium dioxide (TiO/sub 2/) and five titanium pigments with different coating materials in 100 ..mu..g/ml of culture medium. The cell reactions were studied from 1 to 17 days. Titanium particles inhibited the acid phosphatase activity of the cells compared to controls. In comparison to untreated cells the activity of this enzyme increased in most groups studied, being highest in the control cells (2-3.5 times) after 7 days. The titanium pigments did not cause the drastic alterations in these cells as seen with quartz and asbestos particles, but the titanium pigments were not harmless to the mouse peritoneal macrophages with the doses and culture times used.

  4. [Effects of alkaloids from Coptidis Rhizoma on mouse peritoneal macrophages in vitro].

    PubMed

    Zhou, Xia; Peng, Yao-zong; Huang, Tao; Li, Ling; Mou, Shao-xia; Kou, Shu-ming; Li, Xue-gang

    2015-12-01

    This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox. PMID:27141680

  5. Hypoxia enhances lysosomal TNF-alpha degradation in mouse peritoneal macrophages.

    PubMed

    Lahat, Nitza; Rahat, Michal A; Kinarty, Amalia; Weiss-Cerem, Lea; Pinchevski, Sigalit; Bitterman, Haim

    2008-07-01

    Infection, simulated by lipopolysaccharide (LPS), is a potent stimulator of tumor necrosis factor-alpha (TNF-alpha) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1, and PMJ-2R), hypoxia (O(2) < 0.3%) reduces the secretion of LPS-induced TNF-alpha (P < 0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally as TNF-alpha mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF-alpha by twofold (P < 0.01) by enhancing its degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNF-RII, it increased its binding to the secreted TNF-alpha by twofold (P < 0.05). We suggest that these two posttranslational regulatory checkpoints coexist in hypoxia and may partially explain the reduced secretion and diminished biological activity of TNF-alpha in hypoxic peritoneal macrophages. PMID:18434619

  6. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

    PubMed Central

    Angelin, B

    1988-01-01

    The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis. PMID:3202831

  7. Impairment of the oxidative metabolism of mouse peritoneal macrophages by intracellular Leishmania spp.

    PubMed Central

    Buchmüller-Rouiller, Y; Mauël, J

    1987-01-01

    When stimulated in vitro with macrophage-activating factor or lipopolysaccharide, mouse peritoneal macrophages acquire the capacity to develop a strong respiratory burst when they are triggered by membrane-active agents. The presence of intracellular parasites of the genus Leishmania (L. enriettii, L. major) significantly inhibited such activity, as measured by chemiluminescence, reduction of cytochrome c and Nitro Blue Tetrazolium, and hexose monophosphate shunt levels. On the contrary, inert intracellular particles such as latex beads strongly increased the macrophage respiratory burst, suggesting that the Leishmania-linked inhibition resulted from a specific parasite effect. Impairment of macrophage oxidative metabolism by intracellular Leishmania spp. was a function of the number of infecting microorganisms and was more pronounced in macrophages infected with living than with dead parasites. Moreover, the metabolic inhibition was less apparent in L. enriettii-infected macrophages that were exposed to both macrophage-activating factor and lipopolysaccharide, i.e., conditions leading to complete parasite destruction. The mechanisms of respiratory burst inhibition by intracellular Leishmania spp. are unclear, but these observations suggest that such effects may contribute significantly to intracellular survival of the microorganisms. PMID:3546131

  8. Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the arenavirus Pichinde.

    PubMed Central

    Friedlander, A M; Jahrling, P B; Merrill, P; Tobery, S

    1984-01-01

    Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early nonspecific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system, and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose-dependent inhibition of the DNA synthesis induced by macrophage growth factor-colony stimulating factor. At a multiplicity of inoculum of 5, there is a 75 to 95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pretreated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by [3H]thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance. PMID:6690404

  9. Lysosomal glycosidases in mouse peritoneal macrophages stimulated in vitro with soluble and insoluble glycans.

    PubMed

    Bøgwald, J; Johnson, E; Hoffman, J; Seljelid, R

    1984-04-01

    Mouse peritoneal macrophages stimulated with insoluble glycans in vitro release high amounts of acid hydrolases, N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, and beta-D-galactosidase. The most potent of the stimulatory glycans is a beta-1,3-D-glucan isolated from yeast cell walls. Up to 50% of total enzyme activity was found in the medium after stimulation with this glycan for three days. Agarose, another insoluble glycan containing an alternating sequence of the disaccharide beta-1,3-D-galactose-alpha-1,4-3,6-anhydro-L-galactose units was less potent. The soluble beta-1,3-D-glucan laminaran, which also contains small amounts of mannitol, was not able to induce release of acid glycosidases from macrophages. The release was independent of serum since macrophages cultured under serum-free conditions showed nearly the same pattern of enzyme activities, both in the cells and media. There was no increased release of the acid hydrolase alpha-D-mannosidase after stimulation with the insoluble beta-1,3-D-glucan for three days. The release of the lysosomal glycosidases was not due to cell death, since only small amounts of the cytoplasmic enzyme lactate dehydrogenase were found in the culture media. Insoluble polystyrene latex particles were not able to stimulate mouse macrophages to release lysosomal glycosidases. Tritiated glycans (amylose, dextran, laminaran, the insoluble beta-1,3-D-glucan, and agarose) and the p-nitrophenyl-glycopyranoside derivatives were used as substrates to investigate whether the macrophages contained or released glucanases capable of degrading alpha-1,4-D-glucans, alpha-1-6-D-glucans, beta-1,3-D-glucans, and agarose respectively. We conclude that the glycans were not degraded in macrophage cultures during the time period tested nor were the enzymes induced in macrophages by the glycans during in vitro culture for seven days. PMID:6584526

  10. Stimulated arachidonate metabolism during foam cell transformation of mouse peritoneal macrophages with oxidized low density lipoprotein.

    PubMed Central

    Yokode, M; Kita, T; Kikawa, Y; Ogorochi, T; Narumiya, S; Kawai, C

    1988-01-01

    Changes in arachidonate metabolism were examined in mouse peritoneal macrophages incubated with various types of lipoproteins. Oxidized low density lipoprotein (LDL) was incorporated by macrophages and stimulated macrophage prostaglandin E2 (PGE2) and leukotriene C4 syntheses, respectively, 10.8- and 10.7-fold higher than by the control. Production of 6-keto-PGF1 alpha, a stable metabolite of prostacyclin, was also stimulated. No stimulation was found with native LDL, which was minimally incorporated by the cells. Acetylated LDL and beta-migrating very low density lipoprotein (beta-VLDL), though incorporated more efficiently than oxidized LDL, also had no stimulatory effect. When oxidized LDL was separated into the lipoprotein-lipid peroxide complex and free lipid peroxides, most of the stimulatory activity was found in the former fraction, indicating that stimulation of arachidonate metabolism in the cell is associated with uptake of the lipoprotein-lipid peroxide complex. These results suggest that peroxidative modification of LDL could contribute to the progression of atheroma by stimulating arachidonate metabolism during incorporation into macrophages. Images PMID:3125226

  11. Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages.

    PubMed

    Sung, S S; Nelson, R S; Silverstein, S C

    1983-01-01

    We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 mug/ml and 10 mug/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles. PMID:6298248

  12. Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: amastigote infection of resident cells and inflammatory exudate macrophages.

    PubMed Central

    Fortier, A H; Hoover, D L; Nacy, C A

    1982-01-01

    C3HeB/FeJ peritoneal exudate cells elicited by a variety of sterile inflammatory agents were exposed to Leishmania tropica amastigotes in vitro. Cytochemical characterization of cells that contained intracellular parasites suggested that young, peroxidase-positive macrophages were more susceptible to infection by amastigotes than more mature cells. Replication of the parasite in these younger cells, however, was similar to that observed in resident peritoneal macrophages. PMID:7152674

  13. Antibody-dependent cytolysis of chicken erythrocytes by an in vitro-established line of mouse peritoneal macrophages.

    PubMed

    Walker, W S; Demus, A

    1975-02-01

    An in vitro-established line of mouse peritoneal macrophages (IC-21) was tested for its ability to mediate the cytolysis of 51chromiun-labeled chicken erythrocytes. In the presence of specific antibody, but independently of complement, the macrophages phagocytized and lysed labeled erythrocytes. The phagocytic process proved to be functionally distinct from the cytolytic reaction as demonstrated by enhanced cytolysis in the presence of iodoacetate, an inhibitor of phagocytosis. This cell line, because of its effector activity in antibody-dependent cell-mediated immune reactions, will be useful in characterizing the mechanism(s) involved in macrophage-mediated cytolysis. PMID:1167563

  14. Toxicological interactions of silver nanoparticles and organochlorine pesticides in mouse peritoneal macrophages.

    PubMed

    Glinski, Andressa; Liebel, Samuel; Pelletier, Èmilien; Voigt, Carmen Lucia; Randi, Marco Antonio Ferreira; Campos, Sandro Xavier; Oliveira Ribeiro, Ciro Alberto; Filipak Neto, Francisco

    2016-05-01

    Nanotechnology occupies a prominent space in economy and science due to the beneficial properties of nanomaterials. However, nanoparticles may pose risks to living organisms due to their adsorption and pro-oxidative properties. The aim of the current study was to investigate the effects of polymer-coated silver nanoparticles (AgNPs) and organochlorine pesticides (OCPs), as well as their combined effects on mouse peritoneal macrophages. Macrophages were isolated and exposed to three concentrations of AgNPs (groups: N1 = 30, N2 = 300 and N3 = 3000 ng.ml(-1)), two concentrations of OCPs (groups: P1 = 30 and P2 = 300 ng.ml(-1)) and the six possible combinations of these two contaminants for 24 h. AgNPs had irregular shape, Feret diameter of 8.7 ± 7.5 nm and zeta potential of -28.7 ± 3.9 mV in water and -10.7 ± 1.04 mV in culture medium. OCP mixtures and the lower concentrations of AgNPs had no detectable effects on cell parameters, but the highest AgNPs concentration showed high toxicity (trypan blue and MTT assays) resulting in morphological changes, increase of nitric oxide levels and phagocytic index. Foremost, the association of N3 and P2 led to distinct effects from those observed under single exposure. PMID:27001549

  15. Suppression of Mcl-1 induces apoptosis in mouse peritoneal macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Wang, Fei-Yu; Wang, Xin-Min; Wang, Chan; Wang, Xiao-Fang; Zhang, Yu-Qing; Wu, Jiang-Dong; Wu, Fang; Zhang, Wan-Jiang; Zhang, Le

    2016-04-01

    The effect of myeloid cell leukemia-1 (Mcl-1) inhibition on apoptosis of peritoneal macrophages in mice infected with Mycobacterium tuberculosis was investigated and the primary signaling pathway associated with the transcriptional regulation of Mcl-1 was identified. Real-time PCR and western blotting indicated that Mcl-1 transcript and protein expression are upregulated during infection with virulent M. tuberculosis H37Rv and Xinjiang strains but not with attenuated M. tuberculosis strain H37Ra or Bacillus Calmette-Guérin. Mcl-1 transcript and protein expression were downregulated by specific inhibitors of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways (AG490, PD98059 and LY294002, respectively). The strongest inhibitor of Mcl-1 expression was PD98059, the MAPK inhibitor. Flow cytometry demonstrated that the rate of apoptosis in peritoneal macrophages is significantly higher in mice infected with M. tuberculosis and the rate of apoptosis is correlated with the virulence of the strain of M. tuberculosis. Apoptosis was found to be upregulated by AG490, PD98059 and LY294002, whereas inhibition of the MAPK pathway sensitized the infected macrophages to apoptosis. Taken together, these results suggest that specific downregulation of Mcl-1 significantly increases apoptosis of peritoneal macrophages and that the MAPK signaling pathway is the primary mediator of Mcl-1 expression. PMID:26876933

  16. In vitro morphology, viability and cytokine secretion of uterine telocyte-activated mouse peritoneal macrophages.

    PubMed

    Chi, Chi; Jiang, Xiao-Juan; Su, Lei; Shen, Zong-Ji; Yang, Xiao-Jun

    2015-12-01

    Telocytes (TCs), a distinct interstitial cell population, have been identified in the uterus, oviduct and placenta, with multiple proposed potential biological functions. Their unique structure allows them to form intercellular junctions with various immunocytes, both in normal and diseased tissues, suggesting a potential functional relationship with the local immune response. It has been hypothesized that through direct heterocellular junctions or indirect paracrine effects, TCs influence the activity of local immunocytes that are involved in the inflammatory process and in immune-mediated reproductive abnormalities. However, no reliable cytological evidence for this hypothesis is currently available. In this study, we cultured primary murine uterine TCs and collected TC conditioned media (TCM). Mouse peritoneal macrophages (pMACs) were co-cultured for 48 hrs with TCM or with DMEM/F12 or lipopolysaccharide (LPS) as negative and positive controls, respectively. Normal uterine TCs with a typical structure and a CD-34-positive/vimentin-positive/c-kit-negative immunophenotype were observed during culture. Morphologically, TCM-treated pMACs displayed an obvious activation/immunoresponse, in contrast to over-stimulation and cell death after LPS treatment and no sign of activation in the presence of DMEM/F12. Accordingly, a cell counting kit 8 (CCK-8) assay indicated significant activation of pMACs by TCM and LPS compared to DMEM/F12, thus supporting the marked morphological differences among these groups of cells. Furthermore, within a panel of macrophage-derived cytokines/enzymes, interleukin-6 (IL-6) and inducible nitric oxide synthase were significantly elevated in TCM-treated pMACs; tumour necrosis factor α, IL1-R1, and IL-10 were slightly, but significantly, up-regulated; and no changes were observed for transforming growth factor-β1, IL-1β, IL-23α and IL-18. Our results indicate that TCs are not simply innocent bystanders but are rather functional players in

  17. Uptake of remnant like particles (RLP) in diabetic patients from mouse peritoneal macrophages.

    PubMed

    Tomono, S; Kawazu, S; Kato, N; Ono, T; Ishii, C; Ito, Y; Shimizu, M; Shimoyama, M; Nakano, T; Nakajima, K

    1994-01-01

    To investigate whether the remnant like particles (RLP), separated from serum by an immunoaffinity gel mixture of anti-apo B-100 and apo A-I monoclonal antibodies, are relevant to the initiation or progression of atherosclerosis, the incorporation of RLP into mouse macrophages was studied using histochemical and biochemical techniques. Remnant lipoproteins such as RLP are reported to contain a large quantity of chyloniron and very low density lipoprotein (VLDL) remnants, especially in diabetic patients. The RLP separated from the sera of 32 diabetic patients were found to be predominantly taken up into macrophages harvested from mouse abdominal cavities by the staining method applying oil red O. Furthermore, using 14C-oleate to prove the uptake of lipoproteins by macrophages, the uptake of RLP-VLDL, a VLDL fraction of RLP by ultracentrifugation, was the next highest to that of the oxidized LDL, which suggests that RLP-VLDL is also aggressively taken up by macrophages. The degree of uptake of RLP-VLDL by macrophages was positively correlated with HbA1c of these diabetic patients (r = 0.556, p < 0.01), irrespective of the ways of the treatment of diabetes. In conclusion, RLP can contribute to the foaming of macrophages, which in turn may explain the acceleration of atherosclerosis in diabetic patients. PMID:9222876

  18. Effects of Omega-3-Rich Harp Seal Oil on the Production of Pro-Inflammatory Cytokines in Mouse Peritoneal Macrophages.

    PubMed

    Choi, Myungwon; Ju, Jaehyun; Suh, Jae Soo; Park, Kun-Young; Kim, Kwang Hyuk

    2015-06-01

    Omega-3, a polyunsaturated fatty acid, is an essential fatty acid necessary for human health, and it protects against cardiovascular disease, inflammation, autoimmune diseases, and cancer. In the present study, we investigated the effects of omega-3-rich harp seal oil (HSO) on the production of nitric oxide (NO) and cytokines, such as tumor necrosis factor (TNF)-α, interleukin-(IL)-1β, IL-6, and IL-12/IL-23 (p40) in peritoneal macrophages of mice. The culture supernatants of murine macrophages exposed to lipopolysaccharide (LPS), HSO, or HSO+LPS were harvested to assay IL-1β, TNF-α, IL-6, and IL-12/IL-23 (p40) cytokines and NO. TNF-α, IL-1 β, and IL-12/IL-23 (p40) levels, except IL-6, were lower in the culture supernatants of mouse peritoneal macrophages exposed to LPS plus HSO than those of the groups exposed to LPS alone. These observations demonstrate that omega-3-rich harp seal oil downregulates the production of the pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-12/IL-23 (p40). These results suggest that HSO could be potentially used as a preventive agent or as an adjunct in anti-inflammatory therapy, if more research results were accumulated. PMID:26175994

  19. Effects of Omega-3-Rich Harp Seal Oil on the Production of Pro-Inflammatory Cytokines in Mouse Peritoneal Macrophages

    PubMed Central

    Choi, Myungwon; Ju, Jaehyun; Suh, Jae Soo; Park, Kun-Young; Kim, Kwang Hyuk

    2015-01-01

    Omega-3, a polyunsaturated fatty acid, is an essential fatty acid necessary for human health, and it protects against cardiovascular disease, inflammation, autoimmune diseases, and cancer. In the present study, we investigated the effects of omega-3-rich harp seal oil (HSO) on the production of nitric oxide (NO) and cytokines, such as tumor necrosis factor (TNF)-α, interleukin-(IL)-1β, IL-6, and IL-12/IL-23 (p40) in peritoneal macrophages of mice. The culture supernatants of murine macrophages exposed to lipopolysaccharide (LPS), HSO, or HSO+LPS were harvested to assay IL-1β, TNF-α, IL-6, and IL-12/IL-23 (p40) cytokines and NO. TNF-α, IL-1 β, and IL-12/IL-23 (p40) levels, except IL-6, were lower in the culture supernatants of mouse peritoneal macrophages exposed to LPS plus HSO than those of the groups exposed to LPS alone. These observations demonstrate that omega-3-rich harp seal oil downregulates the production of the pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-12/IL-23 (p40). These results suggest that HSO could be potentially used as a preventive agent or as an adjunct in anti-inflammatory therapy, if more research results were accumulated. PMID:26175994

  20. Hypertriglyceridemic very low density lipoproteins induce triglyceride synthesis and accumulation in mouse peritoneal macrophages.

    PubMed

    Gianturco, S H; Bradley, W A; Gotto, A M; Morrisett, J D; Peavy, D L

    1982-07-01

    Triglyceride-rich lipoproteins may be responsible for the lipid accumulation in macrophages that can occur in hypertriglyceridemia. Chylomicrons and very low density lipoproteins (VLDL, total and with flotation constant [S(f)] 100-400) from fasting hypertriglyceridemic subjects induced a massive accumulation of oil red O-positive inclusions in unstimulated peritoneal macrophages. Cell viability was not affected. The predominant lipid that accumulated in cells exposed to hypertriglyceridemic VLDL was triglyceride. Hypertriglyceridemic VLDL stimulated the incorporation of [(14)C]oleate into cellular triglyceride up to ninefold in 16 h, but not into cholesteryl esters. Mass increase in cellular triglyceride was 38-fold. The stimulation of cellular triglyceride formation was dependent on time, temperature, and concentration of hypertriglyceridemic VLDL. By contrast, VLDL, low density, and high density lipoproteins from fasting normolipemic subjects had no significant effect on oleate incorporation into neutral lipids or on visible lipid accumulation.(125)I-Hypertriglyceridemic VLDL (S(f) 100-400) were degraded by macrophages in a dose-dependent manner, with 50 and 100% saturation observed at 3 and 24 mug protein/ml (2.5 and 20 nM), respectively. Hypertriglyceridemic VLDL inhibited the internalization and degradation of (125)I-hypertriglyceridemic VLDL (4 nM) by 50% at 3 nM. Cholesteryl ester-rich VLDL from cholesterol-fed rabbits gave 50% inhibition at 5 nM. Low density lipoproteins (LDL) inhibited by 10% at 5 nM and 40% at 47 nM. Acetyl LDL at 130 nM had no effect. We conclude that the massive triglyceride accumulation produced in macrophages by hypertriglyceridemic VLDL is a direct consequence of uptake via specific receptors that also recognize cholesteryl ester-rich VLDL and LDL but are distinct from the acetyl LDL receptor. Uptake of these triglyceride-rich lipoproteins by monocyte-macrophages in vivo may play a significant role in the pathophysiology of

  1. Eicosanoid production by mouse peritoneal macrophages during Toxoplasma gondii penetration: role of parasite and host cell phospholipases.

    PubMed Central

    Thardin, J F; M'Rini, C; Beraud, M; Vandaele, J; Frisach, M F; Bessieres, M H; Seguela, J P; Pipy, B

    1993-01-01

    The metabolism of endogenous arachidonic acid by mouse resident peritoneal macrophages infected in vitro with Toxoplasma gondii was studied. Prelabeling of macrophages with [5,6,8,9,11,12,14,15-3H]arachidonic acid and challenge with tachyzoites for 15 min resulted in a high mobilization of free labeled arachidonic acid (178%) in the culture medium. The parasites also triggered the synthesis of 6-keto-prostaglandin F1 alpha (47%), prostaglandin E2 (44%), leukotrienes C4 and D4 (33%) and 5-, 12-hydroxyeicosatetraenoic acids (155%). The study indicated that during the intracellular development phase of the parasites, 6-keto-prostaglandin F1 alpha (38%), prostaglandin E2 (31%) leukotrienes C4 and D4 (15%), hydroxyeicosatetraenoic acids (43%), and free arachidonic acid (110%) were secreted into the culture medium. Pretreatment of tachyzoites with phospholipase A2 inhibitors (4-p-bromophenacyl bromide and quinacrine) and no calcium in the culture medium resulted in inhibition of tachyzoite penetration into the macrophages and a decrease of the arachidonic acid metabolism. The triggering of the arachidonic acid cascade by T. gondii was dependent on the active penetration of the parasites into the macrophages, whereas preincubation of the macrophages with phospholipase A2 inhibitors did not affect penetration or free arachidonic acid release, thereby supporting a role for parasite phospholipase in the penetration process and in arachidonic acid mobilization from macrophage membrane phospholipids. Moreover, treatment of macrophages with phospholipase A2 inhibitors decreased the activities of the cyclooxygenase and lipoxygenase pathways, also suggesting an activation of host cell phospholipase A2 by the parasite. PMID:8454347

  2. Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro.

    PubMed

    Eze, M O; Yuan, L; Crawford, R M; Paranavitana, C M; Hadfield, T L; Bhattacharjee, A K; Warren, R L; Hoover, D L

    2000-01-01

    Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucella-macrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement- and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0. 01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 microg/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-gamma). Macrophage activation with IFN-gamma inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-gamma treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N(G)-monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-gamma-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-gamma in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis. PMID:10603396

  3. Rates of utilization of glucose, glutamine and oleate and formation of end-products by mouse peritoneal macrophages in culture.

    PubMed Central

    Newsholme, P; Newsholme, E A

    1989-01-01

    1. The metabolism of mouse thioglycollate-elicited peritoneal macrophages was studied in culture for up to 96 h. 2. The rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 80 h of culture. 3. The rates of glucose and glutamine utilization by cultured macrophages were approx. 500 and 90 nmol/h per mg of protein respectively. This rate of glucose utilization is at least 50% greater than that previously reported for macrophages during 60 min incubation in a shaking flask; and it is now increased by addition of glutamine to the culture medium. The rate of glutamine utilization in culture is similar to that previously reported for macrophages during 60 min incubation. The major end-product of glucose metabolism is lactate, and those of glutamine metabolism are CO2, glutamate, ammonia and alanine. 4. Oleate was utilized by these cells: 14C from [14C]oleate was incorporated into CO2 and cellular lipid. The highest rate of oleate utilization was observed when both glucose and glutamine were present in the culture medium. The presence of oleate in the culture medium did not affect the rates of utilization of either glucose or glutamine. Of the [14C]oleate incorporated into lipid, approx. 80% was incorporated into triacylglycerol and only 18% into phospholipid. 5. The turnover rate for the total ATP content of the macrophage in culture is about 10 times per minute: the value for the perfused isolated maximally working rat heart is 22. This indicates a high metabolic rate for macrophages, and consequently emphasizes the importance of the provision of fuels for their function in an immune response. PMID:2775207

  4. Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors.

    PubMed Central

    Husmann, L K; Johnson, W

    1992-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen of alveolar macrophages. Although previous studies have demonstrated that specific antibody facilitates uptake of L. pneumophila by phagocytic cells, the role of complement has been unclear. Thus, we have examined the relative contributions of Fc gamma- and complement receptor-mediated adherence to guinea pig peritoneal macrophages, U937 human monocytic cells, and J774 mouse macrophage cells. Opsonization of L. pneumophila (Philadelphia 2) with polyclonal immunoglobulin G promoted maximum adherence to guinea pig macrophages. In contrast, incubation in the presence of 20% fresh nonimmune human serum from a single donor did not promote adherence. The results obtained with U937 and J774 cells paralleled those obtained with guinea pig macrophages. In the absence of specific antibody, opsonization with guinea pig complement did not enhance adherence of the Philadelphia 1, Philadelphia 2, or Knoxville strain. However, when complement was added to heat-inactivated, specific antiserum, a fourfold increase in the number of adherent organisms was observed. Blocking studies utilizing membrane receptor-specific monoclonal antibodies demonstrated that both Fc and complement receptors mediated adherence of organisms treated with complement in the presence of specific antibody. These results suggest that complement augments adherence of L. pneumophila only when acting in concert with specific antibody. PMID:1452353

  5. Immunomodulatory action of monosulfated triterpene glycosides from the sea cucumber Cucumaria okhotensis: stimulation of activity of mouse peritoneal macrophages.

    PubMed

    Aminin, Dmitry L; Silchenko, Alexandra S; Avilov, Sergey A; Stepanov, Vadim G; Kalinin, Vladimir I

    2010-12-01

    Six monosulfated triterpene glycosides, frondoside A1 (1), okhotoside B1 (2), okhotoside A1-1 (3), frondoside A (4), okhotoside A2-1 (5) and cucumarioside A2-5 (6), isolated from Cucumaria okhotensis Levin et Stepanov, stimulate spreading and lysosomal activity of mouse macrophages and ROS-formation in the macrophages. The highest macrophage spreading and stimulation of their lysosomal activity was induced by glycosides 1, 4 and 6. All glycosides similarly stimulate ROS formation in macrophages, but glycoside 2 caused minimal stimulation. PMID:21299111

  6. Alpha-Pinene Exhibits Anti-Inflammatory Activity Through the Suppression of MAPKs and the NF-κB Pathway in Mouse Peritoneal Macrophages.

    PubMed

    Kim, Dae-Seung; Lee, Hyun-Ja; Jeon, Yong-Deok; Han, Yo-Han; Kee, Ji-Ye; Kim, Hyun-Jeong; Shin, Hyun-Ji; Kang, JongWook; Lee, Beom Su; Kim, Sung-Hoon; Kim, Su-Jin; Park, Sang-Hyun; Choi, Byung-Min; Park, Sung-Joo; Um, Jae-Young; Hong, Seung-Heon

    2015-01-01

    In this study, we found that alpha-pinene (α-pinene) exhibits anti-inflammatory activity through the suppression of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-κB) pathway in mouse peritoneal macrophages. α-Pinene is found in the oils of many coniferous trees and rosemary. We investigated the inhibitory effects of α-Pinene on inflammatory responses induced by lipopolysaccharide (LPS) using mouse peritoneal macrophages. α-Pinene significantly decreased the LPS-induced production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide (NO). α-Pinene also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in LPS-stimulated macrophages. Additionally, the activations of MAPKs and NF-κB were attenuated by means of α-pinene treatment. These results indicate that α-pinene has an anti-inflammatory effect and that it is a potential candidate as a new drug to treat various inflammatory diseases. PMID:26119957

  7. The toxic effects of indoor atmospheric fine particulate matter collected from allergic and non-allergic families in Wuhan on mouse peritoneal macrophages.

    PubMed

    Yan, Biao; Li, Jinquan; Guo, Junhui; Ma, Ping; Wu, Zhuo; Ling, ZhenHao; Guo, Hai; Hiroshi, Yoshino; Yanagi, U; Yang, Xu; Zhu, Shengwei; Chen, Mingqing

    2016-04-01

    Recent studies have shown that fine particulate matter (PM2.5) is associated with multiple adverse health outcomes and PM2.5-induced oxidative stress is now commonly known as a proposed mechanism of PM2.5-mediated toxicity. However, the association between allergic symptoms in children and exposure to PM2.5 has not been fully elucidated, particularly the role of PM2.5 on the indoor environment involved in allergy or non-allergy is unknown. The aim of the present study was to explore whether indoor PM2.5 from the homes of children with allergic symptoms had more increased risks of allergy than that of healthy ones and then compare the toxicity and inflammatory response of them. In this study, indoor PM2.5 was collected from the homes of schoolchildren with allergic symptoms and those of healthy ones respectively, and components of PM2.5 were analyzed. PM2.5-mediated oxidative damage and inflammatory response were further evaluated in mouse peritoneal macrophages based on its effects on the levels of reactive oxygen species accumulation, lipid peroxidation, DNA damage or cytokine production. It seems that oxidative stress may contribute to PM2.5-induced toxicity, and PM2.5 from the allergic indoor environment produced more serious toxic effects and an inflammatory response on mouse peritoneal macrophages than that from a non-allergic indoor environment. PMID:26304222

  8. Amelioration of oxidative DNA damage in mouse peritoneal macrophages by Hippophae salicifolia due to its proton (H+) donation capability: Ex vivo and in vivo studies

    PubMed Central

    Chakraborty, Mainak; Karmakar, Indrajit; Haldar, Sagnik; Das, Avratanu; Bala, Asis; Haldar, Pallab Kanti

    2016-01-01

    Introduction: The present study evaluates the antioxidant effect of methanol extract of Hippophae salicifolia (MEHS) bark with special emphasis on its role on oxidative DNA damage in mouse peritoneal macrophages. Material and Methods: In vitro antioxidant activity was estimated by standard antioxidant assays whereas the antioxidant activity concluded the H+ donating capacity. Mouse erythrocytes’ hemolysis and peritoneal macrophages’ DNA damage were determined spectrophotometrically. In vivo antioxidant activity of MEHS was determined in carbon tetrachloride-induced mice by studying its effect on superoxide anion production in macrophages cells, superoxide dismutase in the cell lysate, DNA damage, lipid peroxidation, and reduces glutathione. Results: The extract showed good in vitro antioxidant activities whereas the inhibitory concentrations values ranged from 5.80 to 106.5 μg/ml. MEHS significantly (P < 0.05) attenuated the oxidative DNA damage. It also attenuated the oxidative conversion of hemoglobin to methemoglobin and elevation of enzymatic and nonenzymatic antioxidant in cells. Conclusion: The result indicates MEHS has good in vitro-in vivo antioxidant property as well as the protective effect on DNA and red blood cell may be due to its H+ donating property. PMID:27413349

  9. HUMAN ALVEOLAR AND PERITONEAL MACROPHAGES MEDIATE FUNGISTASIS INDEPENDENTLY OF L-ARGININE OXIDATION TO NITRITE OR NITRATE

    EPA Science Inventory

    Human alveolar macrophages (HAM) from 28 normal volunteers were found to inhibit replication of Cryptoccous neoformans. onditions under which fungistasis occurred were different than those required for mouse peritoneal macrophage-mediated fungi stasis. nhibition of fungal replica...

  10. Effect of aflatoxins on rat peritoneal macrophages.

    PubMed Central

    Cusumano, V; Costa, G B; Seminara, S

    1990-01-01

    Phagocytosis, intracellular killing of Candida albicans, and superoxide production by rat peritoneal macrophages exposed to aflatoxins B1, B2, G1, G2, B2a, and M1 at several times and concentrations were analyzed to evaluate the intensity of a depressive effect for each mycotoxin. All aflatoxins used at very low concentrations had a depressive effect on the functions of macrophages. The biggest impairment of phagocytosis, intracellular killing, and spontaneous superoxide production was observed in macrophages exposed to aflatoxins B1 and M1. PMID:2176448

  11. Phagocytic and chemiluminescent responses of mouse peritoneal macrophages to living and killed Salmonella typhimurium and other bacteria.

    PubMed Central

    Tomita, T; Blumenstock, E; Kanegasaki, S

    1981-01-01

    In the presence of luminol, resident as well as thioglycolate-induced and immunized macrophages emitted chemiluminescence more efficiently when the cells were exposed to living Salmonella typhimurium than when they were exposed to the same bacterium killed by ultraviolet light or heat. This phenomenon was observed whether or not the bacterium was opsonized. The different response to living and killed bacteria was also found with Escherichia coli, Pseudomonas aeruginosa, Proteus morganii, and Enterobacter aerogenes, but not with Shigella sonnei, Klebsiella pneumoniae, and Propionibacterium acnes. The results suggest that macrophages respond better to living, motile bacteria than to nonmotile or killed bacteria. The experimental results obtained with motility mutants of S. typhimurium, E. coli, and P. aeruginosa confirm that macrophages exposed to the motile bacteria emit chemiluminescence more efficiently and ingest the motile bacteria at a much faster rate than the nonmotile bacteria. Images PMID:6788707

  12. Stimulation of cholesteryl ester synthesis in mouse peritoneal macrophages by cholesterol-rich very low density lipoproteins from the Watanabe heritable hyperlipidemic rabbit, an animal model of familial hypercholesterolemia

    SciTech Connect

    Kita, T.; Yokode, M.; Watanabe, Y.; Narumiya, S.; Kawai, C.

    1986-05-01

    Cholesterol-rich very low density lipoproteins (VLDL) from the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit induced marked cholesteryl ester accumulation in mouse peritoneal macrophages. This WHHL rabbit, an animal model of human familial hypercholesterolemia, has severe hypercholesterolemia, cutaneous xanthomas, and fulminant atherosclerosis due to the deficiency of the low density lipoprotein (LDL) receptor. When incubated with mouse peritoneal macrophages, the VLDL from WHHL rabbit (WHHL-VLDL) stimulated cholesteryl (/sup 14/C)oleate synthesis 124-fold more than did VLDL from the normal Japanese White rabbit (control-VLDL). The enhancement in cholesteryl ester synthesis and accumulation of WHHL-VLDL was due to the presence of a high affinity binding receptor site on the macrophage cell surface that mediated the uptake and lysosomal degradation of WHHL-VLDL. Competition studies showed that the uptake and degradation of /sup 125/I-WHHL-VLDL was inhibited by unlabeled excess WHHL-VLDL and beta-migrating VLDL (beta-VLDL), but not LDL. Furthermore, the degradation of WHHL-VLDL was not blocked by either fucoidin, polyinosinic acid, or polyguanylic acid, potent inhibitors of the acetylated (acetyl)-LDL binding site, or by acetyl-LDL. These results suggest that macrophages possess a high affinity receptor that recognizes the cholesterol-rich VLDL present in the plasma of the WHHL rabbit and that the receptor which mediates ingestion of WHHL-VLDL seems to be the same as that for beta-VLDL and leads to cholesteryl ester deposition within macrophages. Thus, the uptake of the cholesterol-rich VLDL from the WHHL rabbit by macrophages in vivo may play a significant role in the pathogenesis of atherosclerosis in the WHHL rabbit.

  13. Esculin exhibited anti-inflammatory activities in vivo and regulated TNF-α and IL-6 production in LPS-stimulated mouse peritoneal macrophages in vitro through MAPK pathway.

    PubMed

    Niu, Xiaofeng; Wang, Yu; Li, Weifeng; Zhang, Hailin; Wang, Xiumei; Mu, Qingli; He, Zehong; Yao, Huan

    2015-12-01

    Esculin, a coumarinic derivative found in Aesculus hippocastanum L. (Horse-chestnut), has been reported to have potent anti-inflammatory properties. The present study is designed to investigate the protective effects of esculin on various inflammation models in vivo and in vitro and to clarify the possible mechanism. Induced-animal models of inflammation and lipopolysaccharide (LPS)-challenged mouse peritoneal macrophages were used to examine the anti-inflammatory activity of esculin. In present study, xylene-induced mouse ear edema, carrageenan-induced rat paw edema, and carrageenan-induced mouse pleurisy were attenuated by esculin. In vitro, the pro-inflammatory cytokine levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatant were reduced by esculin. Meanwhile, we found that esculin significantly inhibited LPS-induced activation of mitogen-activated protein kinase (MAPK) pathway in peritoneal macrophages. These results suggest that esculin has potent anti-inflammatory activities in vivo and in vitro, which may involve the inhibition of the MAPK pathway. Esculin may be a promising preventive agent for inflammatory diseases in human. PMID:26391063

  14. Immunoregulation by macrophages II. Separation of mouse peritoneal macrophages having tumoricidal and bactericidal activities and those secreting PGE and interleukin I

    SciTech Connect

    Hopper, K.E.; Cahill, J.M.

    1983-06-01

    Macrophage subpopulations having bactericidal or tumoricidal activities and secreting interleukin I (IL1) or prostaglandin E (PGE) were identified through primary or secondary infection with Salmonella enteritidis and separated by sedimentation velocity. Bactericidal activity was measured by (3H)-thymidine release from Listeria monocytogenes and tumoricidal activity by 51Cr-release from C-4 fibrosarcoma or P815 mastocytoma cells. Macrophages with bactericidal activity were distinguished from those with tumoricidal activity a) during secondary infection when cytolytic activity occurred only at days 1-4 post injection and bactericidal activity remained high throughout and b) after sedimentation velocity separation. Cytolysis was consistently greatest among adherent cells of low sedimentation velocity, whereas cells with bactericidal activity increased in size during the infection. Tumour cytostasis (inhibition and promotion of (3H)-thymidine uptake) differed from cytolysis in that the former was more prolonged during infection and was also detected among large cells. Secretion of immunoregulatory molecules PGE and IL1 occurred maximally among different macrophage subpopulations separated by sedimentation velocity and depending on the type of stimulus used in vitro. There was an inverse correlation between IL1 production and PGE production after stimulation with C3-zymosan or lipopolysaccharide (LPS). The development of immunity during infection may therefore be dependent upon the relative proportions of effector and regulatory macrophage subpopulations and the selective effects of environmental stimuli on these functions.

  15. Anti-Inflammatory Effects of Hyptis albida Chloroform Extract on Lipopolysaccharide-Stimulated Peritoneal Macrophages.

    PubMed

    Sánchez Miranda, Elizabeth; Pérez Ramos, Julia; Fresán Orozco, Cristina; Zavala Sánchez, Miguel Angel; Pérez Gutiérrez, Salud

    2013-01-01

    We examined the effects of a chloroform extract of Hyptis albida (CHA) on inflammatory responses in mouse lipopolysaccharide (LPS) induced peritoneal macrophages. Our findings indicate that CHA inhibits LPS-induced production of tumor necrosis factor (TNF- α ) and interleukin-6 (IL-6). During the process, levels of cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), and nitric oxide (NO) increased in the mouse peritoneal macrophages; however, the extract suppressed them significantly. These results provide novel insights into the anti-inflammatory actions of CHA and support its potential use in the treatment of inflammatory diseases. PMID:23970974

  16. Anti-Inflammatory Effects of Hyptis albida Chloroform Extract on Lipopolysaccharide-Stimulated Peritoneal Macrophages

    PubMed Central

    Sánchez Miranda, Elizabeth; Pérez Ramos, Julia; Fresán Orozco, Cristina; Zavala Sánchez, Miguel Angel; Pérez Gutiérrez, Salud

    2013-01-01

    We examined the effects of a chloroform extract of Hyptis albida (CHA) on inflammatory responses in mouse lipopolysaccharide (LPS) induced peritoneal macrophages. Our findings indicate that CHA inhibits LPS-induced production of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6). During the process, levels of cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), and nitric oxide (NO) increased in the mouse peritoneal macrophages; however, the extract suppressed them significantly. These results provide novel insights into the anti-inflammatory actions of CHA and support its potential use in the treatment of inflammatory diseases. PMID:23970974

  17. Protein kinase C α inhibition prevents peritoneal damage in a mouse model of chronic peritoneal exposure to high-glucose dialysate.

    PubMed

    Wang, Le; Balzer, Michael S; Rong, Song; Menne, Jan; von Vietinghoff, Sibylle; Dong, Lei; Gueler, Faikah; Jang, Mi-Sun; Xu, Gang; Timrott, Kai; Tkachuk, Sergey; Hiss, Marcus; Haller, Hermann; Shushakova, Nelli

    2016-06-01

    Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model of peritoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage and T cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid-induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-β1-induced epithelial-to-mesenchymal transition and peritoneal dialysis fluid-induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients. PMID:27142955

  18. Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride.

    PubMed

    Tian, Yuhu; Huo, Meijun; Li, Guangsheng; Li, Yanyan; Wang, Jundong

    2016-10-01

    F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages. PMID:27421105

  19. Houttuynia cordata Thunb. volatile oil exhibited anti-inflammatory effects in vivo and inhibited nitric oxide and tumor necrosis factor-α production in LPS-stimulated mouse peritoneal macrophages in vitro.

    PubMed

    Li, Weifeng; Fan, Ting; Zhang, Yanmin; Fan, Te; Zhou, Ping; Niu, Xiaofeng; He, Langchong

    2013-11-01

    Houttuynia cordata Thunb. (HC) is a medicinal herb that generally used in traditional Chinese medicine for treating allergic inflammation. The present study investigated the inhibitory effect of the volatile oil from HC Thunb. on animal models of inflammation and the production of inflammatory mediators in vivo and in vitro. In vivo, xylene-induced mouse ear edema, formaldehyde-induced paw edema and carrageenan-induced mice paw edema were significantly decreased by HC volatile oil. HC volatile oil showed pronounced inhibition of prostaglandin (PG) E2 and malondialdehyde production in the edematous exudates. In vitro exposure of mouse resident peritoneal macrophages to 1, 10, 100 and 1000 µg/mL of HC volatile oil significantly suppressed lipopolysaccharide (LPS)-stimulated production of NO and tumor necrosis factor-α (TNF-α) in a dose-dependent manner. Exposure to HC volatile oil had no effect on cell viability and systemic toxicity. Furthermore, HC volatile oil inhibited the production of NO and TNF-α by down-regulating LPS-stimulated iNOS and TNF-α mRNA expression. Western blot analysis showed that HC volatile oil attenuated LPS-stimulated synthesis of iNOS and TNF-α protein in the macrophages, in parallel. These findings add a novel aspect to the biological profile of HC and clarify its anti-inflammatory mechanism. PMID:23280586

  20. β-(1→3)-Glucan of the Southern Bracket Mushroom, Ganoderma australe (Agaricomycetes), Stimulates Phagocytosis and Interleukin-6 Production in Mouse Peritoneal Macrophages.

    PubMed

    de Melo, Renan Henrique; do Amaral, Alex Evangelista; Menolli, Rafael Andrade; Ayala, Thais Soprani; de Cassia Garcia Simao, Rita; de Santana-Filho, Arquimedes Paixao; Sassaki, Guilherme Lanzi; Kadowaki, Marina Kimiko; da Conceicao Silva, Jose Luis

    2016-01-01

    Ganoderma australe was studied to determine the composition of the cell wall, and polysaccharide fraction SK5 was obtained after freeze-thawing an aqueous 5% potassium hydroxide extraction. The monosaccharide composition of the SK5 fraction revealed by gas chromatography-mass spectrometry showed 81.3% glucose, and analyses by 13C nuclear magnetic resonance spectroscopy confirmed a β-glucan with glycosidic links of the (1→3)-β type and most likely 4-O substituted. In addition, the biological effect of the β-glucan from G. australe was evaluated via in vitro cell cultures of peritoneal macrophages isolated from Swiss mice. Biological assays were assessed for toxicity and cell activation, interleukin-6 cytokine concentrations, and the ability to stimulate phagocytic activity. There was an increase in interleukin-6 by approximately 111% with 1.0 µg/mL of polysaccharide, and phagocyte activity was increased in all concentrations examined, obtaining 52.3% with 0.25 µg/mL polysaccharide. The results indicate that a β-(1→3)-glucan isolated from G. australe can be classified as a biological response modifier. PMID:27481297

  1. Anti-inflammatory effects of phenolic extracts from strawberry and mulberry fruits on cytokine secretion profiles using mouse primary splenocytes and peritoneal macrophages.

    PubMed

    Liu, Chieh-Jung; Lin, Jin-Yuarn

    2013-06-01

    This study isolated phenolic-rich extracts from strawberry (ES) and mulberry (EM) fruit juice using 70% ethanol, analyzed the individual phenolics including four flavonoid components using HPLC and assessed their cytokine secretion regulatory activities using murine primary splenocytes and peritoneal macrophages. The results showed that EM was rich in p-coumaric acid (20798±719μg/g dry weight), rutin (1992±26μg/g dry weight) and quercetin (81±5μg/g dry weight), but ES was relatively rich in p-coumaric acid (7475±1219μg/g dry weight), morin (101±68μg/g dry weight) and quercetin (72±42μg/g dry weight). ES and EM administration significantly decreased splenocytes' (IFN-γ+IL-2+IL-12)/IL-10 (Th1/Th2) cytokine secretion ratios in the absence or presence of lipopolysaccharide (LPS) and TNF-α/IL-10 (pro-/anti-inflammatory) cytokine secretion ratios in the presence of LPS in dose-dependent manners. Our results suggest that ES and EM that are rich in p-coumaric acid, rutin, morin or quercetin, may have strong immunomodulatory effects on splenocytes, via decreasing Th1/Th2 and pro-/anti-inflammatory cytokine secretion ratios. PMID:23590821

  2. Sepsis-Induced Potentiation of Peritoneal Macrophage Migration is mitigated by PD-1 Gene Deficiency

    PubMed Central

    Ayala, Alfred; Elphick, Gwendolyn F.; Kim, Ye Sul; Huang, Xin; Carreira-Rosario, Arnaldo; Santos, Sadella C.; Shubin, Nicholas; Chen, Yaping; Reichner, Jonathan; Chung, Chun-Shiang

    2014-01-01

    Programmed cell death receptor (PD)-1’s effect on phagocyte function has not been extensively described. Here we report that experimental mouse sepsis, cecal ligation and puncture (CLP), induced a marked increase in peritoneal macrophage random migration/ motility/ cell spread, but these changes were lost in the absence of PD-1. Alternatively, phagocytic activity was inversely affected. In vitro cell culture imaging studies, with the macrophage cell line J774, documented that blocking PD-1 with antibody led to aggregation of cytoskeletal proteins alphaactinin and F-actin. Further experiments looking at ex vivo peritoneal macrophages from mice illustrated that a similar pattern of alpha-actinin and F-actin was evident on cells from wild-type CLP mice but not PD-1 −/− CLP mouse cells. We also observed that fMLP-induced migration by J774 cells was markedly attenuated using PD-1 blocking antibodies, a non-selective phosphatase inhibitor and a selective Rap1 inhibitor. Finally, peritoneal macrophages derived from CLP as opposed to Sham mice demonstrated aspects of both cell surface co-localization with CD11b and internalization of PD-1 within vacuoles independent of CD11b staining. Together, we believe the data support a role for PD-1 in mediating aspects of innate macrophage immune dysfunction during sepsis, heretofore unappreciated. PMID:24247196

  3. Effects of recombinant human interleukin-1 beta on accumulation of inflammatory peritoneal macrophages in mice treated with pertussis toxin.

    PubMed Central

    Torre, D; Speranza, F; Pugliese, A; Tambini, R

    1990-01-01

    In this study we report that treatment with recombinant human interleukin-1 beta (rIL-1 beta) (10 U per mouse, intraperitoneally) significantly increased the number of inflammatory macrophages in the peritoneal cavity of mice treated with pertussis toxin (PT) (1 micrograms per mouse, intravenously). The administration of rIL-1 beta in a single intraperitoneal dose (10 U per mouse) 1 or 2 days before challenge with PT did not prevent the decrease in the number of inflammatory macrophages in the peritoneal cavity of mice. On the other hand, the simultaneous administration of rIL-1 beta and PT, as well as the administration of rIL-1 beta 24 h after injection of PT, significantly counteracted the inhibitory effect of PT on inflammatory peritoneal macrophages. PMID:2254036

  4. Effect of Kramecyne on the Inflammatory Response in Lipopolysaccharide-Stimulated Peritoneal Macrophages

    PubMed Central

    Sánchez-Miranda, E.; Lemus-Bautista, J.; Pérez, S.; Pérez-Ramos, J.

    2013-01-01

    Kramecyne is a new peroxide, it was isolated from Krameria cytisoides, methanol extract, and this plant was mostly found in North and South America. This compound showed potent anti-inflammatory activity; however, the mechanisms by which this compound exerts its anti-inflammatory effect are not well understood. In this study, we examined the effects of kramecyne on inflammatory responses in mouse lipopolysaccharide- (LPS-) induced peritoneal macrophages. Our findings indicate that kramecyne inhibits LPS-induced production of tumor necrosis factor (TNF-α) and interleukin- (IL-) 6. During the inflammatory process, levels of cyclooxygenase- (COX-) 2, nitric oxide synthase (iNOS), and nitric oxide (NO) increased in mouse peritoneal macrophages; however, kramecyne suppressed them significantly. These results provide novel insights into the anti-inflammatory actions and support its potential use in the treatment of inflammatory diseases. PMID:23573152

  5. Elicitation of macrophages from the peritoneal cavity of channel catfish

    USGS Publications Warehouse

    Jenkins, J.A.; Klesius, P.H.

    1998-01-01

    Four chemicals were evaluated for elicitation of macrophages in peritoneal cavities of 250-300g healthy channel catfish Ictalurus punctatus. Cellular exudates were collected at 3, 5, 7, 10, 14, and 20 d following intraperitoneal injections with squalene, Freund's incomplete adjuvant (FIA), goat serum, thioglycollate, or as a control, phosphate-buffered saline. Injection with either squalene or FIA induced significantly greater (P ??? 0.0001) macrophage recruitment than the other chemicals. The effectiveness of squalene and FIA was compared further by macrophage collection daily for 7 d. Squalene and FIA elicited similarly high macrophage responses (P ??? 0.0450), the highest being 3.43 x 106 macrophages/mL (SE, 2.4 x l06) at 99% purity at day 2 and 2.1 X 106 macrophages/mL (SE, 0.7 x 106) at day 14 at 80% purity, respectively. In both experiments, the time after injection was not statistically significant, nor was there an interaction between time and chemicals. The occurrence of cells other than macrophages decreased with time to yield macrophage recoveries of 47-99% for squalene and 30-80% for FIA. Two subsets of macrophages were observed by means of flow cytometry. As demonstrated by chemiluminescence, the squalene-elicited cells produced high-energy oxygen compounds important to the phagocytic process.

  6. Fate of Mycobacterium tuberculosis inside rat peritoneal macrophages in vitro.

    PubMed

    Vishwanath, V; Meera, R; Puvanakrishnan, R; Narayanan, P R

    1997-10-01

    Rat peritoneal macrophages in vitro were infected with Mycobacterium tuberculosis and the fate of M. tuberculosis inside macrophages was monitored. Alteration in the levels of nitric oxide (NO) measured in terms of nitrite formed, hydrogen peroxide (H2O2) and lysosomal enzymes such as acid phosphatase, cathepsin-D and beta-glucuronidase in macrophages following M. tuberculosis infection was also studied. Elevation in the levels of nitrite were observed from 72 h of M. tuberculosis infection. Irrespective of the time point, M. tuberculosis infected macrophages produced elevated levels of H2O2. Maximum increase in the level of acid phosphatase was observed from 72 h of M. tuberculosis infection, whereas maximum elevation in the level of beta-glucuronidase was observed 48 h after M. tuberculosis infection. However these microbicidal agents did not alter the intracellular viability of M. tuberculosis. PMID:9350049

  7. Transcriptional switching in macrophages associated with the peritoneal foreign body response.

    PubMed

    Mooney, Jane E; Summers, Kim M; Gongora, Milena; Grimmond, Sean M; Campbell, Julie H; Hume, David A; Rolfe, Barbara E

    2014-07-01

    We previously demonstrated that myeloid cells are the source of fibrotic tissue induced by foreign material implanted in the peritoneal cavity. This study utilised the MacGreen mouse, in which the Csf1r promoter directs myeloid-specific enhanced green fluorescent protein (EGFP) expression, to determine the temporal gene expression profile of myeloid subpopulations recruited to the peritoneal cavity to encapsulate implanted foreign material (cubes of boiled egg white). Cells with high EGFP expression (EGFP(hi)) were purified from exudate and encapsulating tissue at different times during the foreign body response, gene expression profiles determined using cDNA microarrays, and data clustered using the network analysis tool, Biolayout Express(3D). EGFP(hi) cells from all time points expressed high levels of Csf1r, Emr1 (encoding F4/80), Cd14 and Itgam (encoding Mac-1) providing internal validation of their myeloid nature. Exudate macrophages (days 4-7) expressed a large cluster of cell cycle genes; these were switched off in capsule cells. Early in capsule formation, Csf1r-EGFP(hi) cells expressed genes associated with tissue turnover, but later expressed both pro- and anti-inflammatory genes alongside a subset of mesenchyme-associated genes, a pattern of gene expression that adds weight to the concept of a continuum of macrophage phenotypes rather than distinct M1/M2 subsets. Moreover, rather than transdifferentiating to myofibroblasts, macrophages contributing to later stages of the peritoneal foreign body response warrant their own classification as 'fibroblastoid' macrophages. PMID:24638066

  8. Identification of mouse and human macrophages as a site of synthesis of hepatic lipase.

    PubMed

    González-Navarro, Herminia; Nong, Zengxuan; Freeman, Lita; Bensadoun, André; Peterson, Katherine; Santamarina-Fojo, Silvia

    2002-05-01

    Hepatic lipase (HL) is synthesized by the liver and is also present in steroidogenic tissues. As both a lipolytic enzyme and a ligand that facilitates the cellular uptake of lipoproteins, HL plays a major role in lipoprotein metabolism and may modulate atherogenic risk. However, HL has not been directly implicated in lesion development. In the present study we demonstrate that HL is also synthesized by mouse and human macrophages. Northern analysis and real time RT-PCR showed that HL mRNA is present in mouse peritoneal macrophages, RAW-264.7, and IC-21 cells. The levels of HL mRNA in mouse peritoneal macrophages were approximately 10-30% that of mouse liver. HL protein was identified by Western blot analyses in human monocyte-derived macrophages, THP, RAW-264.7, and mouse peritoneal macrophages following fractionation by heparin-sepharose affinity chromatography. These combined findings establish that HL is synthesized de novo by macrophages as well as liver, and raises the possibility that HL may have a direct role in the pathogenesis of atherosclerosis. PMID:11971936

  9. Interaction of bioactive glasses with peritoneal macrophages and monocytes in vitro.

    PubMed

    Bosetti, M; Hench, L; Cannas, M

    2002-04-01

    Macrophage activation was analyzed following exposure to pure, crystalline alpha-quartz powders, two bioactive gel-glass powders of different compositions, and a melt-derived glass, 45S5 Bioglass. The release of reactive oxygen metabolites (chemiluminescence test), modifications of cell morphology, the amount of tumor necrosis factor alpha (TNFalpha) secreted, and the amount of TNFalpha mRNA expression were evaluated. The 45S5 Bioglass powders elicited the highest chemiluminescence response while the two solgel glasses had a lower response with less of an oxidative burst difference between them. Particulate bioactive glasses are actively ingested by mouse peritoneal macrophages, and only the 58S solgel glass had a moderate toxic effect on the macrophages. Macrophage cell morphology showed increased size and cell spreading, consistent with the high level of cytokine secretion induced by 45S5 Bioglass. The 45S5 Bioglass powders led to an increased release of TNFalpha and expression of TNFalpha mRNA relative to unstimulated and control treated monocytes. Bioactive glasses (and particularly 45S5 Bioglass) that in vivo induce rapid bone growth appear to activate an autocrine-like process in which the response evoked by the material (for example monocyte and macrophage activation with cytokine production) enhances subsequent interactions with cells in contact with the material. PMID:11835162

  10. A Role for Connexin43 in Macrophage Phagocytosis and Host Survival after Bacterial Peritoneal Infection1

    PubMed Central

    Gribar, Steven C.; Richardson, Ward; Kohler, Jeff W.; Hoffman, Rosemary A.; Branca, Maria F.; Li, Jun; Shi, Xiao-Hua; Sodhi, Chhinder P.; Hackam, David J.

    2016-01-01

    The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection. PMID:19050272

  11. Intra- and extracellular activities of dicloxacillin and linezolid against a clinical Staphylococcus aureus strain with a small-colony-variant phenotype in an in vitro model of THP-1 macrophages and an in vivo mouse peritonitis model.

    PubMed

    Sandberg, Anne; Lemaire, Sandrine; Van Bambeke, Françoise; Tulkens, Paul M; Hughes, Diarmaid; von Eiff, Christof; Frimodt-Møller, Niels

    2011-04-01

    The small-colony-variant (SCV) phenotype of Staphylococcus aureus has been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT) S. aureus strain and its counterpart with an SCV phenotype both in vitro and in vivo, using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player in S. aureus pathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log(10) in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present. PMID:21282430

  12. Estradiol Is a Critical Mediator of Macrophage-Nerve Cross Talk in Peritoneal Endometriosis

    PubMed Central

    Greaves, Erin; Temp, Julia; Esnal-Zufiurre, Arantza; Mechsner, Sylvia; Horne, Andrew W.; Saunders, Philippa T.K.

    2016-01-01

    Endometriosis occurs in approximately 10% of women and is associated with persistent pelvic pain. It is defined by the presence of endometrial tissue (lesions) outside the uterus, most commonly on the peritoneum. Peripheral neuroinflammation, a process characterized by the infiltration of nerve fibers and macrophages into lesions, plays a pivotal role in endometriosis-associated pain. Our objective was to determine the role of estradiol (E2) in regulating the interaction between macrophages and nerves in peritoneal endometriosis. By using human tissues and a mouse model of endometriosis, we demonstrate that macrophages in lesions recovered from women and mice are immunopositive for estrogen receptor β, with up to 20% being estrogen receptor α positive. In mice, treatment with E2 increased the number of macrophages in lesions as well as concentrations of mRNAs encoded by Csf1, Nt3, and the tyrosine kinase neurotrophin receptor, TrkB. By using in vitro models, we determined that the treatment of rat dorsal root ganglia neurons with E2 increased mRNA concentrations of the chemokine C-C motif ligand 2 that stimulated migration of colony-stimulating factor 1–differentiated macrophages. Conversely, incubation of colony-stimulating factor 1 macrophages with E2 increased concentrations of brain-derived neurotrophic factor and neurotrophin 3, which stimulated neurite outgrowth from ganglia explants. In summary, we demonstrate a key role for E2 in stimulating macrophage-nerve interactions, providing novel evidence that endometriosis is an estrogen-dependent neuroinflammatory disorder. PMID:26073038

  13. Activation effect of Ganoderma lucidum polysaccharides liposomes on murine peritoneal macrophages.

    PubMed

    Liu, Zhenguang; Xing, Jie; Huang, Yee; Bo, Ruonan; Zheng, Sisi; Luo, Li; Niu, Yale; Zhang, Yan; Hu, Yuanliang; Liu, Jiaguo; Wu, Yi; Wang, Deyun

    2016-01-01

    The activation of murine peritoneal macrophages by Ganoderma lucidum polysaccharides liposomes (GLPL) was investigated in vitro. After treatment with GLPL, the changes of the nitric oxide (NO) secretion and iNOS (inducible nitric oxide synthase) activity were evaluated. The results showed that NO production and iNOS activity of macrophages were enhanced compared to GLP and BL group. In addition, both the phagocytic activity and levels of cytokines IL-1β, TNF-α and IFN-γ were enhanced in the peritoneal macrophages of mice by stimulation of GLPL. The expression of the major histocompatibility complex class II molecule (MHC II) on the surface of peritoneal macrophages significantly increased. These indicated that GLPL could enhance the activation of peritoneal macrophages and their potential for use as a delivery system of GLP. PMID:26529190

  14. Optimization on conditions of Lycium barbarum polysaccharides liposome by RSM and its effects on the peritoneal macrophages function.

    PubMed

    Bo, Ruonan; Ma, Xia; Feng, Yibo; Zhu, Qian; Huang, Yee; Liu, Zhenguang; Liu, Cui; Gao, Zhenzhen; Hu, Yuanliang; Wang, Deyun

    2015-03-01

    The purpose of this study was to optimize the preparation conditions of Lycium barbarum polysaccharides liposome (LBPL) by response surface methodology (RSM) and to investigate the effect of LBPL activating function of peritoneal macrophages. LBPL was prepared using the reverse-phase evaporation method. The optimal preparation conditions of LBPL by RSM were as follows: the ratio of lipid to drug (w/w) of 25:1, the ultrasound time of 14 min and the ratio of soybean phospholipids to cholesterol (w/w) of 2.4:1. Under these conditions, the experimental encapsulation efficiency of LBPL was 86.37±0.63%, which was close to the predicted value. These indicated that LBPL with high entrapping efficiency and small particle size could be prepared by the reverse-phase evaporation method, which is applied easily. Furthermore, macrophages are the key players in the innate immune system. LBPL could effectively enhance peritoneal macrophages phagocytosis and resulted in inducing NO (nitric oxide) production in mouse peritoneal macrophages. PMID:25498628

  15. Vagal nerve stimulation blocks peritoneal macrophage inflammatory responsiveness after severe burn injury.

    PubMed

    Lopez, Nicole E; Krzyzaniak, Michael; Costantini, Todd W; De Maio, Antonio; Baird, Andrew; Eliceiri, Brian P; Coimbra, Raul

    2012-08-01

    Large surface area burn injuries lead to activation of the innate immune system, which can be blocked by parasympathetic inputs mediated by the vagus nerve. We hypothesized that vagal nerve stimulation (VNS) would alter the inflammatory response of peritoneal macrophages after severe burn injury. Male BALB/c mice underwent right cervical VNS before 30% total body surface area steam burn and were compared with animals subjected to burn alone. Peritoneal macrophages were harvested at several time points following injury and exposed to lipopolysaccharide (LPS) in culture conditions. The inflammatory response of peritoneal macrophages was measured by analyzing changes in nuclear factor κB p65 phosphorylation using flow cytometry. We found that peritoneal macrophages isolated from mice subjected to burn injury were hyperresponsive to LPS challenge, suggesting burn-induced macrophage activation. We identified a protective role for VNS in blocking peritoneal macrophage activation. Analysis of the phosphorylation state of nuclear factor κB pathway mediator, p65 Rel A, revealed a VNS-mediated reduction in p65 phosphorylation levels after exposure to LPS compared with burn alone. In combination, these studies suggest VNS mediates the inflammatory response in peritoneal macrophages by affecting the set point of LPS responsiveness. PMID:22683732

  16. Effect of lipopolysaccharide on protein accumulation by murine peritoneal macrophages: the correlation to activation for macrophage tumoricidal function

    SciTech Connect

    Tannenbaum, C.S.

    1987-01-01

    The protein synthetic patterns of tumoricidal murine peritoneal macrophage populations have been compared to those of non-tumoricidal populations utilizing two dimensional polyacrylamide gel electrophoresis (2D PAGE) of (/sup 35/S)-methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory and activated macrophages had numerous common features which distinguished them from the other normal non-macrophage cell types examined, unique proteins also distinguished each macrophage population from the others. Peritoneal macrophages elicited by treatment with heat killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16h or 72h functional assays, and shared a common protein synthetic profile which differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages.

  17. Ternatin, an anti-inflammatory flavonoid, inhibits thioglycolate-elicited rat peritoneal neutrophil accumulation and LPS-activated nitric oxide production in murine macrophages.

    PubMed

    Rao, V S N; Paiva, L A F; Souza, M F; Campos, A R; Ribeiro, R A; Brito, G A C; Teixeira, M J; Silveira, E R

    2003-09-01

    Ternatin, an anti-inflammatory flavonoid from Egletes viscosa Less., was examined for its possible influence on thioglycolate-elicited neutrophil influx into the rat peritoneal cavity in vivo and nitric oxide production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages ex vivo. The neutrophil influx induced by thioglycolate was found to be significantly lower in ternatin (25 and 50 mg/kg, s. c.) pre-treated rats with a similar magnitude of inhibition produced by dexamethasone (1 mg/kg, s. c.), a known anti-inflammatory agent. Also, peritoneal macrophages from ternatin (25 mg/kg)-treated mice that were exposed to LPS demonstrated significantly less production of nitric oxide (NO). These results suggest that ternatin exerts its anti-inflammatory action, at least in part, through inhibition of neutrophil migration and modulation of macrophage function. PMID:14598213

  18. Expression of the Homeobox Gene HOXA9 in Ovarian Cancer Induces Peritoneal Macrophages to Acquire an M2 Tumor-Promoting Phenotype

    PubMed Central

    Ko, Song Yi; Ladanyi, Andras; Lengyel, Ernst; Naora, Honami

    2015-01-01

    Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8+ tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages. PMID:24332016

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

    PubMed Central

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

    2000-01-01

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

  20. CD36 and Proteoglycan-Mediated Pathways for (n-3) Fatty Acid–Enriched Triglyceride-Rich Particle Blood Clearance in Mouse Models In Vivo and in Peritoneal Macrophages In Vitro1,2

    PubMed Central

    Densupsoontorn, Narumon; Carpentier, Yvon A.; Racine, Radjini; Murray, Faith M.; Seo, Toru; Ramakrishnan, Rajasekhar; Deckelbaum, Richard J.

    2008-01-01

    Because the mechanisms of (n-3) fatty acid–enriched triglyceride-rich particle [(n-3)-TGRP] uptake are not well characterized, we questioned whether (n-3)-TGRP are removed via “nonclassical” pathways, e.g., pathways other than an LDL receptor and/or involving apolipoprotein E (apoE). Chylomicron-sized model (n-3)-TGRP labeled with [3H]cholesteryl ether were injected into wild-type (WT) and CD36 knockout (CD36−/−) mice at low, nonsaturating and high, saturating doses. Blood clearance of (n-3)-TGRP was determined by calculating fractional catabolic rates. At saturating doses, blood clearance of (n-3)-TGRP was slower in CD36−/− mice relative to WT mice, suggesting that in part CD36 contributes to (n-3)-TGRP uptake. To further examine the potential nonclassical clearance pathways, peritoneal-elicited macrophages from WT and CD36−/− mice were incubated with (n-3)-TGRP in the presence of apoE, lactoferrin, and/or sodium chlorate. Cellular (n-3)-TGRP uptake was measured to test the roles of apoE-mediated pathways and/or proteoglycans. ApoE-mediated pathways compensated in part for defective (n-3)-TGRP uptake in CD36−/− cells. Lactoferrin decreased (n-3)-TGRP uptake in the presence of apoE. Inhibition of cell proteoglycan synthesis by chlorate reduced (n-3)-TGRP uptake in both groups of macrophages, and chlorate effects were independent of apoE. We conclude that although CD36 is involved, it is not the primary contributor to the blood clearance of (n-3)-TGRP. The removal of (n-3)-TGRP likely relies more on nonclassical pathways, such as proteoglycan-mediated pathways. PMID:18203888

  1. Beauveriolides, specific inhibitors of lipid droplet formation in mouse macrophages, produced by Beauveria sp. FO-6979.

    PubMed

    Namatame, I; Tomoda, H; Si, S; Yamaguchi, Y; Masuma, R; Omura, S

    1999-01-01

    Beauveria sp. FO-6979, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. A new compound beauveriolide III was isolated along with a known compound beauveriolide I from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography, silica gel column chromatography and preparative HPLC. Beauveriolides I and III caused a reduction in the number and size of cytosolic lipid droplets in macrophages at 10 microM without any cytotoxic effect on macrophages. PMID:10092189

  2. Enantioselective analysis of D- and L-amino acids from mouse macrophages using high performance liquid chromatography.

    PubMed

    Kato, Shiro; Masuda, Yuki; Konishi, Morichika; Oikawa, Tadao

    2015-12-10

    The intrinsic D-amino acid profile of mouse macrophages extracted from the peritoneal cavity was analyzed using high performance liquid chromatography. Six D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu, D-Gln and D-Lys) were detected in cell lysates of mouse macrophages. The content and the D/D+L ratio differed depending on the type of D-amino acid and were approximately 3.5-22 nmol/g cells, and approximately 1-20%, respectively. The D-amino acid composition of RAW 264.7 cells, which is a model macrophage cell line, was similar to that of the mouse macrophage. These results suggest that macrophages and RAW 264.7 cells with macrophage-like functions have a similar D-amino acid profile. PMID:25982753

  3. Expression and preliminary functional analysis of Siglec-F on mouse macrophages*

    PubMed Central

    Feng, Yin-he; Mao, Hui

    2012-01-01

    Sialic acid-binding immunoglobulin-like lectin (Siglec)-F is a mouse functional paralog of human Siglec-8 that induces apoptosis in human eosinophils, and therefore may be useful as the basis of treatments for a variety of disorders associated with eosinophil hyperactivity, such as asthma. The expression pattern and functions of this protein in various cell types remain to be elucidated. The aim of this study was to determine the expression of Siglec-F on mouse macrophages by immunocytochemical staining, and also to investigate the effects of Siglec-F engagement by a Siglec-F antibody on phagocytic activity of macrophages. The results showed that Siglec-F expression was detected on mouse alveolar macrophages, but not on peritoneal macrophages. Furthermore, Siglec-F engagement did not affect the phagocytic activity of alveolar macrophages in the resting state or in the activated state following stimulation by the proinflammatory mediator tumor necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS). Siglec-F expression on alveolar macrophages may be a result of adaptation. Macrophages actively regulate immune responses via production of cytokines. Therefore, further investigation of the effects of Siglec-F engagement on immune mediators or cytokines released by alveolar macrophages is required. PMID:22556177

  4. Electron microscopic study on the interaction between normal guinea pig peritoneal macrophages and Coxiella burnetii.

    PubMed Central

    Kishimoto, R A; Veltri, B J; Canonico, P G; Shirey, F G; Walker, J S

    1976-01-01

    An electron microscopic study was conducted to explore the interaction between normal guinea pig peritoneal macrophages and phase I and II Coxeilla burnetii previously treated with either normal or immune serum. A comparison was made on the efficiency of phagocytosis and subsequent killing of rickettsiae by macrophages. Both phases of rickettsiae previously treated with normal serum multiplied within phagosomes after phagocytosis with resultant destruction of macrophages. In contrast, suspending rickettsiae in immune serum rendered them more susceptible to phagocytosis and potentiated their destruction within macrophages. Images PMID:825466

  5. Differences in peroxidase localization of rabbit peritoneal macrophages after surface adherence.

    PubMed Central

    Bodel, P. T.; Nichols, B. A.; Bainton, D. F.

    1978-01-01

    Unlike resident peritoneal macrophages, which contain peroxidase in the rough endoplasmic reticulum (RER) and perinuclear cisternae (PN), macrophages elicited into the rabbit peritoneal cavity by various stimulants lack the enzyme. Since we had previously found that such peroxidase reactivity rapidly appears in the RER and PN of blood monocytes after surface adherence in vitro, we wondered whether the enzyme could be similarly produced in elicited macrophages by adherence. Cells from peritoneal exudates (96 hours after endotoxin injection) were harvested, suspended in culture medium, and allowed to adhere to fibrin-coated or plastic surfaces. Following culture for various intervals, they were fixed, incubated for peroxidase, and examined by electron microscopy. We observed that these elicited cells, which initially contained no cytochemically detectable peroxidase, acquired peroxidatic activity in the RER and PN within 2 hours after adherence in culture. Thus macrophages, like blood monocytes, may rapidly acquire peroxidase reactivity as a consequence of plasma membrane: external surface interaction. In view of this finding, it would seem unwise to use peroxidase localization as the basis for advocating the existence of two separate lines of peritoneal macrophages, as has been proposed by previous investigators. Images Figure 2 Figure 3 Figure 1 PMID:645814

  6. Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2*

    PubMed Central

    Noor, Shahid; Goldfine, Howard; Tucker, Dawn E.; Suram, Saritha; Lenz, Laurel L.; Akira, Shizuo; Uematsu, Satoshi; Girotti, Milena; Bonventre, Joseph V.; Breuel, Kevin; Williams, David L.; Leslie, Christina C.

    2016-01-01

    Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2α). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (ΔhlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and ΔhlyLM. The attenuated release of arachidonic acid that is observed in TLR2−/− and MyD88−/− macrophages infected with WTLM and ΔhlyLM correlates with diminished MAPK activation. WTLM but not ΔhlyLM increases intracellular calcium, which is implicated in regulation of cPLA2α. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2α+/+ but not cPLA2α−/− macrophages in response to WTLM and ΔhlyLM. Tumor necrosis factor (TNF)-α production is significantly lower in cPLA2α+/+ than in cPLA2α−/− macrophages infected with WTLM and ΔhlyLM. Treatment of infected cPLA2α+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFα production to the level produced by cPLA2α−/− macrophages implicating prostaglandins in TNFα down-regulation. Therefore activation of cPLA2α in macrophages may impact immune responses to L. monocytogenes. PMID:18083708

  7. Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

    SciTech Connect

    Vieira, J.M.B.D.; Seabra, S.H.; Vallim, D.C.; Americo, M.A.; Fracallanza, S.E.L.; Vommaro, R.C.; Domingues, R.M.C.P.

    2009-10-02

    Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

  8. The Immunomodulatory Activity of Jacaric Acid, a Conjugated Linolenic Acid Isomer, on Murine Peritoneal Macrophages

    PubMed Central

    Liu, Wai Nam; Leung, Kwok Nam

    2015-01-01

    This study aims at demonstrating the immunomodulatory property of jacaric acid, a conjugated linolenic acid (CLNA) isomer that is present in jacaranda seed oil, on murine peritoneal macrophages. Our results showed that jacaric acid exhibited no significant cytotoxicity on the thioglycollate-elicited murine peritoneal macrophages as revealed by the neutral red uptake assay, but markedly increased their cytostatic activity on the T-cell lymphoma MBL-2 cells as measured by the fluorometric CyQuant® NF Cell Proliferation Assay Kit. Flow cytometric analysis indicated that jacaric acid could enhance the endocytic activity of macrophages and elevated their intracellular production of superoxide anion. Moreover, jacaric acid-treated macrophages showed an increase in the production of nitric oxide which was accompanied by an increase in the expression level of inducible nitric oxide synthase protein. In addition, the secretion of several pro-inflammatory cytokines, including interferon-γ, interleukin-1β and tumor necrosis factor-α, was up-regulated. Collectively, our results indicated that the naturally-occurring CLNA isomer, jacaric acid, could exhibit immunomodulating activity on the murine peritoneal macrophages in vitro, suggesting that this CLNA isomer may act as an immunopotentiator which can be exploited for the treatment of some immunological disorders with minimal toxicity and fewer side effects. PMID:26629697

  9. Effects of microwave exposure on the hamster immune system. II. Peritoneal macrophage function

    SciTech Connect

    Rama Rao, G.; Cain, C.A.; Lockwood, J.; Tompkins, W.A.

    1983-01-01

    Acute exposure to hamsters to microwave energy (2.45 GHz; 25 mW/cm2 for 60 min) resulted in activation of peritoneal macrophages that were significantly more viricidal to vaccinia virus as compared to sham-exposed or normal (minimum-handling) controls. Macrophages from microwave-exposed hamsters became activated as early as 6 h after exposure and remained activated for up to 12 days. The activation of macrophages by microwave exposure paralleled the macrophage activation after vaccinia virus immunization. Activated macrophages from vaccinia-immunized hamsters did not differ in their viricidal activity when the hamsters were microwave- or sham-exposed. Exposure for 60 min at 15 mW/cm2 did not activate the macrophages while 40 mW/cm2 exposure was harmful to some hamsters. Average maximum core temperatures in the exposed (25 mW/cm2) and sham groups were 40.5 degrees C (+/- 0.35 SD) and 38.4 degrees C (+/- 0.5 SD), respectively. In vitro heating of macrophages to 40.5 degrees C was not as effective as in vivo microwave exposure in activating macrophages to the viricidal state. Macrophages from normal, sham-exposed, and microwave-exposed hamsters were not morphologically different, and they all phagocytosed India ink particles. Moreover, immune macrophage cytotoxicity for virus-infected or noninfected target cells was not suppressed in the microwave-irradiated group (25 mW/cm2, 1 h) as compared to sham-exposed controls, indicating that peritoneal macrophages were not functionally suppressed or injured by microwave hyperthermia.

  10. IL-33 Priming Enhances Peritoneal Macrophage Activity in Response to Candida albicans.

    PubMed

    Tran, Vuvi G; Cho, Hong R; Kwon, Byungsuk

    2014-08-01

    IL-33 is a member of the IL-1 cytokine family and plays a role in the host defense against bacteria, viruses, and fungi. In this study, we investigated the function of IL-33 and its receptor in in vitro macrophage responses to Candida albicans. Our results demonstrate that pre-sensitization of isolated peritoneal macrophages with IL-33 enhanced their pro-inflammatory cytokine production and phagocytic activity in response to C. albicans. These macrophage activities were entirely dependent on the ST2-MyD88 signaling pathway. In addition, pre-sensitization with IL-33 also increased ROS production and the subsequent killing ability of macrophages following C. albicans challenge. These results indicate that IL-33 may increase anti-fungal activity against Candida through macrophage-mediated resistance mechanisms. PMID:25177252

  11. Aging affects the responsiveness of rat peritoneal macrophages to GM-CSF and IL-4.

    PubMed

    Dimitrijević, Mirjana; Stanojević, Stanislava; Blagojević, Veljko; Ćuruvija, Ivana; Vujnović, Ivana; Petrović, Raisa; Arsenović-Ranin, Nevena; Vujić, Vesna; Leposavić, Gordana

    2016-04-01

    Macrophages undergo significant functional alterations during aging. The aim of the present study was to investigate changes of rat macrophage functions and response to M1/M2 polarization signals with age. Therefore, resident and thioglycollate-elicited peritoneal macrophages from young (3-month-old) and aged (18-19-month-old) rats were tested for phagocytic capacity and ability to secrete inflammatory mediators following in vitro stimulation with LPS and GM-CSF, and IL-4, prototypic stimulators for classically (M1) and alternatively activated (M2) macrophages, respectively. Aging increased the frequency of monocyte-derived (CCR7+ CD68+) and the most mature (CD163+ CD68+) macrophages within resident and thioglycollate-elicited peritoneal macrophages, respectively. The ability to phagocyte zymosan of none of these two cell subsets was affected by either LPS and GM-CSF or IL-4. The upregulated production of IL-1β, IL-6 and IL-10 and downregulated that of TGF-β was observed in response to LPS in resident and thioglycollate-elicited macrophages from rats of both ages. GM-CSF elevated production of IL-1β and IL-6 in resident macrophages from aged rats and in thioglycollate-elicited macrophages from young rats. Unexpectedly, IL-4 augmented production of proinflammatory mediators, IL-1β and IL-6, in resident macrophages from aged rats. In both resident and thioglycollate-elicited macrophages aging decreased NO/urea ratio, whereas LPS but not GM-SCF, shifted this ratio toward NO in the macrophages from animals of both ages. Conversely, IL-4 reduced NO/urea ratio in resident and thioglycollate-elicited macrophages from young rats only. In conclusion, our study showed that aging diminished GM-CSF-triggered polarization of elicited macrophages and caused paradoxical IL-4-driven polarization of resident macrophages toward proinflammatory M1 phenotype. This age-related deregulation of macrophage inflammatory mediator secretion and phagocytosis in response to M1/M2

  12. POLY(1):POLY(C)-ENHANCED ALVEOLAR AND PERITONEAL MACROPHAGE PHAGOCYTOSIS: QUANTIFICATION BY A NEW METHOD UTILIZING FLUOROESCENT BEADS

    EPA Science Inventory

    A technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed utilizing fluorescent polystyrene beads. When incorporated into inhalation studies, the technique can be used to determine whether the toxic effect of...

  13. Anti-inflammatory effects of miR-21 in the macrophage response to peritonitis.

    PubMed

    Barnett, Rebecca Elise; Conklin, Daniel J; Ryan, Lindsey; Keskey, Robert C; Ramjee, Vikram; Sepulveda, Ernesto A; Srivastava, Sanjay; Bhatnagar, Aruni; Cheadle, William G

    2016-02-01

    We investigated the role of microRNA-21 in the macrophage response to peritonitis; microRNA-21 expression increases in peritoneal macrophages after lipopolysaccharide stimulation but is delayed until 48 hours after cecal ligation and puncture. MicroRNA-21-null mice and bone marrow-derived cell lines were exposed to cecal ligation and puncture or lipopolysaccharide, and survival, microRNA-21 levels, target messenger RNAs and proteins, and cytokines were assayed. Macrophages were also transfected with microRNA-21 mimics and antagomirs, and similar endpoints were measured. Survival in microRNA-21-null mice was significantly decreased after lipopolysaccharide-induced peritonitis but unchanged after cecal ligation and puncture compared with similarly treated wild-type mice. MicroRNA-21 expression, tumor necrosis factor-α, interleukin 6, and programmed cell death protein 4 levels were increased after lipopolysaccharide addition in peritoneal cells. Pelino1 and sprouty (SPRY) messenger RNAs were similarly increased early, whereas programmed cell death protein 4 messenger RNA was decreased after lipopolysaccharide, and all microR-21 target messenger RNAs were subsequently decreased by 24 hours after lipopolysaccharide. Transfection with mimics and antagomirs led to appropriate responses in microRNA-21 and tumor necrosis factor-α. Knockdown of microRNA-21 in bone marrow-derived cells showed increased tumor necrosis factor-α and decreased interleukin 10 in response to lipopolysaccharide. Target proteins were unaffected by knockdown as was extracellular signal-regulated kinase; however, the nuclear factor κB p65 subunit was increased after lipopolysaccharide in the microRNA-21 knockout cells. In contrast, there was little change in these parameters after cecal ligation and puncture induction between null and wild-type mice. MicroRNA-21 is beneficial to survival in mice following lipopolysaccharide peritonitis. Overexpression of microRNA-21 decreased tumor necrosis factor

  14. Enhanced superoxide anion production in activated peritoneal macrophages from English sole (Pleuronectes vetulus) exposed to PACs

    SciTech Connect

    Clemons, E.; Arkoosh, M.; Casillas, E.

    1995-12-31

    In fish, as in mammals, macrophages play a vital role in the destruction of infective organisms. The purpose of this study was to determine if peritoneal macrophages (M{O}s) from English sole (Pleuronectes vetulus), a marine benthic fish, have an altered ability to produce cytotoxic reactive oxygen intermediates (ROIs) after exposure to polycyclic aromatic compounds (PACs). ROIs are the principle product of M{O}s used to destroy engulfed organisms. Assay conditions, including the concentration of M{O}s, type of in vitro stimulant, tissue culture media, and incubation time were optimized to measure the production of superoxide anion (O{sub 2}{minus}), the progenitor ROI, in English sole M{O}s. English sole were injected with an organic solvent extract of a PAH-contaminated sediment, equivalent to 20g sediment/kg fish, via their dorsal lymphatic sinus, and peritoneal M{O}s were harvested on days 1, 3, 5, 7, and 14 post injection. Activated peritoneal M{O}s from English sole injected with the sediment extract produced significantly more superoxide radicals after stimulation in vitro with either opsonized zymosan (OZ) or phorbol myristate acetate (PMA) than the vehicle injected or control fish. Specifically, activated peritoneal M{O}s stimulated with PMA in vitro produced greater amounts (compared to controls) of O{sub 2}{minus} on days 7 and 14 after exposure, whereas the same cells stimulated with OZ showed heightened production only on day 7 after exposure. No differences in the basal amounts of O{sub 2}{minus} production from activated peritoneal M{O}s between the treatment groups were observed. This study shows that exposure of English sole to PACs altered production of O{sub 2}{minus} by macrophages, however, the consequence to the immunocompetence of exposed fish remains to be elucidated.

  15. In vitro Staphylococcus aureus-induced oxidative stress in mice murine peritoneal macrophages: a duration-dependent approach

    PubMed Central

    Chakraborty, Subhankari Prasad; Roy, Somenath

    2014-01-01

    Objective To evaluate the free radical generation and status of the antioxidant enzymes in murine peritoneal macrophage during in vitro vancomycin sensitive Staphylococcus aureus (VSSA) treatment with different time interval. Methods Peritoneal macrophages were treated with 5×106 CFU/mL VSSA cell suspension in vitro for different time interval (1, 2, 3, 6, 12, and 24 h) and superoxide anion generation, NADPH oxidase activity, myeloperoxidase activity, nitric oxide generation, antioxidant enzyme status and components of glutathione cycle were analyzed. Results Superoxide anion generation, NADPH oxidase activity, myeloperoxidase activity and nitric oxide generation got peak at 3 h, indicating maximum free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during VSSA infection. Reduced glutathione level, glutathione peroxidase, glutathione reductase, and glutathione-s-transferase activity were decreased significantly (P<0.05) with increasing time of VSSA infection. But the oxidized glutathione level was time dependently increased significantly (P<0.05) in murine peritoneal macrophages. All the changes in peritoneal macrophages after 3 h in vitro VSSA treatment had no significant difference. Conclusions From this study, it may be summarized that in vitro VSSA infection not only generates excess free radical but also affects the antioxidant status and glutathione cycle in murine peritoneal macrophages. PMID:25183101

  16. Role of Mitogen-Activated Protein Kinases in Peptidoglycan-Induced Expression of Inducible Nitric Oxide Synthase and Nitric Oxide in Mouse Peritoneal Macrophages: Extracellular Signal-Related Kinase, a Negative Regulator ▿ †

    PubMed Central

    Bhatt, Kunal H.; Sodhi, Ajit; Chakraborty, Rituparna

    2011-01-01

    The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) are important host defense mechanisms against pathogens in mononuclear phagocytes. The objectives of this study were to examine the roles of mitogen-activated protein kinases (MAPKs) and transcription factors (nuclear factor-κB [NF-κB] and activating protein 1 [AP-1]) in peptidoglycan (PGN)-induced iNOS expression and NO production in macrophages. PGN is a cell wall component of Gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. PGN stimulates the activation of all three classes of MAPKs, extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38mapk in macrophages, albeit with differential activation kinetics. Using a selective inhibitor of JNK (SP600125) and JNK1/2 small interfering RNA (siRNA) knocked-down macrophages, it was observed that PGN-induced iNOS and NO expression is significantly inhibited. This suggested that JNK MAPK plays an essential role in PGN-induced iNOS expression and NO production. In contrast, inhibition of the ERK pathway using PD98059 dose dependently enhanced PGN-induced iNOS expression and NO production. PGN-induced ERK activation was attenuated in ERK1/2 siRNA knocked-down macrophages; however, NO and iNOS expression were significantly enhanced. An electrophoretic mobility shift assay showed that SP600125 inhibited PGN-induced NF-κB and AP-1 activation, whereas inhibition of the ERK pathway enhanced NF-κB activation, but with no effect on AP-1. These results indicate that the JNK MAPK positively regulate PGN-induced iNOS and NO expression by activating NF-κB and AP-1 transcription factors, whereas the ERK pathway plays a negative regulatory role via affecting NF-κB activity. PMID:21450974

  17. Macrophages are stimulated by muramyl dipeptide to induce polymorphonuclear leukocyte accumulation in the peritoneal cavities of guinea pigs.

    PubMed

    Nagao, S; Nakanishi, M; Kutsukake, H; Yagawa, K; Kusumoto, S; Shiba, T; Tanaka, A; Kotani, S

    1990-02-01

    N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) injected intraperitoneally significantly increased the number of cells entering the peritoneal cavity of guinea pigs primed with liquid paraffin or thioglycollate. There was a close relationship between peritoneal polymorphonuclear leukocyte (PMN) accumulation and the uptake of glucosamine by macrophages in guinea pigs treated with a variety of bacterial cell surface components such as cell wall peptidoglycan subunits and bacterial or synthetic lipid A. The PMN accumulation was also facilitated by the intraperitoneal transfer of the peritoneal macrophages that had been stimulated by MDP in vitro. Furthermore, cell-free lavage fluids taken from the peritoneum of MDP-treated guinea pigs also initiated the influx of PMNs when introduced into the peritoneal cavities of liquid paraffin-pretreated guinea pigs. These results suggest that a soluble factor which attracts neutrophils is produced by MDP-treated macrophages. Partial characterization of the factor is described. PMID:2298491

  18. Dynasore, a Dynamin Inhibitor, Inhibits Trypanosoma cruzi Entry into Peritoneal Macrophages

    PubMed Central

    Barrias, Emile S.; Reignault, Lissa C.; De Souza, Wanderley; Carvalho, Tecia M. U.

    2010-01-01

    Background Trypanosoma cruzi is an intracellular parasite that, like some other intracellular pathogens, targets specific proteins of the host cell vesicular transport machinery, leading to a modulation of host cell processes that results in the generation of unique phagosomes. In mammalian cells, several molecules have been identified that selectively regulate the formation of endocytic transport vesicles and the fusion of such vesicles with appropriate acceptor membranes. Among these, the GTPase dynamin plays an important role in clathrin-mediated endocytosis, and it was recently found that dynamin can participate in a phagocytic process. Methodology/Principal Findings We used a compound called dynasore that has the ability to block the GTPase activity of dynamin. Dynasore acts as a potent inhibitor of endocytic pathways by blocking coated vesicle formation within seconds of its addition. Here, we investigated whether dynamin is involved in the entry process of T. cruzi in phagocytic and non-phagocytic cells by using dynasore. In this aim, peritoneal macrophages and LLC-MK2 cells were treated with increasing concentrations of dynasore before interaction with trypomastigotes, amastigotes or epimastigotes. We observed that, in both cell lines, the parasite internalization was drastically diminished (by greater than 90% in LLC-MK2 cells and 70% in peritoneal macrophages) when we used 100 µM dynasore. The T. cruzi adhesion index, however, was unaffected in either cell line. Analyzing these interactions by scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences in the stage at which cell entry was blocked. In LLC-MK2 cells, this blockade is observed earlier than it is in peritoneal macrophages. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane. Conclusions/Significance Taken

  19. Biochemical mechanisms underlying the development of radioresistance by cultured peritoneal exudate macrophages

    SciTech Connect

    Lin, H.S.; Hsu, S.

    1989-01-01

    We investigated changes in radiosensitivity of peritoneal exudate macrophage colony-forming cells (PE-CFC) when exudative peritoneal macrophages were cultured in vitro. The change in the shape of the dose-response curve of PE-CFC to ionizing irradiation was partly dependent on the concentration of oxygen in the gas phase of the incubators. When cells were incubated in an environment containing 20% oxygen, the value of both Dq and D0 for PE-CFC increased. The dose-response curve of PE-CFC cultured for 3 days resembled that of alveolar macrophage colony-forming cells (AL-CFC). The changes in radiosensitivity were accompanied by an increase in the level of three antioxidant enzymes: superoxide dismutase, catalase, and glutathione peroxidase. However, when they were cultured in a 6% oxygen environment, only the value of Dq increased. When alveolar macrophages were incubated in vitro, no significant change in the shape of the dose-response curve of AL-CFC was noted whether they were cultured in gas phase containing either 20 or 6% oxygen. It is concluded that the radiosensitivity of PE-CFC changes when they are cultured in vitro. The increase in D0 appears to be related to the intracellular level of antioxidant enzymes.

  20. Decrease in free-radical production with age in rat peritoneal macrophages.

    PubMed Central

    Alvarez, E; Conde, M; Machado, A; Sobrino, F; Santa Maria, C

    1995-01-01

    The respiratory-burst reaction has been studied in rat peritoneal macrophages of different ages (3, 12 and 24 months) using phorbol 12-myristate 13-acetate (PMA) to stimulate NADPH oxidase. Production of O2-. and H2O2 decreased with age (about 50 and 75% respectively); however, no difference in NADPH oxidase activity was found. NO. production was also reduced with age (40%). Furthermore, a progressive and significant decrease in the pentose phosphate flux was detected as a function of age in control and PMA-stimulated macrophages. The NADPH/NADP+ ratio decreased with age in control and PMA-stimulated macrophages. Glucose uptake was lower in middle-aged (12 months) and old (24 months) animals but no differences were found between these groups. PMID:8526870

  1. Specificity and inhibition of glucocorticoid-induced macrocortin secretion from rat peritoneal macrophages.

    PubMed Central

    Blackwell, G. J.

    1983-01-01

    The secretion of the phospholipase A2-inhibitor macrocortin and the binding of dexamethasone were studied in suspensions of rat peritoneal macrophages. Corticosteroid-induced macrocortin secretion was specific for glucocorticoids and did not occur in response to glucocorticoid antagonists or other steroids or in response to non-steroid macrophage activators (formyl-methionyl-leucyl-phenylalanine f-MLP), the calcium ionophore A23187, phorbol myristate acetate (PMA) and lipopolysaccharide-E.-coli(LPS) ). The apparent potency of competition by secretory glucocorticoids for dexamethasone binding to the macrophage parallelled their ability to induce secretion, implying that these binding sites represent the receptors by which macrocortin secretion is initiated. Agents which interfere with microtubule assembly (colchicine, vinblastine and trimethylcolchicinic acid) and prostacyclin and dibutyryl cyclic AMP inhibit macrocortin secretion. Inhibition studies of glucocorticoid-induced macrocortin secretion also suggest dependence upon metabolic energy, a source of Ca2+ and proteolysis and glycosylation prior to secretion. PMID:6317116

  2. Involvement of proton-sensing TDAG8 in extracellular acidification-induced inhibition of proinflammatory cytokine production in peritoneal macrophages.

    PubMed

    Mogi, Chihiro; Tobo, Masayuki; Tomura, Hideaki; Murata, Naoya; He, Xiao-dong; Sato, Koichi; Kimura, Takao; Ishizuka, Tamotsu; Sasaki, Takehiko; Sato, Takashi; Kihara, Yasuyuki; Ishii, Satoshi; Harada, Akihiro; Okajima, Fumikazu

    2009-03-01

    Extracellular acidification inhibited LPS-induced TNF-alpha protein production, which was associated with an inhibition of TNF-alpha mRNA expression, in mouse peritoneal macrophages. The LPS-induced cytokine production was also inhibited by G(s) protein-coupled receptor agonists prostaglandin E(1) and isoproterenol. Among OGR1 family proton-sensing GTP-binding regulatory protein-coupled receptors, TDAG8, OGR1, and G2A are expressed in the cells. The inhibitory action by acidic pH on TNF-alpha production was significantly attenuated in macrophages from TDAG8(Tp/Tp) mice but not in those from OGR1(geo/geo) mice. Moreover, small interfering RNA specific to TDAG8, but not to G2A, clearly attenuated the acidification-induced inhibition of TNF-alpha production. On the other hand, the down-regulation or deficiency of TDAG8 hardly affected prostaglandin E(1)- or isoproterenol-induced actions. LPS-induced IL-6 production was also inhibited by extracellular acidification in a manner that was sensitive to TDAG8 expression. The acidic pH-induced inhibitory action on the cytokine production was significantly reversed either by a small interfering RNA specific to G(s) proteins or by a protein kinase A (PKA)-specific inhibitor H89. Indeed, a PKA-specific cAMP derivative inhibited LPS-induced cytokine production. Moreover, acidification induced cAMP accumulation in a TDAG8-specific way. We conclude that TDAG8, at least partly, mediates the extracellular acidification-induced inhibition of proinflammatory cytokine production through the G(s) protein/cAMP/PKA signaling pathway in mouse macrophages. PMID:19234222

  3. Release of tumor necrosis factor alpha by human peritoneal macrophages in response to toxic shock syndrome toxin-1.

    PubMed

    Buyalos, R P; Rutanen, E M; Tsui, E; Halme, J

    1991-08-01

    We examined the release in vitro of tumor necrosis factor-alpha (TNF-alpha) by peritoneal macrophages and peripheral blood monocytes following incubation with toxic shock syndrome toxin-1 (TSST-1). We obtained peritoneal macrophages from 22 women at laparoscopy and peripheral blood monocytes from four healthy women during both the midfollicular and midluteal phases of the menstrual cycle. The samples were incubated for 24 hours at 37 C with 10(-2)-10(4) ng/mL of TSST-1 or 10(4) ng/mL of bacterial endotoxin. Tumor necrosis factor-alpha activity was determined with a bioassay using an actinomycin D-sensitized WEHI-164 murine fibrosarcoma cell line. Twenty-four-hour incubation with TSST-1 resulted in a dose-dependent release of TNF-alpha by both peritoneal macrophages (maximal response 554 +/- 97 U of activity) and peripheral blood monocytes (maximal response 478 +/- 81 U of activity). We observed enhanced TNF-alpha release by peritoneal macrophages from women with endometriosis, compared with those without endometriosis, at a concentration of 10(4) ng/mL of TSST-1 (704 +/- 134 versus 354 +/- 103 U of activity; P less than .05). These data support the theory that the metabolic and physiologic derangements of perimenstrual toxic shock syndrome may be partially mediated by TNF-alpha released by peritoneal macrophages as a result of exposure to TSST-1. PMID:2067760

  4. Immunocytochemical demonstration of feline infectious peritonitis virus within cerebrospinal fluid macrophages.

    PubMed

    Ives, Edward J; Vanhaesebrouck, An E; Cian, Francesco

    2013-12-01

    A 4-month-old female entire domestic shorthair cat presented with an acute onset of blindness, tetraparesis and subsequent generalised seizure activity. Haematology and serum biochemistry demonstrated a moderate, poorly regenerative anaemia, hypoalbuminaemia and hyperglobulinaemia with a low albumin:globulin ratio. Serology for feline coronavirus antibody was positive with an elevated alpha-1 acid glycoprotein. Analysis of cisternal cerebrospinal fluid (CSF) demonstrated markedly elevated protein and a mixed, predominately neutrophilic pleocytosis. Immunocytochemistry for feline coronavirus was performed on the CSF, with positive staining observed inside macrophages. The cat was subsequently euthanased, and both histopathology and immunohistochemistry were consistent with a diagnosis of feline infectious peritonitis. This is the first reported use of immunocytochemistry for detection of feline coronavirus within CSF macrophages. If this test proves highly specific, as for identification of feline coronavirus within tissue or effusion macrophages, it would be strongly supportive of an ante-mortem diagnosis of feline infectious peritonitis in cats with central nervous system involvement without the need for biopsy. PMID:23744728

  5. Anti-inflammatory action of γ-irradiated genistein in murine peritoneal macrophage

    NASA Astrophysics Data System (ADS)

    Sung, Nak-Yun; Byun, Eui-Baek; Song, Du-Sup; Jin, Yeung-Bae; Park, Jae-Nam; Kim, Jae-Kyung; Park, Jong-Heum; Song, Beom-Seok; Park, Sang-Hyun; Lee, Ju-Woon; Kim, Jae-Hun

    2014-12-01

    This present study was to examine the cytotoxicity and anti-inflammatory activity of gamma (γ)-irradiated genistein in murine peritoneal macrophage. Inflammation to macrophage was induced by adding the lipopolysaccharide (LPS). γ-Irradiated genistein significantly decreased the cytotoxicity to murine peritoneal macrophage in dose ranges from 5 to 10 μM than that of non-irradiated genistein. Anti-inflammatory activity within the doses less than 2 μM showed that γ-irradiated genistein treatment remarkably reduced the lipopolysaccharide-induced inflammation by decreasing the nitric oxide (NO) and cytokines (TNF-α, IL-6) production. In a structural analysis through the high pressure liquid chromatography (HPLC), γ-irradiated genistein showed a new peak production distinguished from main peak of genistein (non-irradiated). Therefore, increase of anti-inflammatory activity may closely mediate with structural changes induced by γ irradiation exposure. Based on the above result, γ-irradiation could be an effective tool for reduction of toxicity and increase of physiological activity of biomolecules.

  6. Phagocytic responses of peritoneal macrophages and neutrophils are different in rats following prolonged exercise

    PubMed Central

    Ferreira, Clílton K O; Prestes, Jonato; Donatto, Felipe F; Verlengia, Rozangela; Navalta, James W; Cavaglieri, Cláudia R

    2010-01-01

    OBJECTIVE: To analyze the effects of exhausting long‐duration physical exercise (swimming) sessions of different durations and intensities on the number and phagocytic capacity of macrophages and neutrophils in sedentary rats. INTRODUCTION: Exercise intensity, duration and frequency are important factors in determining immune response to physical effort. Thus, the effects of exhausting long‐duration exercise are unclear. METHODS: Wistar rats were divided into two groups: an untreated group (macrophage study) and oyster glycogen‐treated rats (neutrophil study). In each group, the animals were subdivided into five groups (10 rats per group): unexercised controls, an unadapted low‐intensity exercise group, an unadapted moderate‐intensity exercise group, a preadapted low‐intensity exercise group and a preadapted moderate‐intensity exercise group. All exercises were performed to exhaustion, and preadaptation consisted of 5, 15, 30 and 45 min sessions. RESULTS: Macrophage study: the number of peritoneal macrophages significantly decreased (9.22 ± 1.78 × 106) after unadapted exercise but increased (21.50 ± 0.63 × 106) after preadapted low‐intensity exercise, with no changes in the moderate‐intensity exercise group. Phagocytic capacity, however, increased by more than 80% in all exercise groups (low/moderate, unadapted/preadapted). Neutrophil study: the number of peritoneal neutrophils significantly decreased after unadapted (29.20 ± 3.34 × 106) and preadapted (50.00 ± 3.53 × 106) low‐intensity exercise but increased after unadapted (127.60 ± 5.14 × 106) and preadapted (221.80 ± 14.85 × 106) moderate exercise. Neutrophil phagocytic capacity decreased by 63% after unadapted moderate exercise but increased by 90% after corresponding preadapted sessions, with no changes in the low‐intensity exercise groups. CONCLUSION: Neutrophils and macrophages of sedentary rats respond differently to exercise‐induced stress. Adaptation sessions reduce

  7. Macrophage Migration Inhibitory Factor Is Involved in Ectopic Endometrial Tissue Growth and Peritoneal-Endometrial Tissue Interaction In Vivo: A Plausible Link to Endometriosis Development

    PubMed Central

    Rakhila, Halima; Girard, Karine; Leboeuf, Mathieu; Lemyre, Madeleine

    2014-01-01

    Pelvic inflammation is a hallmark of endometriosis pathogenesis and a major cause of the disease's symptoms. Abnormal immune and inflammatory changes may not only contribute to endometriosis-major symptoms, but also contribute to ectopic endometrial tissue growth and endometriosis development. A major pro-inflammatory factors found elevated in peritoneal fluid of women with endometriosis and to be overexpressed in peritoneal fluid macrophages and active, highly vascularized and early stage endometriotic lesions, macrophage migration inhibitory factor (MIF) appeared to induce angiogenic and inflammatory and estrogen producing phenotypes in endometriotic cells in vitro and to be a possible therapeutic target in vivo. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngeneic wild type (WT) mice and vice versa, our current study revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant down regulation of the expression of major inflammatory (cyclooxygenease-2), cell adhesion (αv and β3 integrins), survival (B-cell lymphoma-2) and angiogenic (vascular endothelial cell growth) factorsrelevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. Interestingly, cross-experiments revealed that MIF presence in both endometrial and peritoneal host tissues is required for ectopic endometrial tissue growth and pointed to its involvement in endometrial-peritoneal interactions. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis. PMID:25329068

  8. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein kinase C.

    PubMed Central

    De la Fuente, M; Bernaez, I; Del Rio, M; Hernanz, A

    1993-01-01

    The peptides neuropeptide Y (NPY) and peptide YY (PYY) at concentrations from 10(-12) M to 10(-8) M have been shown in this study to stimulate significantly, in vitro, several functions of resting peritoneal macrophages from BALB/c mice: adherence to substrate, chemotaxis, ingestion of inert particles (latex beads) and foreign cells (Candida albicans), and production of superoxide anion measured by nitroblue tetrazolium reduction. A dose-response relationship was observed, with a maximal stimulation of the macrophage functions studied at 10(-10) M. These effects seem to be produced by specific receptors for the neuropeptides studied in peritoneal macrophages. Whereas the two peptides induced no change of intracellular cyclic AMP, they caused a significant stimulation of protein kinase C (PKC) in murine macrophages. These results suggest that NPY and PYY produce their effects on macrophage function through PKC activation. PMID:8262554

  9. Endotoxin-induced enhancement of glucose influx into murine peritoneal macrophages via GLUT1.

    PubMed Central

    Fukuzumi, M; Shinomiya, H; Shimizu, Y; Ohishi, K; Utsumi, S

    1996-01-01

    Hypoglycemia is among the most injurious metabolic disorders caused by endotoxemia. In experimental endotoxemia with lipopolysaccharide (LPS) in animals, a marked glucose consumption is observed in macrophage-rich organs. However, the direct effect of LPS on the uptake of glucose by macrophages has not been fully understood, and the present study was undertaken to shed light on this point. The consumption and uptake of glucose, as measured with 2-deoxy-D-[3H]glucose, by murine peritoneal exudate macrophages in culture were accelerated two- to threefold by stimulation with 3 ng of LPS per ml. The rate of glucose uptake reached a plateau after 20 min of stimulation and remained at the maximum as long as LPS was present. Northern (RNA) blot analysis with cDNA probes for five known isoforms of glucose transporter (GLUT) revealed that the expression of GLUT by macrophages was restricted to the GLUT1 isoform during LPS stimulation and the amount of GLUT1 mRNA was increased by the stimulation. These results suggest that macrophage responses to LPS are supported by a rapid and sustained glucose influx via GLUT1 and that this is a participating factor in the development of systemic hypoglycemia when endotoxemia is prolonged. PMID:8557327

  10. Concanavalin A enhances phagocytosis and killing of Candida albicans by mice peritoneal neutrophils and macrophages.

    PubMed

    Loyola, Wagner; Gaziri, Daniel Augusto; Gaziri, Luis Carlos Jabur; Felipe, Ionice

    2002-07-12

    In this study we tested the hypothesis that after administration of a single intraperitoneal dose of concanavalin A (Con-A) to mice, the proportion of neutrophils and macrophages in the peritoneal exudate and their phagocytic and candidacidal activities should change with time. The number of neutrophils in the peritoneal exudate was greatly increased 6 h after administration of Con-A, and those cells were able to kill both intracellular and extracellular yeast and germ tube forms of Candida albicans. Addition of catalase to the culture medium reduced the killing of C. albicans, suggesting that the candidacidal activity depended on the myeloperoxidase system. The survival of mice pretreated with Con-A and submitted to an inoculum of C. albicans 6 h afterwards was twice higher than that of controls, which suggests that neutrophils were able to clear the experimental infection. One day after the treatment, the population of neutrophils in the exudate was about 45%, but after 2 days it was reduced to only 5% and the candidacidal activity was also reduced. After 4 days the exudate contained over 95% of macrophages, the candidacidal activity reached a maximum, and the phagocytosis mediated by both complement receptors and mannose receptors was increased. Uptake of FITC-mannose-BSA by macrophages was maximal on about the 4th day and was inhibited by mannan, suggesting that treatment with Con-A increased the activity of mannose receptors. These results support the hypothesis that activation of cellular immunity by Con-A occurred in two phases, one dominated by neutrophils, and the other by macrophages expressing increased activity of mannose receptors. PMID:12110482

  11. Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis

    PubMed Central

    Salmanzadeh, Alireza; Kittur, Harsha; Sano, Michael B.; C. Roberts, Paul; Schmelz, Eva M.; Davalos, Rafael V.

    2012-01-01

    Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool. PMID:22536308

  12. BMP4 is increased in the aortas of diabetic ApoE knockout mice and enhances uptake of oxidized low density lipoprotein into peritoneal macrophages

    PubMed Central

    2013-01-01

    Background BMP4, a member of the transforming growth factor-beta superfamily, is upregulated in the aortas of diabetic db/db mice. However, little is known about its role in diabetic atherosclerosis. Therefore, we examined the roles of BMP4 in the formation of diabetic atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and in the uptake of oxidized low density lipoprotein (oxLDL) in peritoneal macrophages of wild-type mice. Methods To induce diabetes, ApoE KO mice were intraperitoneally injected with streptozotocin. Diabetic and non-diabetic ApoE KO mice were then fed a high-fat diet for 4 weeks. Next, to investigate a role of BMP4 in the peritoneal macrophages, we examined the uptake of oxLDL in BMP4-treated macrophages. Results Diabetic ApoE KO mice showed accelerated progression of aortic plaques accompanied by increased luminal plaque area. Western blot analysis showed that BMP4 expression in the whole aorta was greatly increased in diabetic ApoE KO mice, than non-diabetic mice. Western blot analysis showed that the BMP4/SMAD1/5/8 signaling pathway was strongly activated in the aorta from diabetic ApoE KO mice, compared with control ApoE KO mice. Double immunofluorescence staining showed that BMP4 was expressed in MOMA2-labeled macrophage in the aortic lesions of ApoE KO mice. BMP4 significantly increased the uptake of oxLDL into peritoneal macrophages in vitro. Conclusion We show that in the aorta of diabetic ApoE KO mice, BMP4 is increased and activates SMAD1/5/8. Our in vitro findings indicate that BMP4 enhances oxLDL uptake in mouse peritoneal macrophages, suggesting BMP4 may be involved in aortic plaque formation in diabetic ApoE KO mice. Targeting BMP4 may offer a new strategy for inhibition of plaque progression and stabilization of atherosclerotic lesions. PMID:24107300

  13. Stimulation of the ceramide pathway partially mimics lipopolysaccharide-induced responses in murine peritoneal macrophages.

    PubMed Central

    Barber, S A; Detore, G; McNally, R; Vogel, S N

    1996-01-01

    Recent studies have suggested that lipolysaccharide (LPS) stimulates cells by mimicking the second-messenger function of ceramide, a lipid generated in the cell by the action of sphingomyelinase (SMase). To examine this possibility further, we compared the abilities of LPS, SMase, and/or ceramide analogs to induce cytokine secretion, modulate gene expression, and induce endotoxin tolerance in macrophages. SMase and LPS induced secretion of tumor necrosis factor alpha (TNF-alpha) to comparable degrees; however, unlike LPS, SMase failed to stimulate detectable interferon activity. Cell-permeable analogs of ceramide induced the expression of many LPS-inducible genes; however, the expression of interferon-inducible protein 10 (IP-10) and interferon consensus sequence-binding protein (ICSBP) mRNAs was significantly lower than that induced by LPS. Both SMase-induced TNF-alpha secretion and LPS-induced TNF-alpha secretion were inhibited by pretreatment with a serine/threonine phosphatase inhibitor, calyculin A. Macrophages preexposed in vitro to LPS to induce a well-characterized state of endotoxin tolerance secreted little or no TNF-alpha upon secondary challenge with either LPS or SMase, whereas macrophages preexposed to SMase secreted high levels of TNF-alpha upon secondary stimulation with LPS or SMase. Collectively, these results suggest that ceramide activates a subset of LPS-induced signaling pathways in murine peritoneal exudate macrophages. PMID:8757882

  14. Piperine metabolically regulates peritoneal resident macrophages to potentiate their functions against bacterial infection.

    PubMed

    Pan, Hao; Xu, Li-Hui; Huang, Mei-Yun; Zha, Qing-Bing; Zhao, Gao-Xiang; Hou, Xiao-Feng; Shi, Zi-Jian; Lin, Qiu-Ru; Ouyang, Dong-Yun; He, Xian-Hui

    2015-10-20

    Pepper, a daily-used seasoning for promoting appetite, is widely used in folk medicine for treating gastrointestinal diseases. Piperine is the major alkaloid in pepper and possesses a wide range of pharmacological activities. However, the mechanism for linking metabolic and medicinal activities of piperine remains unknown. Here we report that piperine robustly boosts mTORC1 activity by recruiting more system L1 amino acid transporter (SLC7A5/SLC3A2) to the cell membrane, thus promoting amino acid metabolism. Piperine-induced increase of mTORC1 activity in resident peritoneal macrophages (pMΦs) is correlated with enhanced production of IL-6 and TNF-α upon LPS stimulation. Such an enhancement of cytokine production could be abrogated by inhibitors of the mTOR signaling pathway, indicating mTOR's action in this process. Moreover, piperine treatment protected resident pMΦs from bacterium-induced apoptosis and disappearance, and increased their bacterial phagocytic ability. Consequently, piperine administration conferred mice resistance against bacterial infection and even sepsis. Our data highlight that piperine has the capacity to metabolically reprogram peritoneal resident macrophages to fortify their innate functions against bacterial infection. PMID:26439699

  15. Enhancement of carrier-mediated transport after immunologic activation of peritoneal macrophages.

    PubMed

    Bonventre, P F; Straus, D; Baughn, R E; Imhoff, J

    1977-05-01

    Immunologically activated peritoneal macrophages from inbred mice and Hartley strain guinea pigs demonstrate a markedly greater than normal transport of 2-deoxy-D-glucose and L-leucine. The degree of nutrilite transport enhancement was greatest when animals were injected with the appropriate eliciting antigens before harvesting and also, if antigen was included in the tissue culture medium during the initial hours of in vitro culture. Enhanced hexose and amino acid uptake could also be achieved by exposure of macrophages from nonimmunized animals for 48 hr to supernatants of sensitized splenic lymphocyte cultures incubated with specific antigens. The animal systems in which this phenomenon was observed included CBA/J and C57BL/6J mice immunized with Staphylococcus aureus or sub-lethal doses of Listeria monocytogens, and the Hartley strain, albino guinea pig immunized with S. aureus or BCG. In all cases, immunization resulted in a state of delayed hypersensitivity as measured by skin testing or footpad swelling. Splenic cell supernatants contained lymphokines as detected by the presence of macrophage inhibitory factor (MIF), and by the supernatants' capacity to stimulate incorporation of 14C-glucosamine by macrophages in vitro. No increase of glucose or leucine transport by macrophages was observed in the absence of appropriate antigen stimulation in vivo or in vitro. We previously showed that a phagocytic stimulus results in a significant increase in hexose transport by normal macrophages; leucine transport by these same cells was unaltered after phagocytosis. In contrast, immunologically activated macrophages do not transport measurably more 2-deoxy-C-glucose after particle ingestion; activation or the phagocytic stimulus enhance 2-deoxy-C-glucose uptake to approximately the same extent. Analysis of nutrilite transport kinetics revealed that immunologic activation of macrophages increases the initial velocity (V1) and Vmax but does not change the Km values of

  16. Peritonitis

    MedlinePlus

    Acute abdomen ... of blood, body fluids, or pus in the abdomen ( intra-abdominal abscess ). Types of peritonitis are: Spontaneous ... The belly (abdomen) is very painful or tender. The pain may become worse when the belly is touched or when you ...

  17. Effect of aqueous extract of Tinospora cordifolia on functions of peritoneal macrophages isolated from CCl4 intoxicated male albino mice

    PubMed Central

    2011-01-01

    Background The current practice of ingesting phytochemicals for supporting the immune system or fighting infections is based on centuries-old tradition. Macrophages are involved at all the stages of an immune response. The present study focuses on the immunostimulant properties of Tinospora cordifolia extract that are exerted on circulating macrophages isolated from CCl4 (0.5 ml/kg body weight) intoxicated male albino mice. Methods Apart from damaging the liver system, carbon tetrachloride also inhibits macrophage functions thus, creating an immunocompromised state, as is evident from the present study. Such cell functions include cell morphology, adhesion property, phagocytosis, enzyme release (myeloperoxidase or MPO), nitric oxide (NO) release, intracellular survival of ingested bacteria and DNA fragmentation in peritoneal macrophages isolated from these immunocompromised mice. T. cordifolia extract was tested for acute toxicity at the given dose (150 mg/kg body weight) by lactate dehydrogenase (LDH) assay. Results The number of morphologically altered macrophages was increased in mice exposed to CCl4. Administration of CCl4 (i.p.) also reduced the phagocytosis, cell adhesion, MPO release, NO release properties of circulating macrophages of mice. The DNA fragmentation of peritoneal macrophages was observed to be higher in CCl4 intoxicated mice. The bacterial killing capacity of peritoneal macrophages was also adversely affected by CCl4. However oral administration of aqueous fraction of Tinospora cordifolia stem parts at a dose of 40 mg/kg body weight (in vivo) in CCl4 exposed mice ameliorated the effect of CCl4, as the percentage of morphologically altered macrophages, phagocytosis activity, cell adhesion, MPO release, NO release, DNA fragmentation and intracellular killing capacity of CCl4 intoxicated peritoneal macrophages came closer to those of the control group. No acute toxicity was identified in oral administration of the aqueous extract of Tinospora

  18. Candida albicans Quorum Sensing Molecules Stimulate Mouse Macrophage Migration

    PubMed Central

    Hargarten, Jessica C.; Moore, Tyler C.; Petro, Thomas M.; Nickerson, Kenneth W.

    2015-01-01

    The polymorphic commensal fungus Candida albicans causes life-threatening disease via bloodstream and intra-abdominal infections in immunocompromised and transplant patients. Although host immune evasion is a common strategy used by successful human fungal pathogens, C. albicans provokes recognition by host immune cells less capable of destroying it. To accomplish this, C. albicans white cells secrete a low-molecular-weight chemoattractive stimulant(s) of macrophages, a phagocyte that they are able to survive within and eventually escape from. C. albicans opaque cells do not secrete this chemoattractive stimulant(s). We report here a physiological mechanism that contributes to the differences in the interaction of C. albicans white and opaque cells with macrophages. E,E-Farnesol, which is secreted by white cells only, is a potent stimulator of macrophage chemokinesis, whose activity is enhanced by yeast cell wall components and aromatic alcohols. E,E-farnesol results in up to an 8.5-fold increase in macrophage migration in vitro and promotes a 3-fold increase in the peritoneal infiltration of macrophages in vivo. Therefore, modulation of farnesol secretion to stimulate host immune recognition by macrophages may help explain why this commensal is such a successful pathogen. PMID:26195556

  19. Candida albicans Quorum Sensing Molecules Stimulate Mouse Macrophage Migration.

    PubMed

    Hargarten, Jessica C; Moore, Tyler C; Petro, Thomas M; Nickerson, Kenneth W; Atkin, Audrey L

    2015-10-01

    The polymorphic commensal fungus Candida albicans causes life-threatening disease via bloodstream and intra-abdominal infections in immunocompromised and transplant patients. Although host immune evasion is a common strategy used by successful human fungal pathogens, C. albicans provokes recognition by host immune cells less capable of destroying it. To accomplish this, C. albicans white cells secrete a low-molecular-weight chemoattractive stimulant(s) of macrophages, a phagocyte that they are able to survive within and eventually escape from. C. albicans opaque cells do not secrete this chemoattractive stimulant(s). We report here a physiological mechanism that contributes to the differences in the interaction of C. albicans white and opaque cells with macrophages. E,E-Farnesol, which is secreted by white cells only, is a potent stimulator of macrophage chemokinesis, whose activity is enhanced by yeast cell wall components and aromatic alcohols. E,E-farnesol results in up to an 8.5-fold increase in macrophage migration in vitro and promotes a 3-fold increase in the peritoneal infiltration of macrophages in vivo. Therefore, modulation of farnesol secretion to stimulate host immune recognition by macrophages may help explain why this commensal is such a successful pathogen. PMID:26195556

  20. Macrophage Isolation from the Mouse Small and Large Intestine

    PubMed Central

    Harusato, Akihito; Geem, Duke; Denning, Timothy L.

    2016-01-01

    Macrophages play important roles in maintaining intestinal homeostasis via their ability to orchestrate responses to the normal microbiota as well as pathogens. One of the most important steps in beginning to understand the functions of these cells is the ability to effectively isolate them from the complex intestinal environment. Here, we detail methodology for the isolation and phenotypic characterization of macrophages from the mouse small and large intestine. PMID:27246032

  1. Generation and Characterization of Mouse Regulatory Macrophages.

    PubMed

    Carretero-Iglesia, Laura; Hill, Marcelo; Cuturi, Maria Cristina

    2016-01-01

    In the last years, cell therapy has become a promising approach to therapeutically manipulate immune responses in autoimmunity, cancer, and transplantation. Several types of lymphoid and myeloid cells origin have been generated in vitro and tested in animal models. Their efficacy to decrease pharmacological treatment has successfully been established. Macrophages play an important role in physiological and pathological processes. They represent an interesting cell population due to their high plasticity in vivo and in vitro. Here, we describe a protocol to differentiate murine regulatory macrophages in vitro from bone marrow precursors. We also describe several methods to assess macrophage classical functions, as their bacterial killing capacity and antigen endocytosis and degradation. Importantly, regulatory macrophages also display suppressive characteristics, which are addressed by the study of their hypostimulatory T lymphocyte capacity and polyclonal T lymphocyte activation suppression. PMID:26530796

  2. Evidence that Resorption of Bone by Rat Peritoneal Macrophages Occurs in an Acidic Environment

    NASA Technical Reports Server (NTRS)

    Blair, H. C.

    1985-01-01

    Skeletal loss in space, like any form of osteoporosis, reflects a relative imbalance of the activities of cells resorbing (degrading) or forming bone. Consequently, prevention of weightlessness induced bone loss may theoretically be accomplished by (1) stimulating bone formation or (2) inhibiting bone resorption. This approach, however, requires fundamental understanding of the mechanisms by which cells form or degrade bone, information not yet at hand. An issue central to bone resorption is the pH at which resorption takes place. The pH dependent spectral shift of a fluorescent dye (fluorescein isothiocyanate) conjugated to bone matrix was used to determine the pH at the resorptive cell bone matrix interface. Devitalized rat bone was used as the substrate, and rat peritoneal macrophages were used as the bone resorbing cells. The results suggest that bone resorption is the result of generation of an acidic microenvironment at the cell matrix junction.

  3. Peritoneal macrophages from patients with cirrhotic ascites show impaired phagocytosis and vigorous respiratory burst

    PubMed Central

    Ahmed, Abdel Motaal M.; Bomford, Adrian; Nouri-Aria, Kayhan T.; Davies, Ted; Smith, Roger; Williams, Roger

    2011-01-01

    Cirrhotic patients (CPs) are susceptible to spontaneous bacterial peritonitis (SBP). Aim of this study was to examine if this susceptibility was related to peritoneal macrophages' (PMs) altered host defence. Absorbance of phagocytosed particles by PMs from CPs was lower than that of control (31.88% vs. 77.2%). Particle opsonisation increased the absorbance to 41% in CPs' PMs, and this value remains lower than the control; 77.2%. Respiratory burst (RB) was expressed as fluorescence index values, and these were higher in PMs from CPs than in controls (82 vs. 41, 73 vs. 26 and 71 vs. 26). IFN-γ made no further increase of RB values in PMs from CPs. CD14 expression was also higher in CPs' PMs. IFN-γ significantly downregulated CD14 expression in both CPs' PMs and control. Reduced phagocytosis by predominantly CD14-positive PMs from CPs could be related to intense RB. Findings suggest altered host defence that could contribute to susceptibility to SBP. PMID:24371553

  4. Interaction of a mouse macrophage cell line with homologous erythrocytes.

    PubMed

    Singer, J A; Walker, W S; Morrison, M

    1982-06-01

    The interaction of the IC-21 murine macrophage cell line and homologous red blood cells (RBC) was assessed in the absence of exogenous opsonins. These results were used to evaluate this system as a potential model for macrophage-mediated clearance of old or damaged RBC. The binding and ingestion of density-separated and unseparated RBC by IC-21 cells were quantitated in assays that involved both 51Cr-labeled RBC and direct microscopy. The number of unseparated RBC that bound to IC-21 macrophages depended on the number of RBC added. Macrophages phagocytized an appreciable proportion of RBC within 3 hours with the ratio of RBC:macrophage of 10, a point at which the RBC-binding was not rate limiting. The mouse RBC were separated into dense- and less-dense fractions which are presumably enriched for old and young cells, respectively. When these RBC fractions were incubated with the IC-21 macrophage, significantly more of these dense cells were phagocytized. These results show that IC-21 macrophage cell line is a useful model for defining the processes whereby aged or damaged RBC are recognized and removed from circulation by macrophages. PMID:7120230

  5. Antibacterial Responses by Peritoneal Macrophages Are Enhanced Following Vitamin D Supplementation

    PubMed Central

    Bacchetta, Justine; Chun, Rene F.; Gales, Barbara; Zaritsky, Joshua J.; Leroy, Sandrine; Wesseling-Perry, Katherine; Boregaard, Niels; Rastogi, Anjay; Salusky, Isidro B.; Hewison, Martin

    2014-01-01

    Patients with chronic kidney disease (CKD), who usually display low serum 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D), are at high risk of infection, notably those undergoing peritoneal dialysis (PD). We hypothesized that peritoneal macrophages from PD patients are an important target for vitamin D-induced antibacterial activity. Dialysate effluent fluid was obtained from 27 non-infected PD patients. Flow cytometry indicated that PD cells were mainly monocytic (37.9±17.7% cells CD14+/CD45+). Ex vivo analyses showed that PD cells treated with 25D (100 nM, 6 hrs) or 1,25D (5 nM, 6 hrs) induced mRNA for antibacterial cathelicidin (CAMP) but conversely suppressed mRNA for hepcidin (HAMP). PD cells from patients with peritonitis (n = 3) showed higher baseline expression of CAMP (18-fold±9, p<0.05) and HAMP (64-fold±7) relative to cells from non-infected patients. In 12 non-infected PD patients, oral supplementation with a single dose of vitamin D2 (100,000 IU) increased serum levels of 25D from 18±8 to 41±15 ng/ml (p = 0.002). This had no significant effect on PD cell CD14/CD45 expression, but mRNA for HAMP was suppressed significantly (0.5-fold, p = 0.04). Adjustment for PD cell CD14/CD45 expression using a mixed linear statistical model also revealed increased expression of CAMP (mRNA in PD cells and protein in effluent) in vitamin D-supplemented patients. These data show for the first time that vitamin D supplementation in vitro and in vivo promotes innate immune responses that may enhance macrophage antibacterial responses in patients undergoing PD. This highlights a potentially important function for vitamin D in preventing infection-related complications in CKD. PMID:25549329

  6. Naloxone treatment prevents prenatal stress effects on peritoneal macrophage activity in mice offspring.

    PubMed

    Fonseca, Evelise S M; Sakai, Monica; Carvalho-Freitas, Maria Isabel R; Palermo Neto, João

    2005-01-01

    The present study analyzed the effects of maternal stress (PS) and/or naloxone treatment on the activity of peritoneal macrophage in male and female Swiss mice offspring. Pregnant female rats received a daily footshock (0.2 mA) and/or a naloxone injection from gestational day 15 to 19. Experiments were performed on postnatal day 30 on male and female pups. The following results were obtained in male offspring: (1) PS decreased both the index and the percentage of phagocytosis, this decrement being reversed by naloxone treatment, and (2) naloxone alone decreased the percentage of phagocytosis. The following results were obtained in female offspring: (1) PS decreased spontaneous and phorbol myristate acetate-induced macrophage oxidative burst, this decrement being reversed by naloxone pretreatment, and (2) PS decreased both the index and percentage of the phagocytosis, this effect was prevented by naloxone treatment. These data are discussed focussing on a putative neuroimmune interaction involving opioidergic systems during the ontogeny of the central nervous and immune systems. PMID:16210866

  7. Transformation of mouse macrophages by simian virus 40.

    PubMed

    Stone, L B; Takemoto, K K

    1970-11-01

    Studies were undertaken to prove that simian virus 40 (SV40) can transform the mouse macrophage, a cell type naturally restricted from deoxyribonucleic acid (DNA) replication. Balb/C macrophages infected with SV40 demonstrated T-antigen production and induced DNA synthesis simultaneously. In the absence of apparent division, these cells remained T antigen-positive for at least 45 days. SV40 could be rescued from nondividing, unaltered macrophages during the T antigen-producing period. Proliferating transformants appeared at an average of 66 days post-SV40 infection. Established cell lines were T antigen-positive and were negative for infectious virus, but yielded SV40 after fusion with African green monkey kidney cells. Their identity as transformed macrophages was substantiated by evaluation of cellular morphology, phagocytosis, acid phosphatase, beta(1c) synthesis, and aminoacridine incorporation. PMID:4320698

  8. Effects of β-endorphin on the production of reactive oxygen species, IL-1β, Tnf-Α, and IL-10 by murine peritoneal macrophages in vivo.

    PubMed

    Gein, S V; Baeva, T A; Nebogatikov, V O

    2016-07-01

    It has been demonstrated that β-endorphin stimulates the zymosan-induced secretion of reactive oxygen species and suppresses the spontaneous production of IL-1β and IL-10 by murine peritoneal macrophages in vivo. PMID:27595832

  9. Endotoxin suppresses expression of apoprotein E by mouse macrophages in vivo and in culture: a biochemical and genetic study

    SciTech Connect

    Werb, Z.; Chin, J.R.

    1983-09-10

    The synthesis and secretion of apo-E, a component of plasma lipoproteins, are suppressed in mouse macrophages exposed to bacterial lipopolysaccharide endotoxin (LPS) in culture or in vivo. Control mouse macrophages contained intracellular immunofluorescent apo-E, and apo-E represented about 10% of secreted protein. After intraperitoneal injection of LPS, freshly lavaged macrophages neither contained intracellular apo-E nor secreted apo-E. The suppressive effects of LPS and apo-E synthesis in culture were selective, and secretion of many other major macrophage proteins was not affected. When then LPS-elicited macrosphages were cultured for 24-72 h in the absence of LPS, synthesis of apo-E was initiated. Treatment of bone marrow-derived or peritoneal macrophages in culture with less than 1 ng of LPS/ml inhibited apo-E synthesis and secretion by 18 h of treatment. Although LPS stimulates prostaglandin E/sub 2/ synthesis, prostaglandin E/sub 2/ itself did not suppress apo-E synthesis. Macrophages from C3H/HeJ (Lps/sup d//Lps/sup d/) mice, which are resistant to LPS, were neither primed for H/sub 2/O/sub 2/ production nor suppressed for apo-E synthesis in response to LPS in vivo (30 ..mu..g/mouse) or in culture (1..mu../ml), whereas macrophages from the co-isogenic C3H/HeN (Lps/sup n//Lps/sup n/) strain were induced for H/sub 2/O/sub 2/ secretion and had suppressed synthesis of apo-E. Because apo-E serves as a recognition determinant for the receptor-mediated clearance of lipoproteins, the decreased synthesis of apo-E after LPS treatment may in part explain the hyperlipoproteinemia associated with endotoxins in vivo.

  10. Isolation of Mouse and Human Tumor-Associated Macrophages

    PubMed Central

    Cassetta, Luca; Noy, Roy; Swierczak, Agnieszka; Sugano, Gaël; Smith, Harriet; Wiechmann, Lisa; Pollard, Jeffrey W.

    2016-01-01

    The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both “omics” and in vitro studies. PMID:27325269

  11. Isolation of Mouse and Human Tumor-Associated Macrophages.

    PubMed

    Cassetta, Luca; Noy, Roy; Swierczak, Agnieszka; Sugano, Gaël; Smith, Harriet; Wiechmann, Lisa; Pollard, Jeffrey W

    2016-01-01

    The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both "omics" and in vitro studies. PMID:27325269

  12. Alpha-D-galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages.

    PubMed

    Petryniak, J

    1992-04-01

    Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of 125I labelled Evonymus europaea and Griffonia simplicifolia I-B4 lectins. Treatment of the stimulated macrophages with coffee bean alpha-galactosidase abolished binding of the GS I-B4 isolectin and changed the binding pattern of the Evonymus lectin. The affinity (Ka) of Evonymus lectin for alpha-galactosidase-treated macrophages decreased approximately 23-fold, from 1.25 x 10(8) M-1 to 5.5 x 10(6) M-1. Subsequent digestion of alpha-galactosidase-treated macrophages with alpha-L-fucosidase from Trichomonas foetus, further reduced binding of Evonymus lectin. Resident macrophages showed the same pattern of Evonymus lectin binding, with the same affinity, as alpha-galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of the Evonymus lectin which, in the absence of alpha-D-galactosyl groups, requires alpha-L-fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal alpha-L-fucosyl residues. It is also concluded that during macrophage stimulation/activation alpha-D-galactosyl residues are added to this glycoconjugate and that they form part of the receptor for Evonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains alpha-D-galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound to Corynaebacterium parvum activated macrophages. PMID:1344714

  13. The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa shift peritoneal milky spot macrophages towards an M1 phenotype to dampen peritoneal dissemination.

    PubMed

    Miao, Zhi-Feng; Zhao, Ting-Ting; Miao, Feng; Wang, Zhen-Ning; Xu, Ying-Ying; Mao, Xiao-Yun; Gao, Jian; Wu, Jian-Hua; Liu, Xing-Yu; You, Yi; Xu, Hao; Xu, Hui-Mian

    2014-05-01

    Peritoneal dissemination (PD) of tumor cells is the most frequent pattern of gastric cancer recurrence and the leading cause of death. Peritoneal milky spots are deemed as the site of origin of gastric cancer PD wherein the main cellular components are macrophages. A vaccine derived from the mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has exhibit strong immune modulatory properties. In the present study, we tested the hypothesis whether the PA-MSHA vaccine activated peritoneal milky spot macrophages (PMSM) in a manner that would attenuate PD. It was observed that PA-MSHA activated PMSM towards a classical activation phenotype via a toll-like receptor4/9-dependent mechanism, which increased interleukin-12 levels and promoted the expression of co-stimulatory and antigen-presenting molecules like CD80, CD86, and MHC-II (P < 0.05). In addition, PA-MSHA-treated PMSM exhibited strong nonspecific antitumor effects in both contact-dependent and contact-independent modes of action (P < 0.05). In mice treated with PA-MSHA before inoculating gastric cancer cells, we noted alleviated PD toward the untreated mice. In conclusion, our findings demonstrated that PA-MSHA can stimulate PMSM towards an M1 phenotype and that activated PMSM inhibit gastric cancer growth and PD both in vitro and in vivo. The results of the current study provide a mechanistic insight that is relevant to the potential application of PA-MSHA in the treatment of gastric cancer-mediated PD. PMID:24385384

  14. A Mouse Macrophage Lipidome*♦

    PubMed Central

    Dennis, Edward A.; Deems, Raymond A.; Harkewicz, Richard; Quehenberger, Oswald; Brown, H. Alex; Milne, Stephen B.; Myers, David S.; Glass, Christopher K.; Hardiman, Gary; Reichart, Donna; Merrill, Alfred H.; Sullards, M. Cameron; Wang, Elaine; Murphy, Robert C.; Raetz, Christian R. H.; Garrett, Teresa A.; Guan, Ziqiang; Ryan, Andrea C.; Russell, David W.; McDonald, Jeffrey G.; Thompson, Bonne M.; Shaw, Walter A.; Sud, Manish; Zhao, Yihua; Gupta, Shakti; Maurya, Mano R.; Fahy, Eoin; Subramaniam, Shankar

    2010-01-01

    We report the lipidomic response of the murine macrophage RAW cell line to Kdo2-lipid A, the active component of an inflammatory lipopolysaccharide functioning as a selective TLR4 agonist and compactin, a statin inhibitor of cholesterol biosynthesis. Analyses of lipid molecular species by dynamic quantitative mass spectrometry and concomitant transcriptomic measurements define the lipidome and demonstrate immediate responses in fatty acid metabolism represented by increases in eicosanoid synthesis and delayed responses characterized by sphingolipid and sterol biosynthesis. Lipid remodeling of glycerolipids, glycerophospholipids, and prenols also take place, indicating that activation of the innate immune system by inflammatory mediators leads to alterations in a majority of mammalian lipid categories, including unanticipated effects of a statin drug. Our studies provide a systems-level view of lipid metabolism and reveal significant connections between lipid and cell signaling and biochemical pathways that contribute to innate immune responses and to pharmacological perturbations. PMID:20923771

  15. Pathways of retinoid synthesis in mouse macrophages and bone marrow cells.

    PubMed

    Niu, Haixia; Hadwiger, Gayla; Fujiwara, Hideji; Welch, John S

    2016-06-01

    In vivo pathways of natural retinoid metabolism and elimination have not been well characterized in primary myeloid cells, even though retinoids and retinoid receptors have been strongly implicated in regulating myeloid maturation. With the use of a upstream activation sequence-GFP reporter transgene and retrovirally expressed Gal4-retinoic acid receptor α in primary mouse bone marrow cells, we identified 2 distinct enzymatic pathways used by mouse myeloid cells ex vivo to synthesize retinoic acid receptor α ligands from free vitamin A metabolites (retinyl acetate, retinol, and retinal). Bulk Kit(+) bone marrow progenitor cells use diethylaminobenzaldehyde-sensitive enzymes, whereas bone marrow-derived macrophages use diethylaminobenzaldehyde-insensitive enzymes to synthesize natural retinoic acid receptor α-activating retinoids (all-trans retinoic acid). Bone marrow-derived macrophages do not express the diethylaminobenzaldehyde-sensitive enzymes Aldh1a1, Aldh1a2, or Aldh1a3 but instead, express Aldh3b1, which we found is capable of diethylaminobenzaldehyde-insensitive synthesis of all trans-retinoic acid. However, under steady-state and stimulated conditions in vivo, diverse bone marrow cells and peritoneal macrophages showed no evidence of intracellular retinoic acid receptor α-activating retinoids, despite expression of these enzymes and a vitamin A-sufficient diet, suggesting that the enzymatic conversion of retinal is not the rate-limiting step in the synthesis of intracellular retinoic acid receptor α-activating retinoids in myeloid bone marrow cells and that retinoic acid receptor α remains in an unliganded configuration during adult hematopoiesis. PMID:26768478

  16. Enhancement of dengue virus type 2 replication in mouse macrophage cultures by bacterial cell walls, peptidoglycans, and a polymer of peptidoglycan subunits.

    PubMed Central

    Hotta, H; Hotta, S; Takada, H; Kotani, S; Tanaka, S; Ohki, M

    1983-01-01

    The effects of bacterial cell walls, peptidoglycans, and a water-soluble polymer of peptidoglycan subunits on dengue virus type 2 replication in cultured mouse peritoneal macrophages were studied. Pretreatment of macrophage cultures with all of test cell walls isolated from seven bacterial species for 3 days significantly enhanced the virus production in the cultures. Peptidoglycans prepared from four of the above cell walls also exerted the virus production-enhancing effects in a similar manner as the walls. A water-soluble polymer of peptidoglycan subunits which was prepared by treatment of Staphylococcus epidermidis wall peptidoglycan with an interpeptide bridge-splitting enzyme (endopeptidase) also definitely enhanced the virus production in macrophage cultures, although its activity was weaker than that of the original wall and peptidoglycan. Macrophage cultures from athymic nude mice, when treated with cell walls and peptidoglycans of S. epidermidis and Lactobacillus plantarum for 3 days, also showed an increased ability to support dengue virus type 2 replication. The infectious center assay demonstrated that the virus replication enhancement by S. epidermidis cell wall and peptidoglycan was primarily due to an increase in the number of virus-infected cells. This finding did not seem to be in conflict with the observation that macrophages treated with the above cell wall or peptidoglycan phagocytized more latex particles than did untreated macrophages. The conclusions based on the above experiments are that the treatment of mouse peritoneal macrophage cultures with bacterial cell walls and their components increases the take of dengue virus type 2 by macrophages and thus raises the virus production in the macrophage cultures. PMID:6874066

  17. MEK1/2 inhibitors induce interleukin-5 expression in mouse macrophages and lymphocytes.

    PubMed

    Li, Xiaoju; Cao, Xingyue; Zhang, Xiaomeng; Kang, Yanhua; Zhang, Wenwen; Yu, Miao; Ma, Chuanrui; Han, Jihong; Duan, Yajun; Chen, Yuanli

    2016-05-13

    Uptake of oxidized low-density lipoprotein (oxLDL) by macrophages facilitates the formation of foam cells, the prominent part of atherosclerotic lesions. Interleukin-5 (IL-5) is a cytokine regulating interactions between immune cells. It also activates the production of T15/EO6 IgM antibodies in B-1 cells, which can bind oxLDL thereby demonstrating anti-atherogenic properties. We previously reported that inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by mitogen-activated protein kinase kinases 1/2 (MEK1/2) inhibitors can reduce atherosclerosis. In this study, we determined the effects of MEK1/2 inhibitors on IL-5 production both in vitro and in vivo. In vitro, MEK1/2 inhibitors (PD98059 and U0126) substantially inhibited phosphorylation of MEK1/2 and ERK1/2. Associated with inhibition of ERK1/2 phosphorylation both in vitro and in vivo, MEK1/2 inhibitors induced IL-5 protein expression in macrophages (RAW macrophages and peritoneal macrophages) and lymphocytes (EL-4 cells). In vivo, administration of mice with MEK1/2 inhibitors increased serum IL-5 levels, and IL-5 protein expression in mouse spleen and liver. At the mechanistic level, we determined that MEK1/2 inhibitors activated IL-5 mRNA expression and IL-5 promoter activity in the liver X receptor (LXR) dependent manner indicating the induction of IL-5 transcription. In addition, we determined that MEK1/2 inhibitors enhanced IL-5 protein stability. Taken together, our study demonstrates that MEK1/2 inhibitors induce IL-5 production which suggests another anti-atherogenic mechanism of MEK1/2 inhibitors. PMID:27045084

  18. MicroRNA-223 Induced Repolarization of Peritoneal Macrophages Using CD44 Targeting Hyaluronic Acid Nanoparticles for Anti-Inflammatory Effects

    PubMed Central

    Tran, Thanh-Huyen; Krishnan, Swathi; Amiji, Mansoor M.

    2016-01-01

    The aim of this study was to evaluate macrophages repolarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype upon transfection with microRNA-223 (miR-223) duplexes and miR-223 expressing plasmid DNA encapsulated in CD44-targeting hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles (NPs). The HA-PEI/miR-223 NPs with spherical shape and an average diameter of 200 nm were efficiently internalized by J774A.1 alveolar and primary peritoneal macrophages and non-cytotoxic at HA-PEI concentration less than 200 μg/mL. Transfection of HA-PEI/miR-223 NPs in J774A.1 macrophages showed significantly higher miR-223 expression than that with HA-PEI/plasmid DNA expressing miR-223 (pDNA-miR-223). HA-PEI/miR-223 NPs mediated transfection increased miR-223 expression to 90 fold in primary peritoneal macrophages compared to untreated cells. The overexpression of miR-223 in both J774A.1 and peritoneal macrophages induced a phenotypic change from M1 to M2 state as indicated by a decrease in iNOS-2 (M1 marker) and an increase in Arg-1 (M2 marker) levels compared to those in lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-stimulated macrophages (M1). The change in macrophage phenotype by HA-PEI/miR-223 NPs could suppress the inflammation in peritoneal macrophages induced by LPS as evidenced by a significant decrease in pro-inflammatory cytokine levels TNF-α, IL-1β and IL-6, compared to LPS-stimulated peritoneal macrophages without treatment. The results demonstrated that miR-223-encapsulated HA-PEI NPs modulated macrophage polarity toward an anti-inflammatory M2 phenotype, which has potential for the treatment of inflammatory diseases. PMID:27148749

  19. Effect of immunochemotherapy with OK-432 and yeast cell wall on the activities of peritoneal macrophages of mice.

    PubMed

    Mashiba, H; Matsunaga, K; Gojobori, M

    1979-10-01

    The effect of chemotherapy combined with immunostimulants on the activities of macrophages in mice was studied. The number of macrophages and exudate cells in the peritoneal cavity increased 3 days after ip injection with mitomycin-C, cyclophosphamide, and 5-fluorouracil together with OK-432 or yeast cell wall and decreased to normal level after 9 days, while the number of the cells remained decreased in mice receiving multi-drugs alone. Acid phosphatase activity of the macrophages of mice was elevated after the simultaneous injection of yeast cell wall and OK-432, and high activity was preserved in the macrophages of mice receiving yeast cell wall even after 9 days. Spreading of these cells was also enhanced. Macrophage activities examined by these assays were maximal in every respect 6 days after combination therapy. Cytostatic activity of the cells was strengthened after 6 days by combined use of OK-432 or yeast cell wall. Role of the activated macrophages in combination therapy was discussed. PMID:520759

  20. NFATc1 releases BCL6-dependent repression of CCR2 agonist expression in peritoneal macrophages from Saccharomyces cerevisiae infected mice.

    PubMed

    Busch, Rhoda; Murti, Krisna; Liu, Jiming; Patra, Amiya K; Muhammad, Khalid; Knobeloch, Klaus-Peter; Lichtinger, Monika; Bonifer, Constanze; Wörtge, Simone; Waisman, Ari; Reifenberg, Kurt; Ellenrieder, Volker; Serfling, Edgar; Avots, Andris

    2016-03-01

    The link between the extensive usage of calcineurin (CN) inhibitors cyclosporin A and tacrolimus (FK506) in transplantation medicine and the increasing rate of opportunistic infections within this segment of patients is alarming. Currently, how peritoneal infections are favored by these drugs, which impair the activity of several signaling pathways including the Ca(++) /CN/NFAT, Ca(++) /CN/cofilin, Ca(++) /CN/BAD, and NF-κB networks, is unknown. Here, we show that Saccharomyces cerevisiae infection of peritoneal resident macrophages triggers the transient nuclear translocation of NFATc1β isoforms, resulting in a coordinated, CN-dependent induction of the Ccl2, Ccl7, and Ccl12 genes, all encoding CCR2 agonists. CN inhibitors block the CCR2-dependent recruitment of inflammatory monocytes (IM) to the peritoneal cavities of S. cerevisiae infected mice. In myeloid cells, NFATc1/β proteins represent the most prominent NFATc1 isoforms. NFATc1/β ablation leads to a decrease of CCR2 chemokines, impaired mobilization of IMs, and delayed clearance of infection. We show that, upon binding to a composite NFAT/BCL6 regulatory element within the Ccl2 promoter, NFATc1/β proteins release the BCL6-dependent repression of Ccl2 gene in macrophages. These findings suggest a novel CN-dependent cross-talk between NFAT and BCL6 transcription factors, which may affect the outcome of opportunistic fungal infections in immunocompromised patients. PMID:26631626

  1. Miltefosine enhances phagocytosis but decreases nitric oxide production by peritoneal macrophages of C57BL/6 mice.

    PubMed

    Ponte, Charlene Barreto; Alves, Erica Alessandra Rocha; Sampaio, Raimunda Nonata Ribeiro; Urdapilleta, Ada Amalia Ayala; Kückelhaus, Carlos dos Santos; Muniz-Junqueira, Maria Imaculada; Kückelhaus, Selma Aparecida Souza

    2012-05-01

    Miltefosine is an anticancer drug currently used to treat visceral and cutaneous leishmaniasis, also presents a broad-spectrum of fungicidal and antiamoebae activities. It acts on the metabolism of phospholipids and glycoproteins of the membrane of parasites. Our study aimed to evaluate the effects of miltefosine (0.4 to 50.0 μg/mL) on the phagocytosis and nitric oxide production by macrophages of C57BL/6 mice to clarify the immunomodulatory effects of the drug on macrophages of C57BL/6, strain mice that is biased to Th1 response. Peritoneal macrophages were in vitro treated with miltefosine and phagocytosis of sensitized or nonsensitized Saccharomyces cerevisiae was assessed. NO production was evaluated by Griess reaction. In the concentration of 1.6 μg/mL and 50.0 μg/mL, miltefosine increased phagocytosis of non-opsonized S. cerevisiae in 59.7% and 214.3%, respectively. For phagocytosis through opsonin receptors, miltefosine (50.0 μg/mL) increased the phagocytic index in 208.6% (p=0.04, paired t test). Miltefosine (50.0 μg/mL) decreased in 39.3% NO production by macrophages. However, treatment with miltefosine (50.0 μg/mL) after infection of macrophages with Leishmania amazonensis increased NO production in 73.4% (p=0.01, Wilcoxon test). Our data showed that, besides the antimicrobial effect of miltefosine, the drug showed immunomodulatory effects on macrophages of C57BL/6 mice, improving phagocytosis and decreasing NO production, but was able to increase NO production when macrophages were previously infected with L. amazonensis. These results suggest that miltefosine may favor the better evolution of infectious diseases by improving the innate immune response of macrophages. PMID:22465961

  2. Effects of immunomodulatory drugs on TNF-α and IL-12 production by purified epidermal langerhans cells and peritoneal macrophages

    PubMed Central

    2011-01-01

    Background Langerhans cells constitute a special subset of immature dendritic cells localized in the epidermis that play a key role in the skin's immune response. The production of cytokines is a key event in both the initiation and the regulation of immune responses, and different drugs can be used to remove or modify their production by DC and, therefore, alter immune responses in a broad spectrum of diseases, mainly in human inflammatory and autoimmune diseases. In the present study, we examined the effects of prednisone, thalidomide, cyclosporine A, and amitriptyline, drugs used in a variety of clinical conditions, on the production of TNF-α, IL-10, and IL-12 by purified epidermal Langerhans cells and peritoneal macrophages in BALB/c mice. Findings All drugs inhibited TNF-α production by Langerhans cells after 36 hours of treatment at two different concentrations, while prednisone and thalidomide decreased IL-12 secretion significantly, amitriptyline caused a less pronounced reduction and cyclosporine A had no effect. Additionally, TNF-α and IL-12 production by macrophages decreased, but IL-10 levels were unchanged after all treatments. Conclusions Our results demonstrate that these drugs modulate the immune response by regulating pro-inflammatory cytokine production by purified epidermal Langerhans cells and peritoneal macrophages, indicating that these cells are important targets for immunosuppression in various clinical settings. PMID:21276247

  3. Activation of murine peritoneal macrophages by water-soluble extracts of Bursaphelenchus xylophilus, a pine wood nematode.

    PubMed

    Kaji, Hiroaki; Tai, Akihiro; Matsushita, Kazufumi; Kanzaki, Hiroshi; Yamamoto, Itaru

    2006-01-01

    In our previous study, water-soluble extracts from Bursaphelenchus xylophilus (B. xylophilus), a pine wood nematode, were shown to enhance interleukin (IL)-4 plus lipopolysaccharide-induced polyclonal immunoglobulin (Ig) E production in vitro in mice and to increase serum levels of an antigen-nonspecific IgE in vivo. Here we examined whether the nematode extracts stimulate immunofunctions of murine peritoneal macrophages. In both resident and inflammatory macrophages, Fcgamma receptor-mediated phagocytosis was markedly activated by B. xylophilus extracts, while non-specific phagocytosis was not. The enhancement of specific phagocytosis was accompanied by an increase in the formation of IgG-Fcgamma receptor rosettes. B. xylophilus extracts also stimulated IL-1beta production in both types of macrophages, and enhanced NO production and mRNA expression of inflammatory cytokines in inflammatory macrophages. These results indicate that the extracts of B. xylophilus contain an activating substance(s) for immunofunctions in macrophages, besides an enhancing factor for polyclonal IgE production. PMID:16428838

  4. Aging Enhances the Production of Reactive Oxygen Species and Bactericidal Activity in Peritoneal Macrophages by Upregulating Classical Activation Pathways

    SciTech Connect

    Smallwood, Heather S.; López-Ferrer, Daniel; Squier, Thomas C.

    2011-10-07

    Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3–4 months) and aged (14–15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice

  5. Model for high-throughput screening of drug immunotoxicity--study of the anti-microbial G1 over peritoneal macrophages using flow cytometry.

    PubMed

    Tenorio-Borroto, Esvieta; Peñuelas-Rivas, Claudia G; Vásquez-Chagoyán, Juan C; Castañedo, Nilo; Prado-Prado, Francisco J; García-Mera, Xerardo; González-Díaz, Humberto

    2014-01-24

    Quantitative Structure-Activity (mt-QSAR) techniques may become an important tool for prediction of cytotoxicity and High-throughput Screening (HTS) of drugs to rationalize drug discovery process. In this work, we train and validate by the first time mt-QSAR model using TOPS-MODE approach to calculate drug molecular descriptors and Linear Discriminant Analysis (LDA) function. This model correctly classifies 8258 out of 9000 (Accuracy = 91.76%) multiplexing assay endpoints of 7903 drugs (including both train and validation series). Each endpoint correspond to one out of 1418 assays, 36 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). After that, we determined experimentally, by the first time, the values of EC50 = 21.58 μg/mL and Cytotoxicity = 23.6% for the anti-microbial/anti-parasite drug G1 over Balb/C mouse peritoneal macrophages using flow cytometry. In addition, the model predicts for G1 only 7 positive endpoints out 1251 cytotoxicity assays (0.56% of probability of cytotoxicity in multiple assays). The results obtained complement the toxicological studies of this important drug. This work adds a new tool to the existing pool of few methods useful for multi-target HTS of ChEMBL and other libraries of compounds towards drug discovery. PMID:24445280

  6. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality.

    PubMed

    Shaeib, Faten; Khan, Sana N; Thakur, Mili; Kohan-Ghadr, Hamid-Reza; Drewlo, Sascha; Saed, Ghassan M; Pennathur, Subramaniam; Abu-Soud, Husam M

    2016-01-01

    Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes. PMID:26982351

  7. The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality

    PubMed Central

    Shaeib, Faten; Khan, Sana N.; Thakur, Mili; Kohan-Ghadr, Hamid-Reza; Drewlo, Sascha; Saed, Ghassan M.; Pennathur, Subramaniam; Abu-Soud, Husam M.

    2016-01-01

    Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes. PMID:26982351

  8. Methanol extract of Ocimum gratissimum protects murine peritoneal macrophages from nicotine toxicity by decreasing free radical generation, lipid and protein damage and enhances antioxidant protection

    PubMed Central

    Mahapatra, Santanu Kar; Chakraborty, Subhankari Prasad; Das, Subhasis

    2009-01-01

    In the present study, methanol extract of Ocimum gratissimum Linn (ME-Og) was tested against nicotine-induced murine peritoneal macrophage in vitro. Phytochemical analysis of ME-Og shown high amount of flavonoid and phenolic compound present in it. The cytotoxic effect of ME-Og was studied in murine peritoneal macrophages at different concentrations (0.1 to 100 µg/ml) using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) method. To establish the protective role of ME-Og against nicotine toxicity, peritoneal macrophages from mice were treated with nicotine (10 mM), nicotine + ME-Og (1 to 25 µg/ml) for 12 h in culture media. The significantly (p < 0.05) increased super oxide anion generation, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, myeloperoxidase (MPO) activity, lipid peroxidation, protein carbonyls, oxidized glutathione levels were observed in nicotine-treated group as compared to control group; those were significantly (p < 0.05) reduced in ME-Og supplemented groups in concentration dependent manner. More over, significantly (p < 0.05) reduced antioxidant status due to nicotine exposure was effectively ameliorated by ME-Og supplementation in murine peritoneal macrophages. Among the different concentration of ME-Og, maximum protective effect was observed by 25 µg/ml, which does not produce significant cell cytotoxicity in murine peritoneal macrophages. These findings suggest the potential use and beneficial role of O. gratissimum as a modulator of nicotine-induced free radical generation, lipid-protein damage and antioxidant status in important immune cell, peritoneal macrophages. PMID:20716908

  9. Activation of mouse macrophages causes no change in expression and function of phorbol diesters' receptors, but is accompanied by alterations in the activity and kinetic parameters of NADPH oxidase.

    PubMed Central

    Berton, G; Cassatella, M; Cabrini, G; Rossi, F

    1985-01-01

    Mouse peritoneal macrophages activated in vivo by the injection of Corynebacterium parvum release larger amounts of superoxide anion (O2-) than macrophages from control mice when stimulated with phorbol myristate acetate (PMA). The biochemical bases for this enhanced response of activated macrophages have been investigated by studying the expression and function of receptors for the stimulant, and the activity of the enzyme NADPH oxidase which is responsible for the production of O2- in leucocytes. Studies of binding of phorbol dibutyrate, an agent closely related to PMA, showed that the affinity constants (Kds) and the number of binding sites were the same in resident and activated peritoneal macrophages. The activity of the NADPH oxidase was, however, different in the two macrophage populations which differ in their capacity to release O2-. NADPH oxidase activity was studied in macrophage monolayers after lysis with deoxycholate. The main features of this activity were as follows: stimulation of macrophages with PMA or zymosan caused an increase in NADPH-dependent O2- production; NADPH oxidase activity in the lysates followed the same dose-response curve for different concentrations of PMA as O2- release by intact macrophages; O2- release by intact macrophages could be fully accounted for by NADPH-dependent O2- production by macrophage lysates; activity was strictly substrate-specific, in that NADH could not substitute for NADPH; after stimulation with PMA or zymosan, NADPH oxidase activity was higher in lysates of C. parvum-activated macrophages than in lysates of resident macrophages; NADPH oxidase activities of activated and resident macrophages differed markedly in their kinetic parameters. The NADPH oxidase of macrophages activated by C. parvum or trehalose dimycolate of mycobacterial origin displayed a five to seven times lower Km compared to the enzyme in resident macrophages. PMID:2981767

  10. Mouse macrophage innate immune response to chikungunya virus infection

    PubMed Central

    2012-01-01

    Background Infection with Chikungunya alphavirus (CHIKV) can cause severe arthralgia and chronic arthritis in humans with persistence of the virus in perivascular macrophages of the synovial membrane by mechanisms largely ill-characterized. Findings We herein analysed the innate immune response (cytokine and programmed cell death) of RAW264.7 mouse macrophages following CHIKV infection. We found that the infection was restrained to a small percentage of cells and was not associated with a robust type I IFN innate immune response (IFN-α4 and ISG56). TNF-α, IL-6 and GM-CSF expression were upregulated while IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-10 or IL-17 expression could not be evidenced prior to and after CHIKV exposure. Although CHIKV is known to drive apoptosis in many cell types, we found no canonical signs of programmed cell death (cleaved caspase-3, -9) in infected RAW264.7 cells. Conclusion These data argue for the capacity of CHIKV to infect and drive a specific innate immune response in RAW264.7 macrophage cell which seems to be polarized to assist viral persistence through the control of apoptosis and IFN signalling. PMID:23253140

  11. Susceptibility of mouse macrophage J774 to dengue virus infection.

    PubMed

    Moreno-Altamirano, María M B; Sánchez-García, F Javier; Legorreta-Herrera, Martha; Aguilar-Carmona, Israel

    2007-01-01

    The aim of this study was to investigate whether the J774 mouse macrophage cell line could be used as an in vitro model for dengue virus infection (DENV). After 3 days, infection in J774 cells was assessed by detecting dengue virus non-structural protein 1 (NSP-1) production either by dot blot or indirect immunofluorescence assay (IFA) of saponine-permeabilized J774 cells and then confirmed by RT-PCR (171 bp product, corresponding to the DENV-2 core). Based on the presence of NSP-1 in infected but not in non-infected cells by both IFA and dot blot, as well as the amplification of a 171-bp DENV-2-specific RT-PCR product exclusively in the infected cells, the J774 cell line was found to be permissive for dengue virus infection. As far as we know, this is the first report that the J774 mouse macrophage cell line is infected with dengue virus and, thus, that it can be used as an alternative in vitro model for dengue virus infection studies. This finding could help to further elucidate the mechanisms involved in dengue virus infection and pathogenesis. PMID:17356302

  12. Kinetics of tumour necrosis factor and prostaglandin production by murine resident peritoneal macrophages as affected by dietary n-3 polyunsaturated fatty acids.

    PubMed Central

    Hardard'ottir, I; Whelan, J; Kinsella, J E

    1992-01-01

    Cell-associated and secreted tumour necrosis factor (TNF), prostaglandin (PG) E2, and 6-keto PGF1 alpha were monitored at various times following in vitro stimulation of resident peritoneal macrophages with lipopolysaccharide (LPS). Macrophages were obtained from mice maintained on diets containing 1.5 wt% n-3 polyunsaturated fatty acids (PUFA)+ 1.5 wt% n-6 fatty acids; 1.5 wt% n-6 fatty acids; or 3 wt% n-6 fatty acids, for 4 weeks. Cell-associated TNF increased transiently in the resident peritoneal macrophages from mice consuming all diets and decreased after TNF secretion had reached maximum and plateaued. Macrophages from mice consuming the n-3 PUFA contained more cell-associated TNF and secreted more TNF than macrophages from mice consuming diets containing n-6 fatty acids only, at all time-points studied. Macrophages from mice consuming the n-3 PUFA showed an earlier increase in cell-associated and secreted TNF compared with macrophages from mice consuming n-6 fatty acids only. Kinetics of maximum TNF production was not affected by the diets and dietary n-3 PUFA did not cause a prolonged increase in TNF secretion. Macrophages from mice consuming the n-3 PUFA produced less PG than macrophages from mice consuming the n-6 fatty acids only. PG secretion increased following appearance of cell-associated TNF but when PG had accumulated in the medium there was no further increase in TNF production. PMID:1398747

  13. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

    PubMed Central

    Ramprasad, M P; Fischer, W; Witztum, J L; Sambrano, G R; Quehenberger, O; Steinberg, D

    1995-01-01

    We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568176

  14. Eicosanoid production by peritoneal and splenic macrophages in mice depleted of bone marrow by /sup 89/Sr

    SciTech Connect

    Shibata, Y.; Bautista, A.P.; Pennington, S.N.; Humes, J.L.; Volkman, A.

    1987-04-01

    Previous studies showed that the prostaglandin-forming macrophages (M phi) induced in the spleens of CBA/J mice by intraperitoneal administration of Corynebacterium parvum (CP) could not be demonstrated following the depletion of bone marrow and blood monocytes with /sup 89/Sr. The present study compares prostaglandin E2 (PGE2), leukotriene C4 (LTC4), and LTB4 release by splenic and resident peritoneal M phi in /sup 89/Sr-treated mice and /sup 88/Sr controls following in vivo CP and in vitro incubation with zymosan, calcium ionophore A23187, or phorbol ester (PMA). Intraperitoneal administration of CP resulted in the appearance of PGE2- and LTB4-releasing M phi in the spleens of control but not /sup 89/Sr mice. The incorporation and quantitative distribution of 3H-arachidonic acid into membrane lipids, however, were comparable in test and control mice. Neither zymosan nor any of the other stimulatory agents was able to effect significant release of PGE2 in vitro. No release of LTC4 by splenic M phi was detectable under experimental or control conditions. In contrast, the capacity of resident peritoneal M phi to release PGE2, LTC4, and LTB4 was apparently unaffected by /sup 89/Sr-induced bone marrow and monocyte depletion with virtually no demonstrable elicitation. Resident peritoneal M phi removed after CP in such mice showed a dramatic decrease in PGE2 release when incubated in vitro with zymosan, A23187, or PMA. These results, taken with earlier findings, demonstrate characteristically different phenotypic expression of metabolism of certain eicosanoids by splenic M phi from the spleen and the peritoneal cavity and suggest in addition that the induction of PGE2-synthesizing M phi in the spleen by CP is dependent on either an immigrant cell originating in the bone marrow or a regulatory agent derived from a bone marrow cell.

  15. Iron limitation and the gamma interferon-mediated antihistoplasma state of murine macrophages.

    PubMed Central

    Lane, T E; Wu-Hsieh, B A; Howard, D H

    1991-01-01

    The zoopathogenic fungus Histoplasma capsulatum requires iron for growth. Intracellular growth of the fungus within mouse peritoneal macrophages is inhibited by recombinant murine gamma interferon (IFN-gamma). Such treatment of mouse peritoneal macrophages induces a marked downshift in transferrin receptors. We tested whether the antihistoplasma effect of IFN-gamma-treated macrophages is the result of iron deprivation. Treatment of mouse peritoneal macrophages with the intracellular iron chelator deferoxamine inhibits the intracellular growth of H. capsulatum. Exposure of macrophages to holotransferrin antagonizes the effect of both recombinant murine IFN-gamma and deferoxamine treatments. These results suggest that iron restriction may be one of the bases for the IFN-gamma-induced antihistoplasma effect of mouse macrophages. PMID:1904840

  16. Output of peritoneal cells during peritoneal dialysis.

    PubMed Central

    Fakhri, O; Al-Mondhiry, H; Rifaat, U N; Khalil, M A; Al-Rawi, A M

    1978-01-01

    Peritoneal dialysis provides a good source for the collection of macrophages. Six patients with chronic renal failure undergoing peritoneal dialysis for the first time were studied, and maximum cell egress, mostly macrophages, occurred at 24-48 hours and diminished after 48 hours. PMID:670419

  17. A SIRT3/AMPK/autophagy network orchestrates the protective effects of trans-resveratrol in stressed peritoneal macrophages and RAW 264.7 macrophages.

    PubMed

    Duan, Wen-Jun; Li, Yi-Fang; Liu, Fang-Lan; Deng, Jie; Wu, Yan-Ping; Yuan, Wei-Lin; Tsoi, Bun; Chen, Jun-Li; Wang, Qi; Cai, Shao-Hui; Kurihara, Hiroshi; He, Rong-Rong

    2016-06-01

    Resveratrol gains a great interest for its strong antioxidant properties, while the molecular mechanisms underlie the beneficial effects on psychosocial stress remain controversial. In this study, we demonstrated that resveratrol protected peritoneal macrophages and RAW 264.7 cells from stress-induced decrease in the total cell count, phagocytic capability, reactive oxygen species generation, monodansylcadaverine and mitochondrial membrane potential in stressed mice. Resveratrol promoted stress-induced autophagy in both models. Modulation of autophagy by rapamycin or 3-methyladenine regulated the protective effect of resveratrol, suggesting a role of autophagy in the protective mechanisms of resveratrol. The comparison studies revealed that distinct mechanisms were implicated in the protective effect of resveratrol and other antioxidants (vitamin C and edaravone). Resveratrol promoted autophagy via upregulating SIRT3 expression and phosphorylation of AMP-activated protein kinase (AMPK). Knockdown of SIRT3 resulted in decreased autophagy and abolished protective effect of resveratrol. SIRT1 was also involved in the protective mechanism of resveratrol, although its effect on autophagy was unnoticeable. Pharmacological manipulation of autophagy modulated the effects of resveratrol on SIRT3 and AMPK, revealing the engagement of a positive feedback loop. In sharp contrast, vitamin C and edaravone effectively protected macrophages from stress-induced cytotoxicity, accompanied by downregulated SIRT3 expression and AMPK phosphorylation, and decreased level of autophagy response. Taken together, we conclude that a SIRT3/AMPK/autophagy network orchestrates in the protective effect of resveratrol in macrophages. PMID:27021965

  18. Transforming growth factor beta 1 and gamma interferon provide opposing signals to lipopolysaccharide-activated mouse macrophages.

    PubMed Central

    Hausmann, E H; Hao, S Y; Pace, J L; Parmely, M J

    1994-01-01

    Bacterial lipopolysaccharides (LPS) are potent inducers of macrophage activation, leading to the production of a number of proinflammatory mediators. Although several cytokines that prime macrophages for enhanced LPS-triggered responses have been identified, far less is known regarding the role that cytokines play in down-regulating macrophage responses to LPS. This study was designed to determine the effects of recombinant transforming growth factor beta 1 (rTGF-beta 1) on macrophage activation by LPS. Pretreatment of either mouse peritoneal macrophages or cells of the RAW 264.7 macrophage-like cell line with rTGF-beta 1 inhibited their ability to produce both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) in response to LPS. These inhibitory effects were reversed by increasing the concentration of LPS or by priming cells with optimal concentrations of recombinant gamma interferon (rIFN-gamma). Pretreatment of cells with rTGF-beta 1 had only a modest inhibitory effect on the expression of TNF-alpha mRNA. By contrast, the expression of mRNA for the inducible form of nitric oxide synthase (iNOS), which is responsible for NO production in activated macrophages, was significantly inhibited by rTGF-beta 1 pretreatment. Thus, rTGF-beta 1-dependent suppression of macrophage TNF-alpha biosynthesis was manifest at a posttranscriptional level, whereas the inhibition of NO production correlated with a direct effect on iNOS gene expression. Importantly, both of these suppressive effects of rTGF-beta 1 were reversed by exposing the cells to priming concentrations of rIFN-gamma. As with NO production, immunocytochemical analysis of iNOS expression in LPS-stimulated macrophages revealed that rIFN-gamma and rTGF-beta 1 had antagonistic effects, with the former increasing, and the latter reducing, the number of iNOS-expressing cells induced by LPS. These data suggest that a balance between the priming effects of IFN-gamma and the inhibitory effects of TGF-beta 1 can

  19. 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

  20. Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis.

    PubMed

    Lundy, S R; Dowling, R L; Stevens, T M; Kerr, J S; Mackin, W M; Gans, K R

    1990-04-01

    Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids. PMID:2108209

  1. Different Effects of the Immunomodulatory Drug GMDP Immobilized onto Aminopropyl Modified and Unmodified Mesoporous Silica Nanoparticles upon Peritoneal Macrophages of Women with Endometriosis

    PubMed Central

    Antsiferova, Yuliya; Sotnikova, Nataliya

    2013-01-01

    The aim of the present work was to compare in vitro the possibility of application of unmodified silica nanoparticles (UMNPs) and modified by aminopropyl groups silica nanoparticles (AMNPs) for topical delivery of immunomodulatory drug GMDP to the peritoneal macrophages of women with endometriosis. The absence of cytotoxic effect and high cellular uptake was demonstrated for both types of silica nanoparticles. The immobilization of GMDP on the UMNPs led to the suppression of the stimulatory effect of GMDP on the membrane expression of scavenger receptors SR-AI and SR-B, mRNAs expression of NOD2 and RAGE, and synthesis of proteolytic enzyme MMP-9 and its inhibitor TIMP-1. GMDP, immobilized onto AMNPs, enhanced the initially reduced membrane expression of SRs and increased NOD2, RAGE, and MMP-9 mRNAs expression by macrophages. Simultaneously high level of mRNAs expression of factors, preventing undesirable hyperactivation of peritoneal macrophages (SOCS1 and TIMP-1), was observed in macrophages incubated in the presence of GMDP, immobilized onto AMNPs. The effect of AMNPs immobilized GMDP in some cases exceeded the effect of free GMDP. Thus, among the studied types of silica nanoparticles, AMNPs are the most suitable nanoparticles for topical delivery of GMDP to the peritoneal macrophages. PMID:24455738

  2. Different effects of the immunomodulatory drug GMDP immobilized onto aminopropyl modified and unmodified mesoporous silica nanoparticles upon peritoneal macrophages of women with endometriosis.

    PubMed

    Antsiferova, Yuliya; Sotnikova, Nataliya; Parfenyuk, Elena

    2013-01-01

    The aim of the present work was to compare in vitro the possibility of application of unmodified silica nanoparticles (UMNPs) and modified by aminopropyl groups silica nanoparticles (AMNPs) for topical delivery of immunomodulatory drug GMDP to the peritoneal macrophages of women with endometriosis. The absence of cytotoxic effect and high cellular uptake was demonstrated for both types of silica nanoparticles. The immobilization of GMDP on the UMNPs led to the suppression of the stimulatory effect of GMDP on the membrane expression of scavenger receptors SR-AI and SR-B, mRNAs expression of NOD2 and RAGE, and synthesis of proteolytic enzyme MMP-9 and its inhibitor TIMP-1. GMDP, immobilized onto AMNPs, enhanced the initially reduced membrane expression of SRs and increased NOD2, RAGE, and MMP-9 mRNAs expression by macrophages. Simultaneously high level of mRNAs expression of factors, preventing undesirable hyperactivation of peritoneal macrophages (SOCS1 and TIMP-1), was observed in macrophages incubated in the presence of GMDP, immobilized onto AMNPs. The effect of AMNPs immobilized GMDP in some cases exceeded the effect of free GMDP. Thus, among the studied types of silica nanoparticles, AMNPs are the most suitable nanoparticles for topical delivery of GMDP to the peritoneal macrophages. PMID:24455738

  3. Involvement of proton-sensing receptor TDAG8 in the anti-inflammatory actions of dexamethasone in peritoneal macrophages

    SciTech Connect

    He, Xiao-dong; Tobo, Masayuki; Mogi, Chihiro; Nakakura, Takashi; Komachi, Mayumi; Murata, Naoya; Takano, Mutsumi; Tomura, Hideaki; Sato, Koichi; Okajima, Fumikazu

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Glucocorticoid (GC) induced the expression of proton-sensing TDAG8 in macrophages. Black-Right-Pointing-Pointer GC enhanced acidic pH-induced cAMP accumulation and inhibition of TNF-{alpha} production. Black-Right-Pointing-Pointer The enhancement of the GC-induced actions was lost by TDAG8 deficiency. Black-Right-Pointing-Pointer GC-induced anti-inflammatory actions are partly mediated by TDAG8 expression. -- Abstract: Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-{alpha}, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-{alpha} production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.

  4. In vitro immune toxicity of polybrominated diphenyl ethers on murine peritoneal macrophages: apoptosis and immune cell dysfunction.

    PubMed

    Lv, Qi-Yan; Wan, Bin; Guo, Liang-Hong; Zhao, Lixia; Yang, Yu

    2015-02-01

    Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and are often detected in the environment, wildlife, and humans, presenting potential threats to ecosystem and human health. PBDEs can cause neurotoxicity, hepatotoxicity, and endocrine disruption. However, data on PBDE immunotoxicity are limited, and the toxicity mechanisms remain largely unknown. Both immune cell death and dysfunction can modulate the responses of the immune system. This study examined the toxic effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and decabromodiphenyl ether (BDE-209) on the immune system by using peritoneal macrophages as the model. The macrophages were exposed to PBDEs, and cell death was determined through flow cytometry and immunochemical blot. The results showed that after 24h of exposure, BDE-47 (>5 μM) and BDE-209 (>20 μM) induced cell apoptosis, increased intracellular reactive oxygen species (ROS) formation and depleted glutathione. BDE-47 was more potent than BDE-209; the cytotoxic concentrations for BDE-47 and BDE-209 were determined to be 5 μM and 20 μM, respectively, during 24h of exposure. However, pretreatment with n-acetyl-l-cysteine (ROS scavenger) partially reversed the cytotoxic effects. Further gene expression analyses on Caspase-3,-8,-9, TNFR1, and Bax revealed that both intrinsic and extrinsic apoptotic pathways were activated. More importantly, non-cytotoxic concentrations BDE-47 (<2 μM) and BDE-209 (<10 μM) could impair macrophage accessory cell function in a concentration-dependent manner, but no effects were observed on phagocytic responses. These revealed effects of PBDEs on macrophages may shed light on the toxicity mechanisms of PBDEs and suggest the necessity of evaluating cellular functionality during the risk assessment of PBDE immunotoxicity. PMID:25462306

  5. Regulation of the surface expression of the platelet-activating factor receptor in IC-21 peritoneal macrophages. Effects of lipopolysaccharide.

    PubMed

    Liu, H; Chao, W; Olson, M S

    1992-10-15

    The effect of bacterial lipopolysaccharide (LPS) on the expression of the receptor for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; AGEPC) was examined in cultured IC-21 peritoneal macrophages. AGEPC binding to its receptors reached saturation within 20 min at 25 degrees C and was reversible. Scatchard analysis revealed a single class of AGEPC receptors with a Bmax of approximately 170 fmol/mg cellular protein and a Kd of 0.25 nM. Preincubation of IC-21 cells with LPS (0.01-1,000 ng/ml) induced an increase in the surface expression of AGEPC receptors in a time- and concentration-dependent fashion. The maximal effect of LPS on the AGEPC receptor was observed between 5 and 8 h, with a typical increase between 150 and 200%. Scatchard analysis indicated that LPS treatment of IC-21 cells increased the number of AGEPC receptors on the cell surface without any apparent change in the affinity of the receptor for the ligand. The effect of LPS on the surface expression of the AGEPC receptor was nearly abolished by cycloheximide (0.1 mM) and by actinomycin D (3 microM), suggesting the involvement of enhanced receptor protein synthesis and mRNA production in this event. Moreover, LPS treatment increased the capability of the IC-21 cell to respond to AGEPC addition by elevating intracellular free Ca2+ without causing an increase in the basal level of intracellular Ca2+. The present study demonstrates that IC-21 peritoneal macrophages possess high affinity AGEPC receptors and provides the evidence that the number of functional AGEPC receptors on a cell can be increased significantly upon exposure to LPS. PMID:1328211

  6. Generation of a novel mouse model for the inducible depletion of macrophages in vivo.

    PubMed

    Gheryani, Nabeia; Coffelt, Seth B; Gartland, Alison; Rumney, Robin M H; Kiss-Toth, Endre; Lewis, Claire E; Tozer, Gillian M; Greaves, David R; Dear, T Neil; Miller, Gaynor

    2013-01-01

    Macrophages play an essential role in tissue homeostasis, innate immunity, inflammation, and wound repair. Macrophages are also essential during development, severely limiting the use of mouse models in which these cells have been constitutively deleted. Consequently, we have developed a transgenic model of inducible macrophage depletion in which macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline. Induction of the DTA protein in transgenic animals resulted in a significant 50% reduction in CD68+ macrophages of the liver, spleen, and bone over a period of 6 weeks. Pertinently, the macrophages remaining after doxycycline treatment were substantially smaller and are functionally impaired as shown by reduced inflammatory cytokine production in response to lipopolysaccharide. This inducible model of macrophage depletion can now be utilized to determine the role of macrophages in both development and animal models of chronic inflammatory diseases. PMID:22927121

  7. Characterization of mouse peritoneal exudate and associated leukocyte adherence inhibitory activity after intraperitoneal injection of either Bordetella pertussis or Corynebacterium parvum vaccines.

    PubMed Central

    Klein, T W; Pross, S H; Benjamin, W R

    1978-01-01

    Bordetella pertussis and Corynebacterium parvum are commonly used immunopotentiating agents. To explore the inflammatory environment induced by these agents, the peritoneal exudate response in mice following intraperitoneal injection of B. pertussis (PV) and C. parvum (CV) vaccines was investigated. The PV-induced exudate isolated by lavage was characterized by an early neutrophil influx followed by enhanced accumulation of mononuclear cells and fluid protein. The CV exudate was principally mononuclear in nature and displayed fewer numbers of cells and less fluid protein. Both vaccines also enhanced the leukocyte adherence inhibitory activity (LAIA) of peritoneal fluid as measured in vitro. The development of exudate LAIA was T lymphocyte independent. A similar LAIA was demonstrated in nonimmune mouse plasma and serum. Exudate fluid and serum LAIA were heat stable and trypsin sensitive. These studies suggest that significant differences exist in the composition of the local tissue environment following PV and CV injection and that exudate LAIA is serum derived. Further studies in this direction should result in a better understanding of the ways in which inflammatory cells and fluid substances affect lymphocyte-macrophage interaction subsequent to adjuvant administration. PMID:215552

  8. Effect of the native polysaccharide of cashew-nut tree gum exudate on murine peritoneal macrophage modulatory activities.

    PubMed

    Yamassaki, F T; Lenzi, R M; Campestrini, L H; Bovo, F; Seyfried, M; Soldera-Silva, A; Stevan-Hancke, F R; Zawadzki-Baggio, S F; Pettolino, F A; Bacic, A; Maurer, J B B

    2015-07-10

    The native polysaccharide of cashew-nut tree gum exudate (CNTG) and its arabinogalactan-protein component (CNTG-AGP) were tested by using immuno-stimulant and anti-inflammatory in vitro assays of murine peritoneal macrophage activities. In the assay for immuno-stimulant activity (without previous treatment with lipopolysaccharide; LPS), CNTG increased the production of interleukin (IL)-10 and both CNTG and CNTG-AGP decreased the concentrations of IL6. When the macrophages were incubated in the presence of LPS and CNTG a decrease in the levels of nitric oxide (NO(·)) and IFN-γ was observed. The results could explain the popular use of CNTG as an anti-inflammatory. In addition, CNTG is the main component of the cashew-nut tree gum exudate, which has been considered a versatile polymer with potential pharmaceutical and food industry applications. These data may contribute to the study of the immunomodulation activity of plant polysaccharides, as well as encourage future experiments in the field of cashew-nut tree gum exudate applications. PMID:25857980

  9. Mechanisms of glucocorticoid induced suppression of phagocytosis in murine peritoneal macrophage cultures

    SciTech Connect

    Becker, J.L.

    1986-01-01

    Glucocorticoids suppress phagocytosis of heat killed Saccharomyces cerevisiae in macrophage cultures. In order to determine the mechanisms by which this response occurs, this investigation was initiated to examine whether the suppression of phagocytosis is mediated by a steroid induced phagocytosis inhibitory protein (PIP). Furthermore, it is postulated that these suppressive effects may be associated with alterations in macrophage phospholipid metabolism. To assess the association between phospholipid metabolism and phagocytosis, control and 1 ..mu..M dexamethasone treated macrophages were exposed to the phospholipase inhibitor bromophenacylbromide. The enzyme inhibitor suppressed phagocytosis in a time and dose dependent manner. However, supplying dexamethasone treated cultures with arachidonate did not reverse the steroid induced suppression of phagocytosis, whether the arachidonate was supplied alone or together with indomethacin and nordihydroguaiaretic acid. Control cells, prelabeled with /sup 3/H-arachidonate, exhibited an increased percentage of the radiolabeled fatty acid in neutral lipids following phagocytosis, with a corresponding decrease in the percentage associated with phosphatidylcholine.

  10. Epigenetic Alterations Induced by Ambient Particulate Matter in Mouse Macrophages

    PubMed Central

    Miousse, Isabelle R.; Chalbot, Marie-Cécile G.; Aykin-Burns, Nükhet; Wang, Xiaoying; Basnakian, Alexei; Kavouras, Ilias G.; Koturbash, Igor

    2014-01-01

    Respiratory mortality and morbidity has been associated with exposure to particulate matter (PM). Experimental evidence suggests involvement of cytotoxicity, oxidative stress, and inflammation in the development of PM-associated pathological states; however, the exact mechanisms remain unclear. In the current study, we analyzed short-term epigenetic response to PM10 (particles with aerodynamic diameter less than 10 μm) exposure in mouse ascitic RAW264.7 macrophages (BALB/C Abelson murine leukemia virus-induced tumor). Ambient PM10 was collected using a high volume sampler in Little Rock, AR. Analysis revealed that PM10 was composed mainly of Al and Fe, and the water soluble organic fraction was dominated by aliphatic and carbohydrate fragments and minor quantities of aromatic components. Exposure to PM10 compromised the cellular epigenome at concentrations 10–200 μg/ml. Specifically, epigenetic alterations were evident as changes in the methylation and expression of repetitive element-associated DNA and associated DNA methylation machinery. These results suggest that epigenetic alterations, in concert with cytotoxicity, oxidative stress, and inflammation, might contribute to the pathogenesis of PM-associated respiratory diseases. PMID:24535919

  11. Separate Fc-receptors for immunoglogulins IgG2a and IgG2b on an established cell line of mouse macrophages.

    PubMed

    Walker, W S

    1976-04-01

    The specificity of Fe-receptors on IC-21 cells, an established line of mouse peritoneal macrophages with antibody-dependent effector cell activity has been examined. Only IgG2a and IgG2b myeloma proteins bound readily to IC-21 Fc-receptors, the former in nonaggregated as well as aggregated form, the latter only as aggregated complexes. Thus, IgG2a bound in a manner characteristic of classically defined cytophilic antibody, whereas the binding of IgG2b appeared to be mediated by Fc-receptors for antigen-antibody complexes. Evidence is presented in support of the view that IC-21 macrophages possess separate and distinct Fc-receptor sites for these two immunoglobulins. PMID:1254971

  12. Intracellularly survived Staphylococcus aureus after phagocytosis are more virulent in inducing cytotoxicity in fresh murine peritoneal macrophages utilizing TLR-2 as a possible target.

    PubMed

    Nandi, Ajeya; Bishayi, Biswadev

    2016-08-01

    Staphylococcus aureus with high virulence potential is contributing to a current public health crisis in both hospital and community settings. TLR-2 and generation of reactive oxygen species (ROS) by phagocytic cells is thought to be an important component of the host's immunity against S. aureus infection. However, response of S. aureus against modulation of host-derived ROS in absence of TLR-2 during acute staphylococcal infection is still remains unclear. Peritoneal macrophages were pretreated with either inhibitors of superoxide dismutase (SOD) or catalase in presence or absence of anti TLR-2 antibody and were infected with S. aureus strain AG-789. Bacteria were recovered after time dependent phagocytosis; intracellular killing, level and expression of SOD and catalase were measured. Phagocytosed bacteria from respective groups were further used for infection to fresh peritoneal macrophages as well as for in vivo infection. Levels of ROS, cytokine, lysozyme, antioxidant enzymes activity and TLR-2 expression were measured. Results revealed that more bacteria were escaped killing in SOD and catalase inhibitor pretreated TLR-2 neutralized macrophages, found to express more catalase and are antibiotic resistant. Infection of fresh macrophages with S. aureus, recovered from SOD and catalase inhibited TLR-2 neutralized macrophages induced lower ROS, lysozyme and cytokine production and caused increased bacterial count. Furthermore, bacterial antioxidants by modulating host-derived ROS could regulate the cell surface TLR-2 expression in murine peritoneal macrophages. So, in the early phase of infection, TLR-2 participates in the innate immune response and targeting bacterial antioxidants might be useful in the alleviation of Staphylococcus aureus infection. PMID:27270212

  13. A murine platelet-activating factor receptor gene: cloning, chromosomal localization and up-regulation of expression by lipopolysaccharide in peritoneal resident macrophages.

    PubMed Central

    Ishii, S; Matsuda, Y; Nakamura, M; Waga, I; Kume, K; Izumi, T; Shimizu, T

    1996-01-01

    A murine gene encoding a platelet-activating factor receptor (PAFR) was cloned. The gene was mapped to a region of the D2.2 band of chromosome 4 both by fluorescence in situ hybridization and by molecular linkage analysis. Northern blot analysis showed a high expression of the PAFR message in peritoneal macrophages. When C3H/HeN macrophages were treated with bacterial lipopolysaccharide (LPS) or synthetic lipid A, the PAFR gene expression was induced. Bacterial LPS, but not lipid A, induced the level of PAFR mRNA in LPS unresponsive C3H/HeJ macrophages. These induction patterns were parallel to those of tumor necrosis factor-alpha mRNA. Thus the PAFR in macrophages is important in LPS-induced pathologies. PMID:8670084

  14. Effect of triptolide on secretion of inflammatory cellular factors TNF-α and IL-8 in peritoneal macrophages of mice activated by lipopolysaccharide

    PubMed Central

    Yang, Fan; Bai, Xiang-jun; Hu, Duan; Li, Zhan-fei; Liu, Kai-jun

    2010-01-01

    BACKGROUND: Research has been carried out to look for safe and effective anti-inflammation drugs from traditional Chinese herbal medicine. As a powerful research technology of life science, molecular biology has entered many areas of traditional Chinese medicine. This study aimed to investigate the effect of triptolide on tumor necrosis factor-a (TNF-α) and interleukin-8 (IL-8) of peritoneal macrophages activated by lipopolysaccharide (LPS) in mice. METHODS: Peritoneal elicited macrophages were separated, purified and activated by LPS in mice, then cultured in vitro with triptolide at different concentrations. The activity of TNF-α and the level of IL-8 of cellular supernatants were determined by MTT colorimetric assay and ELISA, respectively. RESULTS: The activity of TNF-α in macrophages was significantly inhibited (P<0.01) by triptolide (10-1-101μg/ml) during 4-24 hours in a time- and dose-dependent manner. The level of IL-8 in macrophages was significantly inhibited (P<0.01) by triptolide (10-1-101μg/ml) in 12 hours in a dose-dependent manner. CONCLUSION: Triptolide could inhibit the activity of TNF-α and the level of IL-8 in macrophages activated by LPS. PMID:25214945

  15. Contribution of complement component C3 and complement receptor type 3 to carbohydrate-dependent uptake of oligomannose-coated liposomes by peritoneal macrophages.

    PubMed

    Abe, Yu; Kuroda, Yasuhiro; Kuboki, Noritaka; Matsushita, Misao; Yokoyama, Naoaki; Kojima, Naoya

    2008-11-01

    Peritoneal macrophages (PEMs) preferentially and rapidly take up oligomannose-coated liposomes (OMLs) and subsequently mature to induce a Th-1 immune response following administration of OMLs into the peritoneal cavity. Here, we examine the contributions of complement component C3 and complement receptor type 3 (CR3) to carbohydrate-dependent uptake of OMLs by PEMs. Effective uptake of OMLs into PEMs in vitro was observed only in the presence of peritoneal fluid (PF), and OMLs incubated with PF were incorporated by PEMs in vitro in the absence of PF. These phenomena were inhibited by methyl-alpha-mannoside, N-acetylglucosamine or EDTA, but not by galactose. Pull-down analysis followed by peptide mass fingerprinting of PF-treated OMLs indicated that the OMLs were opsonized with complement fragment iC3b. In vivo uptake of OMLs by PEMs was inhibited by intraperitoneal injection of an antibody against CR3, a receptor for iC3b, and OML uptake by PEMs in the peritoneal cavity was not observed in C3-deficient mice. Thus, our results indicate that OMLs are opsonized with iC3b in a mannose-dependent manner in the peritoneal cavity and then incorporated into PEMs via CR3. PMID:18694897

  16. Direct measurement of intracellular free Ca2+ in rat peritoneal macrophages: correlation with oxygen-radical production.

    PubMed Central

    Hallett, M B; Campbell, A K

    1983-01-01

    A novel method has been developed, based on osmotic lysis of intracellular pinocytotic vesicles, to introduce the Ca2+-activated photoprotein obelin into the cytoplasm of rat peritoneal macrophages. The change in osmolarity of the incubating medium necessary to induce lysis of the pinocytotic vesicles did not significantly affect the viability or responsiveness of the cells. The method enabled on average 3 fl of external medium to be introduced into each cell. Macrophages loaded with photoprotein had a resting intracellular Ca2+ concentration of 0.24 +/- 0.02 microM, calculated from the obelin consumption rate. The calcium ionophore, A23187, induced a prolonged rise in intracellular Ca2+ and also stimulated oxygen-radical production, monitored by luminol-dependent chemiluminescence. The chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, 1 microM, produced a transient increase in cytoplasmic Ca2+ which reached a plateau of 1.2 +/- 0.64 microM (n = 7) and declined with a half-time of approximately 40 sec. Unopsonized particles, latex beads (diameter = 1 micron), did not produce any detectable rise in intracellular Ca2+. Incorporation of a calcium chelator EGTA-ethylene-glycol-bis-(aminoethylether) tetra-acetate--into the cytoplasm abolished the transient intracellular Ca2+ rise induced by chemotactic peptide. Oxygen-radical production was also abolished. However, oxygen radical production induced by unopsonized particles was unaffected by intracellular EGTA. It was concluded that oxygen-radical production detected by chemiluminescence can be triggered by a rise in intracellular Ca2+. Chemotactic peptide induces oxygen-radical production by this mechanism. However, unopsonized particles induce oxygen-radical production by a mechanism independent of a rise in intracellular Ca2+. Images Figure 1 PMID:6414943

  17. Astragalus saponins Inhibits Lipopolysaccharide-Induced Inflammation in Mouse Macrophages.

    PubMed

    Wang, Yue; Ren, Tianjing; Zheng, Lucong; Chen, Hubiao; Ko, Joshua Kashun; Auyeung, Kathy Kawai

    2016-01-01

    Excessive nitric oxide (NO) and pro-inflammatory cytokines are produced during the pathogenesis of inflammatory diseases and cancer. It has been demonstrated that anti-inflammation contributes Astragalus membranaceus saponins (AST)'s beneficial effects in combination of conventional anticancer drugs. However, the immunomodulating property of AST has not been well characterized. In this study, we found that AST suppressed lipopolysaccharide (LPS)-induced generation of NO without causing cytotoxicity in the mouse macrophage RAW264.7. The gene and protein overexpression of inducible NO synthase (iNOS) as well as the production of tumor necrosis factor-[Formula: see text], evoked by LPS, was consistently down-regulated by AST. AST also inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and suppressed nuclear factor (NF)-[Formula: see text]B activation and the associated I[Formula: see text]B[Formula: see text] degradation during LPS insult. Furthermore, AST induced growth inhibition in promyelocytic leukemic HL-60 cells and T-lymphocyte leukemic Jurkat cells, but exerted no cytotoxic effects in normal human peripheral blood mononuclear cells (PBMC). It is known that the chemotherapeutic drug 5-FU can suppress the immune system, which can be identified by a reduced white blood cell count and decreased hematocrit, while the combination of AST and 5-FU can reverse the above hematologic toxicities. To summarize, non-cytotoxic concentrations of AST suppress LPS-induced inflammatory responses via the modulation of p38 MAPK signaling and the inhibition of NO and cytokine release. Importantly, AST can alleviate the hematologic side effects of current chemotherapeutic agents. These findings can facilitate the establishment of AST in the treatment of inflammatory diseases and inflammation-mediated tumor development. PMID:27109155

  18. Diagnostic utility of a direct immunofluorescence test to detect feline coronavirus antigen in macrophages in effusive feline infectious peritonitis.

    PubMed

    Litster, A L; Pogranichniy, R; Lin, T-L

    2013-11-01

    The antemortem diagnosis of feline infectious peritonitis (FIP) remains challenging in clinical practice, since current testing methods have suboptimal diagnostic accuracy. Immunohistochemical testing of biopsy specimens and postmortem examination are the standard diagnostic methods, although direct immunofluorescence (DIF) testing to detect feline coronavirus in macrophages in effusion specimens has been reported to have 100% specificity and has been recommended as an antemortem confirmatory test. The aim of this study was to compare the results of DIF testing in antemortem feline effusions with postmortem results using field samples. Effusion specimens were collected antemortem from 17 cats and tested by DIF, followed by postmortem examination. Histopathological examination of specimens collected at postmortem confirmed FIP in 10/17 cases and ruled out FIP out in 7/17 cases. Antemortem DIF testing was positive in all 10 cases confirmed as FIP at postmortem examination. In the seven cats where FIP was ruled out at postmortem examination, DIF was negative in five cases and positive in the remaining two cases. The calculated sensitivity of DIF testing was 100% and the specificity was 71.4%. Duplicate effusion specimens from eight cats that were initially DIF positive were stored refrigerated (4 °C) or at room temperature (22-25 °C) and subjected to serial DIF testing to determine the duration of positive results. DIF-positive specimens stored at both temperatures retained their positive status for at least 2 days. PMID:24076123

  19. Evaluation of the Leishmanicidal Activity of Rutaceae and Lauraceae Ethanol Extracts on Golden Syrian Hamster (Mesocricetus auratus) Peritoneal Macrophages

    PubMed Central

    Chávez Enciso, N. A.; Coy-barrera, E. D.; Patiño, O. J.; Cuca, L. E.; Delgado, Gabriela

    2014-01-01

    Traditional medicine has provided a number of therapeutic solutions for the control of infectious agents, cancers, and other diseases. After screening a wide variety of Colombian plant extracts, we have identified promising antileishmanial activity in ethanol extracts from Ocotea macrophylla (Lauraceae) and Zanthoxyllum monophyllum (Rutaceae). In this study, we evaluated the in vitro activity of two ethanol extracts, one from Ocotea macrophylla and the other from Zanthoxyllum monophyllum and one alkaloid fraction of ethanol extract of Zanthoxyllum monophyllum, on peritoneal macrophages isolated from golden Syrian hamsters (Mesocricetus auratus) infected with Leishmania panamensis and Leishmania major promastigotes. All of the extracts studied displayed promising (≥2) selectivity indices (S/I), the most significant of which were for ethanol extract of Zanthoxyllum monophyllum against Leishmania panamensis (S/I=12) and alkaloid fraction of ethanol extract of Zanthoxyllum monophyllum against Leishmania major (S/I=11). These results support the use of ethanol extracts and alkaloid fractions isolated from Ocotea macrophylla and Zanthoxyllum monophyllum, respectively; as therapeutic options for cutaneous leishmaniasis. PMID:25035529

  20. Evaluation of the Leishmanicidal Activity of Rutaceae and Lauraceae Ethanol Extracts on Golden Syrian Hamster (Mesocricetus auratus) Peritoneal Macrophages.

    PubMed

    Chávez Enciso, N A; Coy-Barrera, E D; Patiño, O J; Cuca, L E; Delgado, Gabriela

    2014-05-01

    Traditional medicine has provided a number of therapeutic solutions for the control of infectious agents, cancers, and other diseases. After screening a wide variety of Colombian plant extracts, we have identified promising antileishmanial activity in ethanol extracts from Ocotea macrophylla (Lauraceae) and Zanthoxyllum monophyllum (Rutaceae). In this study, we evaluated the in vitro activity of two ethanol extracts, one from Ocotea macrophylla and the other from Zanthoxyllum monophyllum and one alkaloid fraction of ethanol extract of Zanthoxyllum monophyllum, on peritoneal macrophages isolated from golden Syrian hamsters (Mesocricetus auratus) infected with Leishmania panamensis and Leishmania major promastigotes. All of the extracts studied displayed promising (≥2) selectivity indices (S/I), the most significant of which were for ethanol extract of Zanthoxyllum monophyllum against Leishmania panamensis (S/I=12) and alkaloid fraction of ethanol extract of Zanthoxyllum monophyllum against Leishmania major (S/I=11). These results support the use of ethanol extracts and alkaloid fractions isolated from Ocotea macrophylla and Zanthoxyllum monophyllum, respectively; as therapeutic options for cutaneous leishmaniasis. PMID:25035529

  1. Spacious phagosome formation within mouse macrophages correlates with Salmonella serotype pathogenicity and host susceptibility.

    PubMed Central

    Alpuche-Aranda, C M; Berthiaume, E P; Mock, B; Swanson, J A; Miller, S I

    1995-01-01

    Light microscopic studies indicated a correlation between the virulence for mice of different Salmonella serotypes and the ability to form or maintain spacious phagosomes (SP) within mouse macrophages. Although Salmonella typhimurium induced membrane ruffling, macropinocytosis, and SP formation in macrophages from BALB/c mice, serotypes which are nonpathogenic for mice produced markedly fewer SP. SP formation correlated with both serotype survival within mouse macrophages and reported lethality for mice. Time-lapse video microscopy demonstrated that the human pathogen S. typhi induced generalized macropinocytosis and SP formation in human monocyte-derived macrophages, indicating a similar morphology for the initial phases of this host-pathogen interaction. In contrast to bone marrow-derived macrophages from BALB/c mice, macrophages from S. typhimurium-resistant outbred (CD-1) and inbred (CBA/HN) mice did not initiate generalized macropinocytosis after bacterial infection and formed markedly fewer SP. These deficiencies were not due to the Ihy resistance genotype of these mice, as macrophages from mice that were congenic except for the Ihy locus demonstrated equal SP formation in response to S. typhimurium. The observation that S. typhimurium-resistant CD-1 and CBA/HN mice are deficient in the ability to form and/or maintain SP indicates that a variable host component is important for SP formation and suggests that the ability to induce or form SP affects susceptibility to S. typhimurium. When serotypes nonpathogenic for mice were used to infect BALB/c macrophages, or when CD-1 or CBA/HN mouse macrophages were infected by S. typhimurium, some of the SP that formed shrank within seconds. This rapid shrinkage suggests that SP maintenance is also important for S. typhimurium survival within macrophages. These studies indicate that both host and bacterial factors contribute to SP formation and maintenance, which correlate with Salmonella intracellular survival and the

  2. Investigating the role of macrophages in tumor formation using a MaFIA mouse model.

    PubMed

    Clifford, A B; Elnaggar, A M; Robison, R A; O'Neill, K

    2013-08-01

    Tumor-associated macrophages (TAMs) interact with tumors in their development, growth and metastatic activities. Using a transgenic mouse model that allows for the selective depletion of macrophages we were able to access the macrophage's potential to facilitate metastasis. In the MaFIA (Macrophage Fas-Induced Apoptosis) mouse, transgene-expressing cells of the myeloid lineage undergo death by apoptosis in the presence of the drug AP20187. Enhanced green fluorescent protein (EGFP) was fused to the suicide gene to allow identification of transgene-expressing cells. Tumor induction was accomplished by subdermal and intravenous injections of B16-F10 melanoma cells. Metastasis in mice with depleted macrophages was compared to metastasis in normal control mice. The lungs and kidneys were examined for metastatic cells. The macrophage-depleted groups showed significantly less metastasis (P>0.001) compared to the control groups. We theorize that macrophages may aid the metastatic process by fusing with melanoma cells. Using appropriate cell markers and fluorescence-activated cell sorting, we were able to detect a small population of double-positive cells. We confirmed cell fusion by microscopic analysis, visualizing the cell's morphology by both immunohistochemistry and immunofluorescence. The presence of double-positive cells suggests macrophage/cancer cell fusion could be a possible mechanism for metastasis. PMID:23722325

  3. How Mouse Macrophages Sense What Is Going On

    PubMed Central

    Ley, Klaus; Pramod, Akula Bala; Croft, Michael; Ravichandran, Kodi S.; Ting, Jenny P.

    2016-01-01

    Macrophages are central to both innate and adaptive immunity. With few exceptions, macrophages are the first cells that sense trouble and respond to disturbances in almost all tissues and organs. They sense their environment, inhibit or kill pathogens, take up apoptotic and necrotic cells, heal tissue damage, and present antigens to T cells. Although the origins (yolk sac versus monocyte-derived) and phenotypes (functions, gene expression profiles, surface markers) of macrophages vary between tissues, they have many receptors in common that are specific to one or a few molecular species. Here, we review the expression and function of almost 200 key macrophage receptors that help the macrophages sense what is going on, including pathogen-derived molecules, the state of the surrounding tissue cells, apoptotic and necrotic cell death, antibodies and immune complexes, altered self molecules, extracellular matrix components, and cytokines, including chemokines. PMID:27313577

  4. The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation

    PubMed Central

    2012-01-01

    Background The development of ascites in cirrhotic patients generally heralds a deterioration in their clinical status. A differential gene expression profile between alcohol- and hepatitis C virus (HCV)-related cirrhosis has been described from liver biopsies, especially those associated with innate immune responses. The aim of this work was to identify functional differences in the inflammatory profile of monocyte-derived macrophages from ascites in cirrhotic patients of different etiologies in an attempt to extrapolate studies from liver biopsies to immune cells in ascites. To this end 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (n = 15) or alcohol (n = 30) were studied. Cytokines and the cell content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation in peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined. Results A different pattern of leukocyte migration to the peritoneal cavity and differences in the primed status of macrophages in cirrhosis were observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral blood cell subpopulations could be observed, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in the HCV than the alcohol etiology. HCV-related cirrhosis etiology was associated with a decreased inflammatory profile in ascites compared with the alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were observed in ascitic fluid from the HCV group. Isolated peritoneal monocyte-derived macrophages maintained their primed status in vitro throughout the 24 h culture period. The level of ERK1/2 phosphorylation was higher in ALC peritoneal macrophages at baseline than in HCV patients, although the addition of LPS induced a greater increase in ERK1/2 phosphorylation in HCV than in ALC patients. Conclusions The

  5. Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse.

    PubMed

    Hopkinson-Woolley, J; Hughes, D; Gordon, S; Martin, P

    1994-05-01

    Macrophages play a pivotal role in the adult inflammatory response to wounding. They are directly responsible for cellular debridement and, by providing a source of growth factors and cytokines, they recruit other inflammatory and fibroblastic cells and influence cell proliferation and tissue remodelling. In this paper we investigate the role of macrophages in clearing areas of programmed cell death in the developing embryo and also their role in embryonic and foetal wound healing. Immunocytochemistry using the monocyte/macrophage-specific monoclonal antibody, F4/80, reveals a close association between areas of programmed cell death in the remodelling interdigital regions of the mouse footplate and of F4/80-positive cells, suggesting that monocyte-derived macrophages, and not locally recruited fibroblastic cells, as previously believed, are responsible for phagocytosing and clearing areas of interdigital apoptosis. Our studies of wound healing reveal that macrophages are not recruited to, and therefore cannot be playing an active role in the healing of, excisional wounds made in the mouse embryo at any stage up until E14.5. Beyond this transition stage we see a significant recruitment of macrophages within 12 hours of wounding. We find that macrophages can be attracted to wounds in earlier embryos if the wound results in significant cell death such as after burning. PMID:7929625

  6. Peritoneal "melanosis".

    PubMed

    Chang, Ea-sle; Bachul, Piotr; Szura, Mirosław; Szpor, Joanna; Okoń, Krzysztof; Walocha, Jerzy A

    2015-09-01

    A case of a23 year old female with peritoneal melanosis associated with adenocarcinoma of the rectum is reported. During laparoscopic anterior resection of the rectum, diffuse black pigmentations on the parietal peritoneum, greater omentum, mesenteric lymph nodes and ovaries were discovered. The histopathological findings revealed the presence of macrophages packed with black pigment. These results together with clinical data excluded metastatic melanoma and confirmed the diagnosis of the race condition called peritoneal melanosis. Due to the begin character of the lesions the laparoscopic treatment was continued. There were no remissions or progression of the reported in English literature and this is the second case of peritoneal melanosis that has been associated with adenocarcinoma of the large intestine. PMID:26619112

  7. Intracellular activity of antibiotics against Staphylococcus aureus in a mouse peritonitis model.

    PubMed

    Sandberg, Anne; Hessler, Jonas H R; Skov, Robert L; Blom, Jens; Frimodt-Møller, Niels

    2009-05-01

    Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log(10) number of CFU/ml (Delta log(10) CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Delta log(10) CFU/ml) > cefuroxime (3.56 Delta log(10) CFU/ml) > rifampin (1.86 Delta log(10) CFU/ml) > gentamicin (0.61 Delta log(10) CFU/ml) > azithromycin (0.21 Delta log(10) CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs. PMID

  8. The action of Clofazimine on the level of lysosomal enzymes of cultured macrophages.

    PubMed Central

    Sarracent, J; Finlay, C M

    1982-01-01

    Mouse peritoneal and calf alveolar macrophage cultures were exposed to various concentrations of Clofazimine, 3 (p-chloroanilino)-10-p-Chlorophenyl 2, 10-dihydro-2-isopropylimino, for 120 hr and an increase of four lysosomal enzymes were found with 0 . 3 micrograms/ml of the drug. In mouse peritoneal macrophage cultures, higher concentrations were toxic. Cycloheximide inhibited the lysosomal enzyme activity increase found. No change in enzymatic activity was observed when a lysosomal enriched granular fraction was incubated with various drug concentrations. Our results strongly suggest that Clofazimine at concentrations close to therapeutic serum levels induces de novo synthesis of lysosomal enzymes in macrophage cultures. PMID:7083642

  9. Increasing cytotoxic activity and production of reactive oxygen and nitrogen intermediates by peritoneal macrophages during the development of multiple organ dysfunction syndrome in mice.

    PubMed

    Jansen, M J; Hendriks, T; Huyben, C M; Tax, W J; van der Meer, J W; Goris, R J

    1996-10-01

    A major problem in the intensive care unit nowadays is the development of multiple organ dysfunction syndrome (MODS), a cumulative sequence of progressive deterioration of organ functions. While the pathogenic pathways of MODS remain to be elucidated, it is assumed that cells of the host defence system, especially the macrophages, are altered in their function. During the development of MODS it is assumed that macrophages are overactivated and that an exaggerated inflammatory response may contribute to its pathogenesis. In order to gain insight into the alterations of the functional status of the macrophage during the development of MODS, a series of macrophage functions was measured in the subsequent phases of zymosan induced generalized inflammation in mice. Male C57BL/6 mice received a single dose of zymosan intraperitoneally and groups of animals were killed after 2, 5, 8, and 12 days. Peritoneal macrophages were collected for in vitro assessment of the ADCC, the production of superoxide (O2-) and nitric oxide (NO), and complement mediated phagocytosis and intracellular killing of Staphylococcus aureus. A single intraperitoneal injection with zymosan resulted in a three-phase illness. During the third phase the animals developed MODS-like symptoms. Peritoneal cells from control animals produced very low to non-detectable amounts of O2- and NO, and the cytotoxic activity was also low. During the development of MODS, from day 7 onwards, the ability to produce O2- and NO2- became strongly elevated, as did the cytotoxic activity. These findings are in parallel with the development of MODS whereas the phagocytic and killing capacity remained essentially unaltered. The changes found could be detrimental for the organism, thus possibly contributing to the onset and development of MODS. PMID:8845029

  10. Lipopolysaccharide Attenuates the Cytotoxicity of Resveratrol in Transformed Mouse Macrophages.

    PubMed

    Achy-Brou, Christelle A Adiabouah; Billack, Blase

    2016-09-01

    Resveratrol and pterostilbene are natural products that are present in plants and have been incorporated into various dietary supplements. Numerous beneficial pharmacologic effects have been reported for these stilbenes; however, the mechanism by which these compounds exert a cytotoxic effect in RAW 264.7 macrophages has not been well characterized. We have previously described that resveratrol is toxic to these tumor-derived macrophages and that stimulation with lipopolysaccharide (LPS) reduces resveratrol toxicity via a mechanism that involves activation of toll like receptor 4. In the present work, we examined the cellular and molecular effects of resveratrol and the related compound pterostilbene by determining cell viability and caspase 3 activity in control and LPS-stimulated RAW 264.7 macrophages incubated with these stilbenes for 24 h. We found that LPS stimulation reduced the cytotoxicity of resveratrol but not of pterostilbene in these cells. When examined for effects on caspase 3 activation after a 24 h incubation, resveratrol and pterostilbene were each found to separately and significantly increase caspase 3 activity in these cells. LPS stimulation prevented caspase 3 activation by pterostilbene and reduced caspase 3 activation by resveratrol in RAW 264.7 macrophages. The data presented here indicate that LPS induces a phenotype switch in tumor-derived RAW 264.7 macrophages in which cells experiencing LPS in the presence of resveratrol or pterostilbene become less likely to activate the pro-apoptotic factor caspase 3. PMID:27277074

  11. Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production

    PubMed Central

    Yang, Kun; Wu, Yongjian; Xie, Heping; Li, Miao; Ming, Siqi; Li, Liyan; Li, Meiyu; Wu, Minhao; Gong, Sitang; Huang, Xi

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection. PMID:27251437

  12. Structure elucidation of fungal beauveriolide III, a novel inhibitor of lipid droplet formation in mouse macrophages.

    PubMed

    Namatame, I; Tomoda, H; Tabata, N; Si, S; Omura, S

    1999-01-01

    The structure of fungal beauveriolide III, an inhibitor of lipid droplet formation in mouse macrophages, was elucidated to be cyclo-[(3S,4S)-3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl- D-allo-isoleucyl] by spectral analyses and chemical degradation. PMID:10092190

  13. Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production.

    PubMed

    Yang, Kun; Wu, Yongjian; Xie, Heping; Li, Miao; Ming, Siqi; Li, Liyan; Li, Meiyu; Wu, Minhao; Gong, Sitang; Huang, Xi

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection. PMID:27251437

  14. Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages

    NASA Astrophysics Data System (ADS)

    Xie, Qiao-Wen; Cho, Hearn J.; Calaycay, Jimmy; Mumford, Richard A.; Swiderek, Kristine M.; Lee, Terry D.; Ding, Aihao; Troso, Tiffany; Nathan, Carl

    1992-04-01

    Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

  15. Identification of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using RAW 264.7 mouse macrophages

    PubMed Central

    Huang, Nan; Rizshsky, Ludmila; Hauck, Cathy; Nikolau, Basil J.; Murphy, Patricia A.; Birt, Diane F.

    2011-01-01

    Hypericum perforatum (St. John’s wort) is an herb widely used as supplement for mild to moderate depression. Our prior studies revealed synergistic anti-inflammatory activity associated with 4 bioactive compounds in a fraction of H. perforatum ethanol extract. Whether these 4 compounds also contributed to the ethanol extract activity was addressed in the research reported here. Despite the popularity of H. perforatum, other Hypericum species with different phytochemical profiles could have their anti-inflammatory potentials attributed to these or other compounds. In the current study, ethanol extracts of different Hypericum species were compared for their inhibitory effect on LPS-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production in RAW 264.7 mouse macrophages. Among these extracts, those made from H. perforatum and H. gentianoides demonstrated stronger overall efficacy. LC-MS analysis indicated the 4 compounds in H. perforatum extract and pseudohypericin in all active fractions. The 4 compounds accounted for a significant part of the extract’s inhibitory activity on PGE2, NO, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7 as well as peritoneal macrophages. Pseudohypericin was the most important contributor of the anti-inflammatory potential among the 4 compounds. The lipophilic fractions of H. gentianoides extract, which did not contain the previously identified active constituents, decreased PGE2 and NO potently. These fractions were rich in acylphloroglucinols, including uliginosin A that accounted for a proportion of the anti-inflammatory activity observed with the active fractions. Overall, the current study revealed a different group of major anti-inflammatory constituents in H. gentianoides, while showing that a previously identified 4 compounds combination was important for H. perforatum’s anti-inflammatory potential. PMID:21855951

  16. Combined influence of teichoic acids from Staphylococcus aureus and heterometallik Cu/Cd ethylenediamine complex on peritoneal macrophages and tumor cells.

    PubMed

    Nikulina, V V; Garmanchuk, L V; Senchylo, N V; Nikolaenko, T V; Dzhus, O I; Ostapchenko, L I; Khranovska, N M

    2014-01-01

    We investigated the effects of teichoic acid (TA) from Staphylococcus aureus Wood 46 on tumor growth and metastasis of the experimental Lewis lung carcinoma (LLC) in mice. Intranasal administration of TA alone aggravated both tumor growth and metastasis, whereas combined administration of TA with a synthetic bimetallic (copper : cadmium) ethylene diamine complex PO244 resulted in pronounced antitumor and antimetastatic effects. The group of animals subjected to the combined treatment with TA and PO244 manifested the highest degree of lymphocyte infiltration into the tumor tissue, compared to the control group and those exposed to TA or PO244 alone. Moreover, the combined treatment negatively affected the adhesive properties of peritoneal macrophages in the LLC bearing mice. Co-cultivation of the isolated macrophages with primary LLC cultures revealed significant (p < 0.05) cytotoxic and cytostatic effects, detected as an increased level of apoptosis and a reduced fraction of replicating cells. PMID:25536823

  17. Pharmacodynamics of Glycopeptides in the Mouse Peritonitis Model of Streptococcus pneumoniae or Staphylococcus aureus Infection

    PubMed Central

    Knudsen, Jenny Dahl; Fuursted, Kurt; Raber, Susan; Espersen, Frank; Frimodt-Møller, Niels

    2000-01-01

    The emergence of resistance to various antibiotics in pneumococci leaves the glycopeptides as the only antibiotics against which pneumococci have no resistance mechanism. This situation has led to a renewed interest in the use of glycopeptides. It has not yet been possible to conclude which one or more of the pharmacokinetic or pharmacodynamic (PK/PD) parameters are the most important and best predictors for the effects of treatment with glycopeptides in animal models or in humans. We used the mouse peritonitis model with immunocompetent mice and with Staphylococcus aureus and Streptococcus pneumoniae as infective organisms. A wide spectrum of different treatment regimens with vancomycin and teicoplanin was tested to study the pharmacodynamics of these drugs. In studies in which the single dose that protected 50% of lethally infected mice (ED50) was given as one dose or was divided into two doses, survival was significantly decreased when the dose was divided. The only statistically significant correlations between the percentage of survival of the mice after 6 days and each of the PK/PD parameters were for peak concentration (Cmax)/MIC and S. aureus and for the free fraction of Cmax (Cmax-free)/MIC and S. pneumoniae. For S. pneumoniae, the ED50 for different dosing regimens increased with the number of doses given; e.g., the single-dose ED50s for vancomycin and teicoplanin were 0.65 and 0.45 mg/kg, respectively, but the ED50s for dosing regimens with 2-h doses given for 48 h were 6.79 and 5.67 mg/kg, respectively. In experiments with 39 different vancomycin dosing regimens and 40 different teicoplanin dosing regimens against S. pneumoniae, the different PK/PD parameters were analyzed using logistic regression. The Cmax-free/MIC was one of two parameters that best explained the effect for both drugs; for vancomycin, the other important parameter was the AUC/MIC, and for teicoplanin, the other parameter was the time the free fraction of the drug is above the MIC

  18. Pharmacodynamics of glycopeptides in the mouse peritonitis model of Streptococcus pneumoniae or Staphylococcus aureus infection.

    PubMed

    Knudsen, J D; Fuursted, K; Raber, S; Espersen, F; Frimodt-Moller, N

    2000-05-01

    The emergence of resistance to various antibiotics in pneumococci leaves the glycopeptides as the only antibiotics against which pneumococci have no resistance mechanism. This situation has led to a renewed interest in the use of glycopeptides. It has not yet been possible to conclude which one or more of the pharmacokinetic or pharmacodynamic (PK/PD) parameters are the most important and best predictors for the effects of treatment with glycopeptides in animal models or in humans. We used the mouse peritonitis model with immunocompetent mice and with Staphylococcus aureus and Streptococcus pneumoniae as infective organisms. A wide spectrum of different treatment regimens with vancomycin and teicoplanin was tested to study the pharmacodynamics of these drugs. In studies in which the single dose that protected 50% of lethally infected mice (ED(50)) was given as one dose or was divided into two doses, survival was significantly decreased when the dose was divided. The only statistically significant correlations between the percentage of survival of the mice after 6 days and each of the PK/PD parameters were for peak concentration (C(max))/MIC and S. aureus and for the free fraction of C(max) (C(max-free))/MIC and S. pneumoniae. For S. pneumoniae, the ED(50) for different dosing regimens increased with the number of doses given; e.g., the single-dose ED(50)s for vancomycin and teicoplanin were 0.65 and 0. 45 mg/kg, respectively, but the ED(50)s for dosing regimens with 2-h doses given for 48 h were 6.79 and 5.67 mg/kg, respectively. In experiments with 39 different vancomycin dosing regimens and 40 different teicoplanin dosing regimens against S. pneumoniae, the different PK/PD parameters were analyzed using logistic regression. The C(max-free)/MIC was one of two parameters that best explained the effect for both drugs; for vancomycin, the other important parameter was the AUC/MIC, and for teicoplanin, the other parameter was the time the free fraction of the drug is

  19. Modulation of Leishmania (L.) amazonensis Growth in Cultured Mouse Macrophages by Prostaglandins and Platelet Activating Factor

    PubMed Central

    Lonardoni, M. V. C.; Barbieri, C. L.; Russo, M.

    1994-01-01

    The role of endogenously synthesized PAF and prostaglandins on the infection of mouse macrophages by Letsbmanta (L.) amazonensis was investigated, as well as the possible correlation between the effects of these inflammatory mediators with nitric oxide production. It was found that pretreatment of macrophages with 10−5 M of the PAF antagonists, BN-52021 or WEB-2086, increased macrophage infection by 17 and 59%, respectively. The cyclooxygenase inhibitor, indomethacin (10 μg/ml), induced a significant inhibition which was reversed by addition of PGE (10-3 M) to the culture medium. These results suggested that the infection of macrophages by leisbmanla is inhibited by PAF and enhanced by prostaglandins and that these mediators are produced by macrophages during this infection. This was confirmed by addition of these mediators to the culture medium before infection; PAF (10−6, 10−9 and 10−12M) reduced significantly the infection whereas PGE2 (10−5 M) induced a marked enhancement. This effect of exogenous PAF on macrophage infection was reversed by the two PAF antagonists used in this study as well as by the inhibitor of nitric oxide synthesis, L-arginine methyl ester (100 mM). Taken together the data suggest that endogenous production of PAF and PGE2 exert opposing effects on Lesbmana–macrophage interaction and that nitric oxide may be involved in the augmented destruction of parasites induced by PAF. PMID:18472932

  20. Macrophage Depletion Attenuates Extracellular Matrix Deposition and Ductular Reaction in a Mouse Model of Chronic Cholangiopathies.

    PubMed

    Best, Jan; Verhulst, Stefaan; Syn, Wing-Kin; Lagaisse, Kimberly; van Hul, Noemi; Heindryckx, Femke; Sowa, Jan-Peter; Peeters, Liesbeth; Van Vlierberghe, Hans; Leclercq, Isabelle A; Canbay, Ali; Dollé, Laurent; van Grunsven, Leo A

    2016-01-01

    Chronic cholangiopathies, such as primary and secondary sclerosing cholangitis, are progressive disease entities, associated with periportal accumulation of inflammatory cells, encompassing monocytes and macrophages, peribiliary extracellular matrix (ECM) deposition and ductular reaction (DR). This study aimed to elucidate the relevance of macrophages in the progression of chronic cholangiopathies through macrophage depletion in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse model. One group of mice received a single i.p. injection of Clodronate encapsulated liposomes (CLOLipo) at day 7 of a 14 day DDC treatment, while control animals were co-treated with PBSLipo instead. Mice were sacrificed after 7 or respectively 14 days of treatment for immunohistochemical assessment of macrophage recruitment (F4/80), ECM deposition (Sirius Red, Laminin) and DR (CK19). Macrophage depletion during a 14 day DDC treatment resulted in a significant inhibition of ECM deposition. Porto-lobular migration patterns of laminin-rich ECM and ductular structures were significantly attenuated and a progression of DR was effectively inhibited by macrophage depletion. CLOLipo co-treatment resulted in a confined DR to portal regions without amorphous cell clusters. This study suggests that therapeutic options selectively directed towards macrophages might represent a feasible treatment for chronic cholestatic liver diseases. PMID:27618307

  1. Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

    PubMed

    Almeida-Souza, Fernando; de Souza, Celeste da Silva Freitas; Taniwaki, Noemi Nosomi; Silva, João José Mendes; de Oliveira, Renata Mondêgo; Abreu-Silva, Ana Lúcia; Calabrese, Kátia da Silva

    2016-08-31

    Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 μg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 μg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes. PMID:27328771

  2. In the Absence of Endogenous Gamma Interferon, Mice Acutely Infected with Neospora caninum Succumb to a Lethal Immune Response Characterized by Inactivation of Peritoneal Macrophages

    PubMed Central

    Nishikawa, Yoshifumi; Tragoolpua, Khajornsak; Inoue, Noboru; Makala, Levi; Nagasawa, Hideyuki; Otsuka, Haruki; Mikami, Takeshi

    2001-01-01

    Following infection with Neospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-γ)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-γ-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-γ-deficient mice died within 9 days of infection with N. caninum, whereas those treated with exogenous IFN-γ lived longer. Although N. caninum invaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-γ-deficient mice were activated by exogenous IFN-γ associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-γ-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-γ-deficient mice did not. However, in vivo treatment with exogenous IFN-γ induced up-regulated MHC class II expression in IFN-γ-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-γ and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-γ-deficient mice. The levels of IL-12 in IFN-γ-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-γ production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses against N. caninum. PMID:11427432

  3. Stimulation of IFN-γ production by garlic lectin in mouse spleen cells: involvement of IL-12 via activation of p38 MAPK and ERK in macrophages.

    PubMed

    Dong, Qing; Sugiura, Tsutomu; Toyohira, Yumiko; Yoshida, Yasuhiro; Yanagihara, Nobuyuki; Karasaki, Yuji

    2011-02-15

    Several lectins, present in beans and edible plant products, have immuno-potentiating and anti-tumor activities. We here report the effects of garlic lectin purified from garlic bulbs on the production of cytokines such as interleukin-12 (IL-12) and interferon-γ (IFN-γ) in the mouse. Garlic lectin induced IFN-γ production in spleen cells in a bell-shaped time (24-60 h)- and concentration (0.25-2.0 mg/ml)-dependent manner. The maximal enhancement was observed at 36 h with 0.5 mg/ml of garlic lectin. The stimulatory effect of garlic lectin on IFN-γ production was completely inhibited by both actinomycin D and cycloheximide, an inhibitor of ribosomal protein synthesis and DNA-dependent RNA polymerase, respectively, and was associated with an increase in IFN-γ mRNA level. Garlic lectin also induced IL-12 production in mouse peritoneal macrophages in a concentration (0.25-1.0 mg/ml)- and bell-shaped time (3-24 h)-dependent manner. The lectin increased the phosphorylation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) in macrophages. Furthermore, specific pharmacological inhibitors of ERK kinase (U0126) and p38 MAPK (SB203580) also suppressed the production of IL-12 induced by garlic lectin. The present findings suggest that garlic lectin induces IL-12 production via activation of p38 MAPK and ERK in mouse macrophages, which, in turn, stimulates IFN-γ production through an increase in IFN-γ mRNA in the spleen cells. PMID:20724126

  4. A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

    PubMed Central

    Yamamoto, N; Naraparaju, V R

    1996-01-01

    Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice. PMID:8881764

  5. CpG ODN enhances uptake of bacteria by mouse macrophages

    PubMed Central

    UTAISINCHAROEN, P; KESPICHAYAWATTANA, W; ANUNTAGOOL, N; CHAISURIYA, P; PICHYANGKUL, S; KRIEG, A M; SIRISINHA, S

    2003-01-01

    Unmethylated CpG motif in synthetic oligodeoxynucleotide (CpG ODN) or bacterial DNA is well recognized for its role in innate immunity, including enhancing production of NO and cytokines by macrophages. In the present study, we demonstrated the effect of CpG ODN on the phagocytic uptake of bacteria by macrophages. Flow cytometric analysis of mouse macrophages (RAW 264·7) incubated with fluorescein isothiocyanate (FITC)-labelled Burkholderia pseudomallei, Salmonella enterica serovar Typhi or Escherichia coli showed that CpG ODN increased the uptake of these bacteria by mouse macrophages. The enhancement of bacterial uptake by CpG ODN was concentration-dependent. The increase of bacterial uptake by CpG ODN-activated macrophages shown above is consistent with the result of bacteria internalization study using a standard antibiotic protection assay. There was also an increase in the rate and degree of multi-nucleated giant cell formation, phenomena which have been shown previously to be unique when the cells were infected with B. pseudomallei. These observations may provide significant insights for future investigation into host cell–pathogen interaction. PMID:12653838

  6. Ginsenoside Rd alleviates mouse acute renal ischemia/reperfusion injury by modulating macrophage phenotype

    PubMed Central

    Ren, Kaixi; Jin, Chao; Ma, Pengfei; Ren, Qinyou; Jia, Zhansheng; Zhu, Daocheng

    2015-01-01

    Background Ginsenoside Rd (GSRd), a main component of the root of Panax ginseng, exhibits anti-inflammation functions and decreases infarct size in many injuries and ischemia diseases such as focal cerebral ischemia. M1 Macrophages are regarded as one of the key inflammatory cells having functions for disease progression. Methods To investigate the effect of GSRd on renal ischemia/reperfusion injury (IRI) and macrophage functional status, and their regulatory role on mouse polarized macrophages in vitro, GSRd (10–100 mg/kg) and vehicle were applied to mice 30 min before renal IRI modeling. Renal functions were reflected by blood serum creatinine and blood urea nitrogen level and histopathological examination. M1 polarized macrophages infiltration was identified by flow cytometry analysis and immunofluorescence staining with CD11b+, iNOS+/interleukin-12/tumor necrosis factor-α labeling. For the in vitro study, GSRd (10–100 μg/mL) and vehicle were added in the culture medium of M1 macrophages to assess their regulatory function on polarization phenotype. Results In vivo data showed a protective role of GSRd at 50 mg/kg on Day 3. Serum level of serum creatinine and blood urea nitrogen significantly dropped compared with other groups. Reduced renal tissue damage and M1 macrophage infiltration showed on hematoxylin–eosin staining and flow cytometry and immunofluorescence staining confirmed this improvement. With GSRd administration, in vitro cultured M1 macrophages secreted less inflammatory cytokines such as interleukin-12 and tumor necrosis factor-α. Furthermore, macrophage polarization-related pancake-like morphology gradually changed along with increasing concentration of GSRd in the medium. Conclusion These findings demonstrate that GSRd possess a protective function against renal ischemia/reperfusion injury via downregulating M1 macrophage polarization. PMID:27158241

  7. Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

    PubMed Central

    Fink, Avner; Hassan, Musa A.; Okan, Nihal A.; Sheffer, Michal; Camejo, Ana; Saeij, Jeroen P. J.

    2016-01-01

    ABSTRACT Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background. PMID:26980837

  8. Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences.

    PubMed

    Martinez, Fernando O; Helming, Laura; Milde, Ronny; Varin, Audrey; Melgert, Barbro N; Draijer, Christina; Thomas, Benjamin; Fabbri, Marco; Crawshaw, Anjali; Ho, Ling Pei; Ten Hacken, Nick H; Cobos Jiménez, Viviana; Kootstra, Neeltje A; Hamann, Jörg; Greaves, David R; Locati, Massimo; Mantovani, Alberto; Gordon, Siamon

    2013-02-28

    The molecular repertoire of macrophages in health and disease can provide novel biomarkers for diagnosis, prognosis, and treatment. Th2-IL-4–activated macrophages (M2) have been associated with important diseases in mice, yet no specific markers are available for their detection in human tissues. Although mouse models are widely used for macrophage research, translation to the human can be problematic and the human macrophage system remains poorly described. In the present study, we analyzed and compared the transcriptome and proteome of human and murine macrophages under resting conditions (M0) and after IL-4 activation (M2). We provide a resource for tools enabling macrophage detection in human tissues by identifying a set of 87 macrophage-related genes. Furthermore, we extend current understanding of M2 activation in different species and identify Transglutaminase 2 as a conserved M2 marker that is highly expressed by human macrophages and monocytes in the prototypic Th2 pathology asthma. PMID:23293084

  9. Efficient replication of pneumonia virus of mice (PVM) in a mouse macrophage cell line

    PubMed Central

    Dyer, Kimberly D; Schellens, Ingrid MM; Bonville, Cynthia A; Martin, Brittany V; Domachowske, Joseph B; Rosenberg, Helene F

    2007-01-01

    Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-α (TNF-α), interferon-β (IFN-β), macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems. PMID:17547763

  10. Geniposide, from Gardenia jasminoides Ellis, inhibits the inflammatory response in the primary mouse macrophages and mouse models.

    PubMed

    Fu, Yunhe; Liu, Bo; Liu, Jinhua; Liu, Zhicheng; Liang, Dejie; Li, Fengyang; Li, Depeng; Cao, Yongguo; Zhang, Xichen; Zhang, Naisheng; Yang, Zhengtao

    2012-12-01

    Geniposide, a main iridoid glucoside component of gardenia fruit, has been known to exhibit antibacterial, anti-inflammatory and other important therapeutic activities. The objective of this study was to investigate the protective effects of geniposide on inflammation in lipopolysaccharide (LPS) stimulated primary mouse macrophages in vitro and LPS induced lung injury model in vivo. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Toll-like receptor 4 (TLR4) were determined by Western blot. Further analysis was carried out in mTLR4 and mMD-2 co-transfected HEK293 cells. The results showed that geniposide markedly inhibited the LPS-induced TNF-α, IL-6 and IL-1β production both in vitro and in vivo. Geniposide blocked the phosphorylation of IκBα, p65, p38, ERK and JNK in LPS stimulated primary mouse macrophages. Furthermore, geniposide inhibited the expression of TLR4 in LPS stimulated primary mouse macrophages and inhibited the LPS-induced IL-8 production in HEK293-mTLR4/MD-2 cells. In vivo study, it was also observed that geniposide attenuated lung histopathologic changes in the mouse models. These results suggest that geniposide exerts an anti-inflammatory property by down-regulating the expression of TLR4 up-regulated by LPS. Geniposide is highly effective in inhibiting acute lung injury and may be a promising potential therapeutic reagent for acute lung injury treatment. PMID:22878137

  11. Effect of swainsonine on processing and turnover of two lysosomal glycoproteins from mouse peritoneal macrophages

    SciTech Connect

    Tropea, J.E.

    1987-01-01

    No effect of the inhibitor on the relative rates of synthesis of the precursor form of either enzyme was observed with either (/sup 35/S)methionine or 2-(/sup 3/H)mannose as the labeled tracer. On the other hand, processing of ..beta..-galactosidase and ..beta..-glucuronidase was markedly altered by swainsonine as manifested by a number of differences in the processed forms. (1) The altered forms had slightly greater electrophoretic mobility than normal forms. (2) Endo-..beta..-N-acetylglucosaminidase H digestion decreased the molecular sizes of the altered forms much more than those of the normal forms. (3) On isoelectric separation of the charge isoforms of ..beta..-glucuronidase, the three most negatively charged forms of the normal processed enzyme were absent in the altered material. This difference was eliminated by neuraminidase treatment of the processed form. These findings are consistent with a blockage by swainsonine of the removal of the ..cap alpha..-1,3 and ..cap alpha..-1,6 linked mannose residues that occurs in normal processing.

  12. Immunostaining of macrophages, endothelial cells and smooth muscle cells in the atherosclerotic mouse aorta

    PubMed Central

    Menon, Prashanthi; Fisher, Edward A.

    2016-01-01

    The atherosclerotic mouse aorta consists of a heterogeneous population of cells, including macrophages, endothelial cells (EC) and smooth muscle cells (SMC), that play critical roles in cardiovascular disease. Identification of these vascular cells in the vessel wall is important to understanding their function in pathological conditions. Immunohistochemistry is an invaluable technique used to detect the presence of cells in different tissues. Here, we describe immunohistochemical techniques commonly used for the detection of the vascular cells in the atherosclerotic mouse aorta using cell specific markers. PMID:26445786

  13. Impact of Omega-6 Polyunsaturated Fatty Acid:Eicosapentaenoic acid (EPA)+Docosahexaenoic Acid (DHA) Ratios in LDL Receptor Knockout (LDLr-/-) Mice on Atherosclerotic Lesion Formation and Elicited Peritoneal Macrophage Inflamm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Very long chain omega-3 fatty acids have been associated with decreased risk of CVD. LDL receptor knockout mice were used to assess the effect of different omega-6:EPA+DHA ratios on atherosclerotic lesion formation and elicited peritoneal macrophage inflammatory response. Methods and R...

  14. Carrageenan-induced inflammation promotes ROS generation and neutrophil extracellular trap formation in a mouse model of peritonitis.

    PubMed

    Barth, Cristiane R; Funchal, Giselle A; Luft, Carolina; de Oliveira, Jarbas R; Porto, Bárbara N; Donadio, Márcio V F

    2016-04-01

    Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular proteins, such as neutrophil elastase (NE). NETs are released in the extracellular space in response to different stimuli. Carrageenan is a sulfated polysaccharide extracted from Chondrus crispus, a marine algae, used for decades in research for its potential to induce inflammation in different animal models. In this study, we show for the first time that carrageenan injection can induce NET release in a mouse model of acute peritonitis. Carrageenan induced NET release by viable neutrophils with NE and myeloperoxidase (MPO) expressed on DNA fibers. Furthermore, although this polysaccharide was able to stimulate reactive oxygen species (ROS) generation by peritoneal neutrophils, NADPH oxidase derived ROS were dispensable for NET formation by carrageenan. In conclusion, our results show that carrageenan-induced inflammation in the peritoneum of mice can induce NET formation in an ROS-independent manner. These results may add important information to the field of inflammation and potentially lead to novel anti-inflammatory agents targeting the production of NETs. PMID:26786873

  15. Increased NHC Cells in the Peritoneal Cavity of Plasmacytoma Susceptible BALB/c Mouse.

    PubMed

    Sánchez-González, Berenice; García-Vázquez, Francisco Javier; Farfán-Morales, José Eduardo; Jiménez-Zamudio, Luis Antonio

    2015-01-01

    BALB/c strain mice are unique in that they develop murine plasmacytoma (MPC) as a consequence of the inflammation induced by pristane oil injection in the peritoneal cavity. In this work the Treg, Th17, B1, B2, and NHC lymphocyte populations from the peritoneal environment of BALB/c, the susceptible strain, and C57BL/6 mice, which do not develop MPC after oil treatment, were studied. Both oil-treated strains showed decreased levels of Th17 lymphocytes, no significant variation in Treg lymphocytes, and a drastic decrease of all B lymphocyte populations. However, only oil-induced BALB/c showed increased levels of natural helper cells (NHC) which could be important in the myeloma induction. PMID:26504358

  16. Increased NHC Cells in the Peritoneal Cavity of Plasmacytoma Susceptible BALB/c Mouse

    PubMed Central

    Sánchez-González, Berenice; García-Vázquez, Francisco Javier; Farfán-Morales, José Eduardo; Jiménez-Zamudio, Luis Antonio

    2015-01-01

    BALB/c strain mice are unique in that they develop murine plasmacytoma (MPC) as a consequence of the inflammation induced by pristane oil injection in the peritoneal cavity. In this work the Treg, Th17, B1, B2, and NHC lymphocyte populations from the peritoneal environment of BALB/c, the susceptible strain, and C57BL/6 mice, which do not develop MPC after oil treatment, were studied. Both oil-treated strains showed decreased levels of Th17 lymphocytes, no significant variation in Treg lymphocytes, and a drastic decrease of all B lymphocyte populations. However, only oil-induced BALB/c showed increased levels of natural helper cells (NHC) which could be important in the myeloma induction. PMID:26504358

  17. Deletion of scavenger receptor A gene in mice resulted in protection from septic shock and modulation of TLR4 signaling in isolated peritoneal macrophages

    PubMed Central

    Drummond, Robert; Cauvi, David M; Hawisher, Dennis; Song, Donghuan; Niño, Diego F; Coimbra, Raul; Bickler, Stephen; De Maio, Antonio

    2015-01-01

    Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(−/−) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(−/−) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMϕs) were isolated from Sra(−/−) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMϕs isolated from B6 mice. No differences were found in LPS binding between PMϕs derived from Sra(−/−) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity. PMID:22751446

  18. In vitro macrophage cytotoxicity of five calcium silicates.

    PubMed Central

    Skaug, V; Davies, R; Gylseth, B

    1984-01-01

    Five calcium silicate minerals (two naturally occurring and three synthetic compounds) with defined morphology and chemical composition were compared for their cytotoxic and lysosomal enzyme releasing effects on unstimulated mouse peritoneal macrophages in vitro. One synthetic material, a fibrous tobermorite, was cytotoxic towards the cells, and two naturally occurring wollastonites induced selective release of beta-glucuronidase from the cells. Images PMID:6318798

  19. Enhancer Turnover Is Associated with a Divergent Transcriptional Response to Glucocorticoid in Mouse and Human Macrophages.

    PubMed

    Jubb, Alasdair W; Young, Robert S; Hume, David A; Bickmore, Wendy A

    2016-01-15

    Phenotypic differences between individuals and species are controlled in part through differences in expression of a relatively conserved set of genes. Genes expressed in the immune system are subject to especially powerful selection. We have investigated the evolution of both gene expression and candidate enhancers in human and mouse macrophages exposed to glucocorticoid (GC), a regulator of innate immunity and an important therapeutic agent. Our analyses revealed a very limited overlap in the repertoire of genes responsive to GC in human and mouse macrophages. Peaks of inducible binding of the GC receptor (GR) detected by chromatin immunoprecipitation-Seq correlated with induction, but not repression, of target genes in both species, occurred at distal regulatory sites not promoters, and were strongly enriched for the consensus GR-binding motif. Turnover of GR binding between mice and humans was associated with gain and loss of the motif. There was no detectable signal of positive selection at species-specific GR binding sites, but clear evidence of purifying selection at the small number of conserved sites. We conclude that enhancer divergence underlies the difference in transcriptional activation after GC treatment between mouse and human macrophages. Only the shared inducible loci show evidence of selection, and therefore these loci may be important for the subset of responses to GC that is shared between species. PMID:26663721

  20. Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

    PubMed

    Manna, Pulak R; Sennoune, Souad R; Martinez-Zaguilan, Raul; Slominski, Andrzej T; Pruitt, Kevin

    2015-08-14

    Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease. PMID:26119689

  1. Extrathyroidal release of thyroid hormones from thyroglobulin by J774 mouse macrophages.

    PubMed Central

    Brix, K; Herzog, V

    1994-01-01

    Thyroglobulin appears in the circulation of vertebrates at species-specific concentrations. We have observed that the clearance of thyroglobulin from the circulation occurs in the liver by macrophages. Here we show that the thyroid hormones T3 and T4 were released by incubation of mouse macrophages (J774) with thyroglobulin. Thyroid hormone release was a fast process, with an initial rate of approximately 20 pmol T4/mg per min and approximately 0.6 pmol T3/mg per min, indicating that macrophages preferentially release T4. The bulk of released thyroid hormones appeared after 5 min of incubation of macrophages with thyroglobulin, whereas degradation of the protein was detectable only after several hours. During internalization of thyroglobulin, endocytic vesicles and endosomes were reached at 5 min and lysosomes at 60 min. T4 release started extracellularly by secreted proteases and continued along the endocytic pathway of thyroglobulin, whereas T3 release occurred mainly intracellularly when thyroglobulin had reached the lysosomes. This shows that the release of both hormones occurred at distinct cellular sites. Our in vitro observations suggest that macrophages in situ represent an extrathyroidal source for thyroid hormones from circulating thyroglobulin. Images PMID:8163643

  2. Search for potent modulators of cytokine production by macrophages.

    PubMed

    Nikitin, A A; Abidov, M T; Kovalevskaya, E O; Kalyuzhin, O V

    2004-09-01

    We compared the effects of Tamerit, Polyoxidony, and Licopid on spontaneous and lipopolysaccharide-stimulated production of interleukin-1 and tumor necrosis factor by mouse peritoneal macrophages in vitro. The test preparations were equally potent in stimulating nonactivated cells. Licopid produced a costimulatory effect on macrophages primed with endotoxin. Tamerit in different doses suppressed cytokine production by cells. Polyoxidony in low doses activated, but in high doses suppressed this process. PMID:15665918

  3. Isolation and partial characterization of a pectic polysaccharide from the fruit pulp of Spondias cytherea and its effect on peritoneal macrophage activation.

    PubMed

    Iacomini, Marcello; Serrato, Rodrigo V; Sassaki, Guilherme L; Lopes, Luciana; Buchi, Dorly F; Gorin, Phillip A J

    2005-12-01

    The total carbohydrate content of the intact pulp of Spondias cytherea was 41%. Polysaccharides were obtained via hot aqueous extraction after defatting with organic solvents. The aqueous extract was treated with excess ethanol to form a precipitate, which was then solubilized in water. The material precipitated upon acidification when HCl was removed. The resulting supernatant fraction was submitted to freeze-thawing treatment yielding a soluble fraction (sFTS). This fraction had Ara, Rha, Gal and GalA in its structure as determined by GC-MS. 13C NMR analysis showed signals assigned to alpha-L-Araf, beta-D-Galp, alpha-D-GalpA and alpha-L-Rhap units, in addition to galacturonic acid units, which were present also as methyl ester. These results suggest a type I rhamnogalacturonan with arabinogalactan branches. Cell eliciting activity in a dose-depending pattern was observed in vitro on peritoneal macrophages treated with sFTS. PMID:16239076

  4. Sulforaphane promotes immune responses in a WEHI‑3‑induced leukemia mouse model through enhanced phagocytosis of macrophages and natural killer cell activities in vivo.

    PubMed

    Shih, Yung-Luen; Wu, Lung-Yuan; Lee, Ching-Hsiao; Chen, Yung-Liang; Hsueh, Shu-Ching; Lu, Hsu-Feng; Liao, Nien-Chieh; Chung, Jing-Gung

    2016-05-01

    Sulforaphane (SFN) is an isothiocyanate, inducing cytotoxic effects in various human cancer cells, including leukemia cells through cell cycle arrest and apoptosis. However, the effect of SFN on the immune responses in a leukemia mouse model remains to be investigated. The present study investigated whether SFN has an effect on the immune responses in a WEHI‑3‑induced leukemia mouse model in vivo. Normal BALB/c mice were injected with WEHI‑3 cells to generate the leukemia mouse model, and were subsequently treated with placebo or SFN (0, 285, 570 and 1,140 mg/kg) for 3 weeks. Following treatment, all mice were weighted and blood samples were collected. In addition, liver and spleen samples were isolated to determine cell markers, phagocytosis and natural killer (NK) cell activities, and cell proliferation was examined using flow cytometry. The results indicated that SFN treatment had no significant effect on the spleen weight, however it decreased liver and body weight. Furthermore, SFN treatment increased the percentage levels of CD3 (T cells) and CD19 (B cell maker), however had no effect on the levels of CD11b (monocytes) or Mac‑3 (macrophages), compared with the WEHI‑3 control groups. The administration of SFN increased the phagocytosis of macrophages from peripheral blood mononuclear cells and peritoneal cavity, and increased the activity of NK cells from splenocytes. Administration of SFN promoted T and B cell proliferation following stimulation with concanavalin A and lipopolysaccharide, respectively. PMID:27035756

  5. Immunomodulatory effects of sulfated polysaccharides of pine pollen on mouse macrophages.

    PubMed

    Geng, Yue; Xing, Li; Sun, Mengmeng; Su, Fangchen

    2016-10-01

    This study was undertaken to explore the effects of sulfated polysaccharide (SPPM60-D) from masson pine pollen on [Ca(2+)]i and immune function of RAW264.7 macrophages. SPPM60-D was obtained by subjecting Masson pine pollen to boiling water and alcohol precipitation, 60% ethanol grading precipitation, Sephacryl S-400HR purification, and chlorosulfonic acid-pyridine method sulfation. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the effect of SPPM60-D on relative activity and proliferation of RAW264.7 cells, and a fluorescence spectrophotometer was used to determine [Ca(2+)]i. Phagocytosis of neutral red was used to determine phagocytosis capacity. Adherence, scratch healing, and transwell assays were used to assess migration and adhesion abilities of macrophages. An enzyme-linked immuno sorbent assay (ELISA) assay was used to assess the secretion of cytokines and inflammatory mediators. A dexamethasone (DEX) inhibition method was used to measure the recovery of RAW264.7 immune activity by SPPM60-D. SPPM60-D significantly increased relative activity, proliferation, and [Ca(2+)]i levels of mouse RAW264.7 cells. It also significantly enhanced the immune function of macrophages from normal and immune-suppressed mouse. The results showed that SPPM60-D mainly bound to TLR4 on macrophages. This activated the TLR4-PI3K-PLC-IP3R signaling pathway, leading to the opening of calcium release-activated calcium channels (CRAC), increasing [Ca(2+)]i and activating the macrophages, and thus improving immunity. PMID:27288698

  6. Apigenin Induces the Apoptosis and Regulates MAPK Signaling Pathways in Mouse Macrophage ANA-1 Cells

    PubMed Central

    Liao, Yuexia; Shen, Weigan; Kong, Guimei; Lv, Houning; Tao, Wenhua; Bo, Ping

    2014-01-01

    Apigenin is a naturally occurring plant flavonoid that possesses antioxidant, anti-cancer and anti-inflammatory properties. However, there are few reports has been done on the ability of apigenin to induce apoptosis in macrophages. In this study, mouse macrophage ANA-1 cells were incubated with different concentrations of apigenin. The cell viability was determined by an MTT assay. The cell apoptosis were analyzed by flow cytometric analysis. Apoptosis were also analyzed using a TUNEL assay and a DNA ladder. The level of intracellular ROS was detected using a dichlorofluorescein -diacetate probe. The expression levels of apoptosis-related proteins were detected by western blot analysis. The results showed that apigenin decreased the viability of ANA-1 cells and induced apoptosis in a dose- and time-dependent manner. Apigenin increased the level of intracellular ROS, downregulated the expression of Bcl-2 and upregulated the expression of caspase-3 and caspase-8 in ANA-1 cells. Furthermore, apigenin downregulated the expression of phospho-ERK and phospho-JNK, upregulated the expression of phospho-p38 and had no significant effect on the expression of Bax, ERK, JNK and p38. The results suggested that apigenin induced cell apoptosis in mouse macrophage ANA-1 cells may via increasing intracellular ROS, regulating the MAPK pathway, and then inhibiting Bcl-2 expression. PMID:24646936

  7. Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs

    NASA Astrophysics Data System (ADS)

    Krenkel, Martin; Markus, Andrea; Bartels, Matthias; Dullin, Christian; Alves, Frauke; Salditt, Tim

    2015-05-01

    We have performed x-ray phase-contrast tomography on mouse lung tissue. Using a divergent x-ray beam generated by nanoscale focusing, we used zoom tomography to produce three-dimensional reconstructions with selectable magnification, resolution, and field of view. Thus, macroscopic tissue samples extending over several mm can be studied in sub-cellular-level structural detail. The zoom capability and, in particular, the high dose efficiency are enabled by the near-perfect exit wavefront of an optimized x-ray waveguide channel. In combination with suitable phase-retrieval algorithms, challenging radiation-sensitive and low-contrast samples can be reconstructed with minimal artefacts. The dose efficiency of the method is demonstrated by the reconstruction of living macrophages both with and without phagocytized contrast agents. We also used zoom tomography to visualize barium-labelled macrophages in the context of morphological structures in asthmatic and healthy mouse lung tissue one day after intratracheal application. The three-dimensional reconstructions showed that the macrophages predominantly localized to the alveoli, but they were also found in bronchial walls, indicating that these cells might be able to migrate from the lumen of the bronchi through the epithelium.

  8. Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs

    PubMed Central

    Krenkel, Martin; Markus, Andrea; Bartels, Matthias; Dullin, Christian; Alves, Frauke; Salditt, Tim

    2015-01-01

    We have performed x-ray phase-contrast tomography on mouse lung tissue. Using a divergent x-ray beam generated by nanoscale focusing, we used zoom tomography to produce three-dimensional reconstructions with selectable magnification, resolution, and field of view. Thus, macroscopic tissue samples extending over several mm can be studied in sub-cellular-level structural detail. The zoom capability and, in particular, the high dose efficiency are enabled by the near-perfect exit wavefront of an optimized x-ray waveguide channel. In combination with suitable phase-retrieval algorithms, challenging radiation-sensitive and low-contrast samples can be reconstructed with minimal artefacts. The dose efficiency of the method is demonstrated by the reconstruction of living macrophages both with and without phagocytized contrast agents. We also used zoom tomography to visualize barium-labelled macrophages in the context of morphological structures in asthmatic and healthy mouse lung tissue one day after intratracheal application. The three-dimensional reconstructions showed that the macrophages predominantly localized to the alveoli, but they were also found in bronchial walls, indicating that these cells might be able to migrate from the lumen of the bronchi through the epithelium. PMID:25966338

  9. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide.

    PubMed Central

    Stuehr, D J; Marletta, M A

    1985-01-01

    Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H/He and LPS-resistant C3H/HeJ mice. Intraperitoneal injection of 15 micrograms of LPS led to a temporary 5- to 6-fold increase in blood nitrate concentration in the C3H/He strain. Levels of nitrate excreted in the urine were also increased. In contrast, no increase was observed in the C3H/HeJ strain with LPS injections up to 175 micrograms. Furthermore, thioglycolate-elicited peritoneal macrophages from C3H/He, but not from C3H/HeJ mice, produced nitrite (60%) and nitrate (40%) when cultured with LPS (10 micrograms/ml). T-lymphocyte addition/depletion experiments showed the presence of T cells enhanced this response. However, LPS did not cause nitrite or nitrate production in cultures of spleen lymphocytes from either strain. LPS-induced nitrate synthesis was also observed with nude mice and CBA/N mice, indicating that neither functional T lymphocytes nor LPS-responsive B lymphocytes were required for the response in vivo. This was consistent with the in vitro results showing macrophages alone were competent. Mycobacterium bovis infection of C3H/He and C3H/HeJ mice resulted in a large increase in nitrate production over the course of the infection for both strains, suggesting T-lymphocyte-mediated activation of macrophages as a potent stimulus for nitrate biosynthesis. The synthesis of nitrite is significant in that it can directly participate in the endogenous formation of nitrosamines and may also be involved in some aspect of the chemistry of cytotoxicity. PMID:3906650

  10. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide

    SciTech Connect

    Stuehr, D.J.; Marletta, M.A.

    1985-11-01

    Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H/He and LPS-resistant C3H/HeJ mice. Intraperitoneal injection of 15 ..mu..g of LPS led to a temporary 5- to 6-fold increase in blood nitrate concentration in the C3H/He strain. Levels of nitrate excreted in the urine were also increased. In contrast, no increase was observed in the C3H/HeJ strain with LPS injections up to 175 ..mu..g. Furthermore, thioglycolate-elicited peritoneal macrophages from C3H/He, but not from C3H/HeJ mice, produced nitrite (60%) and nitrate (40%) when cultured with LPS (10 ..mu..g/ml). T-lymphocyte addition/depletion experiments showed the presence of T cells enhanced this response. However, LPS did not cause nitrite or nitrate production in cultures of spleen lymphocytes from either strain. LPS-induced nitrate synthesis was also observed with nude mice and CBA/N mice, indicating that neither functional T lymphocytes nor LPS-responsive B lymphocytes were required for the response in vivo. This was consistent with the in vitro results showing macrophages alone were competent. Mycobacterium bovis infection of C3H/He and C3H/HeJ mice resulted in a large increase in nitrate production over the course of the infection for both strains, suggesting T-lymphocyte-mediated activation of macrophages as a potent stimulus for nitrate biosynthesis. The synthesis of nitrite is significant in that it can directly participate in the endogenous formation of nitrosamines and may also be involved in some aspect of the chemistry of cytotoxicity.

  11. Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways.

    PubMed

    Cárdeno, A; Sánchez-Hidalgo, M; Aparicio-Soto, M; Sánchez-Fidalgo, S; Alarcón-de-la-Lastra, C

    2014-06-01

    Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree Olea europaea L. Phenolic compounds present in EVOO have recognized anti-oxidant and anti-inflammatory properties. However, the activity of the total phenolic fraction extracted from EVOO and the action mechanisms involved are not well defined. The present study was designed to evaluate the potential anti-inflammatory mechanisms of the polyphenolic extract (PE) from EVOO on LPS-stimulated peritoneal murine macrophages. Nitric oxide (NO) production was analyzed by the Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. Moreover, changes in the protein expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), as well as the role of nuclear transcription factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signalling pathways, were analyzed by Western blot. PE from EVOO reduced LPS-induced oxidative stress and inflammatory responses through decreasing NO and ROS generation. In addition, PE induced a significant down-regulation of iNOS, COX-2 and mPGES-1 protein expressions, reduced MAPK phosphorylation and prevented the nuclear NFκB translocation. This study establishes that PE from EVOO possesses anti-inflammatory activities on LPS-stimulated murine macrophages. PMID:24740524

  12. Activation of calcium-insensitive phospholipase A(2) (iPLA(2)) by P2X(7) receptors in murine peritoneal macrophages.

    PubMed

    El Ouaaliti, M; Seil, M; Dehaye, J P

    2012-12-01

    Free fatty acid releases are triggered by PLA2 activation and are substrates for many enzymes such as cyclooxygenases. These reactions are responsible for the production of many prostaglandins implicated in the inflammation yet many purinergic receptors have been implicated in diseases characterised by chronic inflammation. The role of P2X receptors was evaluated in LPS-primed murine peritoneal macrophages which were labelled with either [(3)H]-oleic acid or [(3)H]-arachidonic acid. Ten μmolar thapsigargin and 1mM ATP stimulated the release of both unsaturated acids. ATP had no effect at 10 μM and ivermectin had no effect on the response to ATP. The response to ATP was inhibited by magnesium and was not observed with cells from P2X(7)(-/-) mice. The response to ATP was not affected by the removal of extracellular calcium and was inhibited by arachidonyltrifluoromethyl ketone and bromoenol lactone but not by pyrrophenone. The release of the [(3)H]-fatty acids by ATP and thapsigargin was diminished by PD-98058, an inhibitor of MEK-1. It was concluded that in LPS-primed macrophages, P2X(7) receptors, not P2X(4) receptors, activated an iPLA(2) and promoted the release of unsaturated fatty acids secondary to the activation of a kinase. This response might contribute to the inflammation provoked by extracellular ATP. PMID:23041292

  13. Transcriptional regulation of mouse granulocyte-macrophage colony-stimulating factor/IL-3 locus

    SciTech Connect

    Osborne, C.S.; Vadas, M.A.; Cockerill, P.N.

    1995-07-01

    Granulocyte-macrophage (GM)-CSF and IL-3 are hemopoietic growth factors whose genes are closely linked in both humans and mice. In humans, the GM-CSF and IL-3 genes are regulated by a cyclosporin A-inhibitable enhancer located 3 kb upstream of the GM-CSF gene that is inducible by signals that mimic TCR activation. To search for a murine homologue of this enhancer we probed mouse genomic DNA and located a 400-bp element 2 kb upstream of the mouse GM-CSF gene that was 76% homologous with the human GM-CSF enhancer. Like the human GM-CSF enhancer, this element formed a cyclosporin A-inhibitable DNase I-hypersensitive site in the murine T cell line EL4 upon activation with phorbol ester and calcium ionophore. Transient transfection assays showed that this homologue of the human enhancer acted as an inducible enhancer of the thymidine kinase promoter, the mouse IL-3 promoter, and the human GM-CSF promoter. We observed, however, that the mouse GM-CSF promoter was significantly more active than the human GM-CSF promoter and found that it supported a level of activity equivalent to the combination of the human GM-CSF promoter and the human GM-CSF enhancer. Consequently, the activity of mouse GM-CSF promoter was not significantly elevated in the presence of the mouse GM-CSF enhancer. Because the mouse GM-CSF enhancer is considerably less active than its human homologue we suggest that the mouse GM-CSF gene has evolved with less dependence upon the upstream enhancer for its activation. 53 refs., 6 figs.

  14. Effects of red clover extract on the activation and proliferation of mouse T lymphocytes and the NO secretion of mouse macrophages.

    PubMed

    Yang, Zhi; Huang, Xiu-yan; Zeng, Yao-ying

    2008-10-01

    The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide (NO) secretion of mouse macrophages stimulated by lipopolysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had little cell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators (NO, CD69, CD25, CD71), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3+ T lymphocytes. These data suggested that RCE might exhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages. PMID:19127865

  15. Intracellular survival of wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse populations of macrophages.

    PubMed

    Buchmeier, N A; Heffron, F

    1989-01-01

    Salmonella typhimurium survives within macrophages and causes a fatal infection in susceptible strains of mice. A number of S. typhimurium mutants that contain Tn10 insertions in genes which are necessary for survival within the macrophage have been isolated. To demonstrate the importance of each gene in intracellular survival, the mutations were transduced into a smooth-strain background and the ability to survive intracellularly was assayed in five different populations of macrophages. The majority of the original macrophage-sensitive mutants retained the macrophage-sensitive phenotype in the smooth-strain background. The ability to survive or grow within macrophages varied with both the source of macrophages and the individual mutants. S. typhimurium grew best in the macrophage-like cell line J774, survived at moderate levels in splenic and bone marrow-derived macrophages, and was killed most efficiently in peritoneal macrophages. Macrophage-sensitive mutants transduced into a smooth background were also less virulent than the parent, with a 50% lethal dose of 2 to 5 logs greater than that of the parental strain. These experiments demonstrate that survival of S. typhimurium within macrophages varies with the source of cells, with a distinct ability to survive in macrophages from mouse spleens, where S. typhimurium grows rapidly. These experiments also demonstrate the heterogeneity in intracellular survival among the various macrophage-sensitive mutants, which may reflect the relative importance of the individual mutated genes in survival within macrophages. PMID:2642463

  16. A dual enzymatic-biosensor for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages of diabetic mice: evaluation of the diabetes-accelerated atherosclerosis risk.

    PubMed

    Huang, Qilin; An, Yarui; Tang, Linlin; Jiang, Xiaoli; Chen, Hua; Bi, Wenji; Wang, Zhongchuan; Zhang, Wen

    2011-11-30

    In this paper, a novel dual enzymatic-biosensor is described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. The biosensor was constructed by a three-step method. First, a poly-thionine (PTH) film was assembled on the surface of glassy carbon electrode by cyclic voltammetric electropolymerization of thionine, which serves as an electron transfer mediator (ETM). Second, gold nanoparticles (GNPs) were covered on the surface of PTH facilitating the electron transfer between glucose oxidase (GOx), cholesterol oxidase (ChOx) and electrode. Finally, the enzymes, GOx, cholesterol esterase (ChE), and ChOx, were covalently attached to the PTH layer through a chitosan (CH) linker. The PTH coupled with GNPs provides good selectivity, high sensitivity and little crosstalk for the dual enzymatic-biosensor. The developed biosensor had good electrocatalytic activity toward the oxidations of glucose and cholesterol, exhibiting a linear range from 0.008 mM to 6.0 mM for glucose with a detection limit of 2.0 μM, and a linear range from 0.002 mM to 1.0 mM for cholesterol with a detection limit of 0.6 μM. The results of the diabetic mice demonstrated that the cholesterol level did not change obviously with the increase of glucose level in serum, while the cholesterol level was induced with the increase of the glucose level in PMs. Previous studies have shown that the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, which is the hallmark of early atherosclerosis. This study provides useful further evidences for the development of diabetes-accelerated atherosclerosis. PMID:22027130

  17. Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response

    PubMed Central

    Ojeda-Fernández, Luisa; Recio-Poveda, Lucía; Aristorena, Mikel; Lastres, Pedro; Blanco, Francisco J.; Sanz-Rodríguez, Francisco; Gallardo-Vara, Eunate; de las Casas-Engel, Mateo; Corbí, Ángel; Arthur, Helen M.; Bernabeu, Carmelo; Botella, Luisa M.

    2016-01-01

    Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Engfl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Engfl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients. PMID:27010826

  18. Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.

    PubMed

    Braza, Faouzi; Dirou, Stéphanie; Forest, Virginie; Sauzeau, Vincent; Hassoun, Dorian; Chesné, Julie; Cheminant-Muller, Marie-Aude; Sagan, Christine; Magnan, Antoine; Lemarchand, Patricia

    2016-07-01

    Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti-inflammatory therapy in allergic asthma. However, the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)-induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f-sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and decreased ex vivo carbachol-induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To evaluate in vivo MSC survival, MSCs were labeled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digested to obtain single-cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26(+) lung cells expressed specific macrophage markers in control and Der f mice, respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26(+) macrophages expressed M2 phenotype, while the innate PKH26(-) macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26(+) MSCs expressed 10- to 100-fold more COX-2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. Stem Cells 2016;34:1836-1845. PMID:26891455

  19. ROCK inhibition impedes macrophage polarity and functions.

    PubMed

    Liu, Yianzhu; Tejpal, Neelam; You, Junping; Li, Xian C; Ghobrial, Rafik M; Kloc, Malgorzata

    2016-02-01

    Macrophages play an important role in immune responses including allograft rejection and they are one of the potential targets of anti-rejection therapies in organ transplantation. Macrophage alloreactivity relies on their phenotype/polarity, motility, phagocytosis and matrix degradation, which in turn depend on proper functioning of actin cytoskeleton and its regulators, the small GTPase RhoA and its downstream effector the Rho-associated protein kinase (ROCK). Several laboratories showed that administration of ROCK inhibitor Y-27632 to the graft recipient inhibits chronic rejection or rodent cardiac allografts. Here we studied the effect of Y-27632 on mouse peritoneal macrophage structure, polarity and functions in in vitro assays. We show that Y-27632 inhibitor affects macrophage phenotype/polarity, phagocytosis, migration, and matrix degradation. These novel findings suggest that the impediment of macrophage structure and function via interference with the RhoA/ROCK pathway has a potential to be therapeutically effective in organ transplantation. PMID:26711331

  20. Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes

    SciTech Connect

    Bozza, M.; Gerard, C.; Kolakowski, L.F. Jr.

    1995-06-10

    Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5{prime} untranslated region of the cDNA, a 3{prime} poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. 46 refs., 5 figs., 1 tab.

  1. Characterization of cellular response to thiol-modified gold surfaces implanted in mouse peritoneal cavity.

    PubMed

    Nygren, H; Kanagaraja, S; Braide, M; Eriksson, C; Lundström, I

    1999-05-01

    The early inflammatory reaction in vivo to three well defined surfaces-gold, gold coated with glutathione (GSH), and 3-mercapto-1, 2-propanediol (MG)-was assessed as manifested by the adherence and activation of inflammatory cells during implantation intraperitoneally in mice. Evaluation of cell adhesion and activation was done by immunohistochemistry using specific monoclonal antibodies directed against cell differentiation antigens CD11b/CD18, CD74, and CD25 or by measurement by chemoluminescence of reactive oxygen radical species produced by adhering cells. Cell recruitment and activation was slow on the GSH-coated gold surfaces. These surfaces also had the highest percentage of adhering cells with an intact cell membrane. The MG-coated surfaces, on the other hand, rapidly recruited and activated cells and also caused cell membrane leakage to propidium iodide, suggesting cell membrane damage or cell death. The respiratory burst of adhering cells was stimulated by phorbol-myristate acetate on the GSH-coated surface but not on the MG-coated surface and by opsonized zymosan on the Mg-coated surface but only to a small degree on the GSH-coated surface. The respiratory burst following zymosan activation of cells adhering to the MG-coated surface was inhibited by treatment with 2. 3-diphosphoglycerate, a phospholipase D inhibitor. The presented data suggest that peritoneal leukocytes adhering to foreign materials may raise a respiratory burst response via a phospholipase D-dependent and protein kinase C-independent pathway. PMID:10397965

  2. Sorting nexin 10 acting as a novel regulator of macrophage polarization mediates inflammatory response in experimental mouse colitis

    PubMed Central

    You, Yan; Zhou, Chun; Li, Dong; Cao, Zhong-Lian; Shen, Weixing; Li, Wan-Zhen; Zhang, Sulin; Hu, Bin; Shen, Xiaoyan

    2016-01-01

    In response to changes in microenvironment, macrophages polarize into functionally distinct phenotypes, playing a crucial role in the pathogenesis of inflammatory bowel disease (IBD). Here, we investigated the effects of sorting nexin 10 (SNX10), a protein involved in endosomal trafficking and osteoclast maturation, on regulation of macrophage polarization and progression of mouse colitis. Our results revealed that SNX10 deficiency increased the population of M2-type monocytes/macrophages, and protected against colonic inflammation and pathological damage induced by dextran sulfate sodium (DSS). By in vitro study, we showed that deficiency of SNX10 polarized macrophages derived from mouse bone marrow or human peripheral blood mononuclear cells (PBMCs) towards an anti-inflammatory M2 phenotype, which partially reversed by SNX10 plasmid transfection. Adoptive transfer of SNX10−/− macrophages ameliorated colitis in WT mice. However, transfer of WT macrophages exacerbated colitis in SNX10−/− mice. Our data disclose a crucial role and novel function for SNX10 in macrophage polarization. Loss of SNX10 function may be a potential promising therapeutic strategy for IBD. PMID:26856241

  3. TIGIT negatively regulates inflammation by altering macrophage phenotype.

    PubMed

    Chen, Xi; Lu, Pu-Han; Liu, Lei; Fang, Ze-Min; Duan, Wu; Liu, Zhe-Long; Wang, Cong-Yi; Zhou, Ping; Yu, Xue-Feng; He, Wen-Tao

    2016-01-01

    Macrophages function as an essential component of innate immune system, contributing to both the initiation and appropriate resolution of inflammation. The exposure of macrophages to the microbial products, such as lipopolysaccharide (LPS), can strongly shift the balance between tissue homeostasis and inflammation in favor of causing systemic damage, in which macrophage M1 polarization play important roles. Strategies aiming at restoring the balance of macrophage polarization remain to be further explored. Herein, we have demonstrated that poliovirus receptor (PVR), the receptor of TIGIT, was dramatically upregulated on the surface of mouse peritoneal macrophages when exposed to LPS. TIGIT-Fc fusion protein not only inhibited the macrophage activation, but also skewed M1/M2 balance toward an anti-inflammatory profile, especially enhanced the secretion of IL-10. The activation of TIGIT/PVR pathway in macrophages correlated with increased nuclear translocation of c-Maf, which promotes IL-10 transcription. Treatment with fibroblasts stably secreting TIGIT-Fc fusion protein significantly reversed the lethal and sublethal endotoxic shock, which facilitated peritoneal macrophages to switch towards anti-inflammatory M2 cytokine profiles. These findings highlight a novel role of the TIGIT/PVR pathway in macrophage M2 polarization and suggest that TIGIT may have the potential to optimize the treatment of macrophage-involved inflammatory diseases. PMID:26307002

  4. Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells.

    PubMed

    Rafi, Mohamed M; Yadav, Prem Narayan; Reyes, Marynell

    2007-01-01

    Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 microM) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene. PMID:17995901

  5. Effects of Bothrops asper snake venom on the expression of cyclooxygenases and production of prostaglandins by peritoneal leukocytes in vivo, and by isolated neutrophils and macrophages in vitro.

    PubMed

    Moreira, Vanessa; Gutiérrez, José María; Amaral, Rafaela Bacci; Zamunér, Stella Regina; Teixeira, Catarina de Fátima Pereira

    2009-01-01

    In this study, the ability of Bothrops asper snake venom (BaV) to increase the production of prostaglandins PGE(2) and PGD(2) was assessed in a mouse model in vivo and in inflammatory cells in vitro. In addition, the expressions of COX-1 and COX-2 were assessed. BaV induced an increment in the in vivo synthesis of PGE(2) and PGD(2), together with an enhanced expression of COX-2, but not of COX-1. However, enzymatic activities of COX-1 and COX-2 were increased. Incubation of isolated macrophages and neutrophils with a sub-cytotoxic concentration of BaV in vitro resulted in increased release of PGE(2) and PGD(2) by macrophages and PGE(2) by neutrophils, concomitantly with an increment in the expression of COX-2, but not of COX-1 by both cell types. Our results demonstrate the ability of BaV to promote the expression of COX-2 and to induce the synthesis of proinflammatory prostaglandins. Macrophages and neutrophils may be important targets for this venom under in vivo situation. PMID:19155166

  6. Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease

    PubMed Central

    Vinchi, Francesca; Costa da Silva, Milene; Ingoglia, Giada; Petrillo, Sara; Brinkman, Nathan; Zuercher, Adrian; Cerwenka, Adelheid; Tolosano, Emanuela

    2016-01-01

    Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease. PMID:26675351

  7. Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease.

    PubMed

    Vinchi, Francesca; Costa da Silva, Milene; Ingoglia, Giada; Petrillo, Sara; Brinkman, Nathan; Zuercher, Adrian; Cerwenka, Adelheid; Tolosano, Emanuela; Muckenthaler, Martina U

    2016-01-28

    Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease. PMID:26675351

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

    PubMed

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

    2016-01-01

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

  9. Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages

    PubMed Central

    Li, Ning; Sun, Jing; Benet, Zachary L.; Wang, Ze; Al-Khodor, Souhaila; John, Sinu P.; Lin, Bin; Sung, Myong-Hee; Fraser, Iain D. C.

    2015-01-01

    Macrophages play a critical role in the innate immune response to pathogen infection, but few tools exist for systematic dissection of these responses using modern genome-wide perturbation methods. To develop an assay platform for high-throughput analysis of macrophage activation by pathogenic stimuli, we generated reporter systems in human and mouse macrophages with dynamic readouts for NF-κB and/or TNF-α responses. These reporter cells show responsiveness to a broad range of TLR ligands and to gram-negative bacterial infection. There are significant challenges to the use of RNAi in innate immune cells, including efficient small RNA delivery and non-specific immune responses to dsRNA. To permit the interrogation of the macrophage pathogen response pathways with RNAi, we employed the stably expressed reporter genes to develop efficient siRNA delivery protocols for maximal target gene silencing with minimal activation of the innate macrophage response to nucleic acids. We demonstrate the utility of these macrophage cell systems for siRNA screening of pathogen responses by targeting components of the human and mouse TLR pathways, and observe species-specific perturbation of signaling and cytokine responses. Our approach to reporter cell development and siRNA delivery optimization provides an experimental paradigm with significant potential for developing genetic screening platforms in mammalian cells. PMID:25831078

  10. Echinacea Species and Alkamides Inhibit Prostaglandin E2 Production in RAW264.7 Mouse Macrophage Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of prostaglandin E2 production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells was assessed with an enzyme immunoassay following treatments with Echinacea extracts or synthesized alkamides. Results indicated that ethanol extracts from E. angustifolia, E. pallida, E. simu...

  11. A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

    PubMed

    Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N

    1994-05-15

    Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages. PMID:8176226

  12. A Novel Polysaccharide in Insects Activates the Innate Immune System in Mouse Macrophage RAW264 Cells

    PubMed Central

    Ohta, Takashi; Ido, Atsushi; Kusano, Kie; Miura, Chiemi; Miura, Takeshi

    2014-01-01

    A novel water-soluble polysaccharide was identified in the pupae of the melon fly (Bactrocera cucurbitae) as a molecule that activates the mammalian innate immune response. We attempted to purify this innate immune activator using nitric oxide (NO) production in mouse RAW264 macrophages as an indicator of immunostimulatory activity. A novel acidic polysaccharide was identified, which we named “dipterose”, with a molecular weight of 1.01×106 and comprising nine monosaccharides. Dipterose was synthesized in the melon fly itself at the pupal stage. The NO-producing activity of dipterose was approximately equal to that of lipopolysaccharide, a potent immunostimulator. Inhibition of Toll-like receptor 4 (TLR4) led to the suppression of NO production by dipterose. Furthermore, dipterose induced the expression of proinflammatory cytokines and interferon β (IFNβ) and promoted the activation of nuclear factor kappa B (NF-κB) in macrophages, indicating that it stimulates the induction of various cytokines in RAW264 cells via the TLR4 signaling pathway. Our results thus suggest that dipterose activates the innate immune response against various pathogenic microorganisms and viral infections. This is the first identification of an innate immune-activating polysaccharide from an animal. PMID:25490773

  13. Effect of inhaled alpha-emitting nuclides on mouse alveolar macrophages

    SciTech Connect

    Talbot, R.J.; Nicholls, L.; Morgan, A.; Moores, S.R. )

    1989-08-01

    The effects of inhaled alpha emitters on the free cell population of the mouse lung were investigated up to 100 days after exposure. Groups of mice inhaled aerosols of {sup 238}PuO{sub 2}, {sup 239}PuO{sub 2}, or {sup 241}Am(NO{sub 3}){sub 3} to give alveolar deposits resulting in lung-averaged cumulative absorbed doses of about 20 Gy by the end of the study. Initially, with {sup 238}Pu most of the activity was associated with relatively few pulmonary alveolar macrophages (PAM), whereas with {sup 241}Am, all pulmonary alveolar macrophages were labeled and a substantial fraction was extracellular. The free cell population of the lung was sampled using bronchoalveolar lavage. The main parameters investigated were (a) the recovery and total numbers of free cells, including PAM, lymphocytes, and neutrophils; (b) the incidence of nuclear abnormalities in PAM (cells with more than one nucleus or with micronuclei); and (c) metabolic activation of PAM from measurements of their size and associated beta-glucuronidase activity. All three actinides produced depletions in total numbers of PAM, increased incidences of nuclear abnormalities, and metabolic activation of PAM, without a marked infiltration of inflammatory cells. Americium-241, which is distributed relatively uniformly in PAM, produced the most marked changes in that population and {sup 238}Pu, which gave the most inhomogeneous distribution of activity, produced the least.

  14. Colony stimulating factor-1 receptor signaling networks inhibit mouse macrophage inflammatory responses by induction of microRNA-21

    PubMed Central

    Caescu, Cristina I.; Guo, Xingyi; Tesfa, Lydia; Bhagat, Tushar D.; Verma, Amit; Zheng, Deyou

    2015-01-01

    Macrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721–regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721–regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21–regulated messenger RNAs revealed that 80% of the CSF-1–regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R–mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R–regulated miR-21 network that modulates macrophage polarization. PMID:25573988

  15. Coumarin or warfarin treatment of mice does not increase the microbicidal or tumoricidal capacities of macrophages.

    PubMed Central

    Filice, G. A.; Remington, J. S.

    1981-01-01

    Benzopyrones have been shown to affect several functions of macrophages. We examined the effects of two benzopyrones, coumarin and warfarin, on the capacity of mouse macrophages to inhibit microorganisms and tumour target cells. Mice were treated with daily i.v. doses of either drug. Then the mice were challenged with lethal doses of Toxoplasma gondii or peritoneal macrophages from these mice were challenged in vitro with T. gondii or tumour target cells Survival of coumarin or warfarin-treated mice challenged with T. gondii was similar to that of control mice. Multiplication of T gondii and growth of tumour target cells were similar in preparations of macrophages from coumarin-treated, warfarin-treated, or control mice and were inhibited in preparations of activated macrophages from Corynebacterium parvum-treated mice that served as positive controls. Under our experimental conditions, benzopyrones did not activate mouse macrophages. PMID:7236495

  16. Effect of bone marrow depletion on prostaglandin E-producing suppressor macrophages in mouse spleen

    SciTech Connect

    Shibata, Y.; Volkman, A.

    1985-12-01

    The i.p. injection of Corynebacterium parvum (CP) into CBA/J mice effected increases in macrophage colony-forming cells (M-CFC) when spleen cells were cultured with L cell culture filtrate as a source of colony-stimulating factor. Significant increases in phagocytic macrophages (M phi) with Fc receptors for IgG2a and IgG2b immune complexes were additionally noted among the spleen cells in these mice. These M phi effectively inhibited Con A-induced lymphocyte proliferation, probably reflecting a 10-fold increase above normal controls in prostaglandin E to 47 ng/3 X 10(6) spleen cells/ml. To determine whether the suppressor M phi are immediate derivatives of splenic M-CFC, we tried to induce suppressor M phi by the injection of CP into mice depleted of bone marrow M-CFC by the earlier administration of the bone-seeking isotope, 89Sr. This procedure reduced M-CFC in the bone marrow to less than 1% of normal for more than 30 days. Monocytes in the blood fell to 5% of normal by day 10 and were 30% on day 30. Levels of resident peritoneal M phi showed relatively little change in this period. By contrast, splenic M-CFC increased to 20-fold higher than the cold 88Sr controls. CP-induced suppressor M phi activity, however, was sharply reduced in 89Sr marrow-depleted mice on day 10, despite the striking increase in M-CFC. There was a threefold increase in the number of phagocytic M phi binding IgG2a immune complexes, with no significant increase in IgG2b binding M phi. The kinetics of recovery of suppressor M phi activity showed that on days 20, 30, and 50 after 89Sr injection the activities reached 20%, 30%, and 70% of the cold control, respectively, and correlated with the recovery of significant levels of M-CFC in the bone marrow. Taken together, these observations suggest that splenic M-CFC are not an immediate source of PGE-suppressor M phi in vivo.

  17. 3AE8: monoclonal antibody defining inflammatory macrophages in three species.

    PubMed

    Chen, Z; Yen, S E; Walker, W S

    1984-01-01

    A mouse monoclonal antibody (MAb 3AE8) of the IgG1 isotype was prepared against rabbit splenocytes and was found by indirect immunofluorescence and direct binding assays to react, in the rabbit, primarily with oil-induced peritoneal exudate macrophages (PEM phi). This MAb did not bind to rabbit T cells, B cells, polymorphonuclear leukocytes, or resident alveolar or peritoneal M phi but it did bind to a subpopulation of rabbit splenocytes with surface characteristics of null cells. The antibody also recognized mouse and rat PEM phi as well as the murine M phi cell lines P388D1 and IC-21. Consistent with findings in the rabbit, it did not bind to M phi obtained from the peritoneal cavities of rats or mice. The addition of MAb 3AE8 to mouse PEM phi caused a marked enhancement in the phagocytic uptake of erythrocyte target cells sensitized with a mouse antierythrocyte antiserum. PMID:6480022

  18. Macrophage permissiveness for Legionella pneumophila growth modulated by iron.

    PubMed Central

    Gebran, S J; Newton, C; Yamamoto, Y; Widen, R; Klein, T W; Friedman, H

    1994-01-01

    We have investigated the modulation of iron in two populations of macrophages which differ in susceptibility to Legionella pneumophila intracellular proliferation. Previously, we reported that thioglycolate-elicited peritoneal macrophages obtained from the inbred A/J mouse strain readily support the intracellular growth of L. pneumophila, while resident macrophages from the same strain do not. In this study, we show that A/J elicited macrophages exhibit markedly higher expression of transferrin receptor and intracellular iron content than A/J resident macrophages. Furthermore, apotransferrin and desferrioxamine inhibited the intracellular proliferation of L. pneumophila in elicited macrophages, and this suppression was reversed by the additions of Fe-transferrin or ferric nitrilotriacetate. Fe-transferrin and ferric nitrilotriacetate did not further increase the intracellular proliferation of L. pneumophila in thioglycolate-elicited macrophages. However, ferric citrate and ferric nitrilotriacetate stimulated in a dose-dependent manner the growth of L. pneumophila in resident macrophages. Furthermore, equimolar concentrations of desferrioxamine reversed the stimulatory effect of iron in these resident cells. These data provide evidence supporting the hypothesis that differences in susceptibility to L. pneumophila growth between permissive elicited macrophages and nonpermissive resident macrophages from the A/J mouse strain are due to intracellular availability of iron. PMID:8300214

  19. IL-33 enhances lipopolysaccharide-induced inflammatory cytokine production from mouse macrophages by regulating lipopolysaccharide receptor complex.

    PubMed

    Espinassous, Quentin; Garcia-de-Paco, Elvira; Garcia-Verdugo, Ignacio; Synguelakis, Monique; von Aulock, Sonja; Sallenave, Jean-Michel; McKenzie, Andrew N J; Kanellopoulos, Jean

    2009-07-15

    Bacterial LPS triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. However, once exposed to LPS, they become hyporesponsive to a subsequent endotoxin challenge. This phenomenon is defined as LPS desensitization or tolerance. Previous studies have identified some components of the biochemical pathways involved in negative modulation of LPS responses. In particular, it has been shown that the IL-1R-related protein ST2 could be implicated in LPS tolerance. The natural ligand of ST2 was recently identified as IL-33, a new member of the IL-1 family. In this study, we investigated whether IL-33 triggering of ST2 was able to induce LPS desensitization of mouse macrophages. We found that IL-33 actually enhances the LPS response of macrophages and does not induce LPS desensitization. We demonstrate that this IL-33 enhancing effect of LPS response is mediated by the ST2 receptor because it is not found in ST2 knockout mice. The biochemical consequences of IL-33 pretreatment of mouse macrophages were investigated. Our results show that IL-33 increases the expression of the LPS receptor components MD2 (myeloid differentiation protein 2) and TLR-4, the soluble form of CD14 and the MyD88 adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR. PMID:19553541

  20. Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

    PubMed Central

    Ebermann, Linda; Ruzsics, Zsolt; Guzmán, Carlos A.; van Rooijen, Nico; Casalegno-Garduño, Rosaely; Koszinowski, Ulrich; Čičin-Šain, Luka

    2012-01-01

    The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system. PMID:23271968

  1. Anti-inflammatory effects of oroxylin A on RAW 264.7 mouse macrophages induced with polyinosinic-polycytidylic acid

    PubMed Central

    LEE, JI YOUNG; PARK, WANSU

    2016-01-01

    Oroxylin A (5,7-dihydroxy-6-methoxy-2-phenylchromen-4-one; Baicalein 6-methyl ether) is an active flavonoid compound originally isolated from Scutellaria radix, which has been used to treat pulmonary infection in Korea, China, and Japan. Oroxylin A is known to possess dopamine reuptake inhibitor activity. However, the effects of oroxylin A on virus-induced macrophages has not been fully elucidated. In the present study, the anti-inflammatory effects of oroxylin A on double-stranded RNA-induced macrophages were examined. Production of nitric oxide (NO), various cytokines, as well as calcium release and the mRNA expression of signal transducer and activator of transcription 1 (STAT1) in dsRNA polyinosinic-polycytidylic acid (PIC)-induced RAW 264.7 mouse macrophages were evaluated. Oroxylin A restored the cell viability in PIC-induced RAW 264.7 mouse macrophages at concentrations of up to 50 µM. Additionally, oroxylin A significantly inhibited the production of nitric oxide (NO), interleukin (IL)-1α, IL-1β, IL-6, IL-10, interferon gamma-induced protein 10, granulocyte-colony stimulating factor (CSF), granulocyte macrophage-CSF, leukemia inhibitory factor (IL-6 class cytokine), lipopolysaccharide-induced CXC chemokine (LIX), monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T Expressed and Secreted, tumor necrosis factor-α, and vascular endothelial growth factor as well as calcium release and the mRNA expression of STAT1 in PIC-induced RAW 264.7 cells (P<0.05). Thus, the present results suggest that oroxylin A has anti-inflammatory properties, associated with its inhibition of NO, cytokines, chemokines and growth factors in PIC-induced macrophages via the calcium-STAT pathway. PMID:27347031

  2. Bacillus Calmette Guerin Induces Fibroblast Activation Both Directly and through Macrophages in a Mouse Bladder Cancer Model

    PubMed Central

    Lodillinsky, Catalina; Langle, Yanina; Guionet, Ariel; Góngora, Adrián; Baldi, Alberto; Sandes, Eduardo O.; Casabé, Alberto; Eiján, Ana María

    2010-01-01

    Background Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. Methodology/Principal Findings We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. Conclusions/Significance Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy. PMID:21042580

  3. NAC Attenuates LPS-Induced Toxicity in Aspirin-Sensitized Mouse Macrophages via Suppression of Oxidative Stress and Mitochondrial Dysfunction

    PubMed Central

    Raza, Haider; John, Annie; Shafarin, Jasmin

    2014-01-01

    Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects. PMID:25075522

  4. Sperm-macrophage interaction in the mouse: a quantitative assay in vitro using 111indium oxine-labeled sperm

    SciTech Connect

    Olive, D.L.; Weinberg, J.B.; Haney, A.F.

    1987-12-01

    The role of reproductive tract macrophages in contraception and reproductive failure has become widely recognized. However, in vitro analysis of sperm phagocytosis by macrophages has relied upon a semi-quantitative method of sperm counting that is of limited accuracy and reproducibility. We have developed an assay using murine sperm labeled with /sup 111/indium oxine, and results indicate the labeling to be rapid and efficient. Incorporation of /sup 111/indium into sperm increased the dose and sperm concentration and reached 90% maximal uptake after 15 min incubation, with maximal uptake occurring at 30 min. No decrease in sperm motility was noted with levels of oxine in excess of those required for significant labeling. Maximal labeling efficiency occurred in phosphate-buffered saline (PBS), with Dulbecco's modified Eagle's medium (DMEM) + 10% adult bovine serum (ABS) producing significantly less uptake. Label dissociation was detectable in PBS at room temperature, but at 37 degrees C in DMEM + 10% ABS, loss of label occurred at a rate of 23.5%/h. Addition of labeled sperm to murine macrophage monolayers under optimal conditions resulted in uptake of /sup 111/indium by macrophages, while free label was unincorporated. Results indicated assay specificity for macrophage-limited uptake, with insignificant label uptake by nonphagocytic murine fibroblasts and better sensitivity than sperm counting. Macrophages from Bacillus Calmette-Guerin (BCG)-infected mice resulted in a decrease in sperm uptake. Female macrophages showed greater capacity for sperm uptake than those of the male mouse. These initial studies demonstrated the utility of this model system in enhancing the understanding of sperm-macrophage interaction in the female reproductive tract.

  5. Blocking macrophage migration inhibitory factor activity alleviates mouse acute otitis media in vivo.

    PubMed

    Zhang, Jin; Xu, Min; Zheng, Qingyin; Zhang, Yan; Ma, Weijun; Zhang, Zhaoqiang

    2014-11-01

    This study was to investigate the role of macrophage migration inhibitory factor (MIF) in mouse acute otitis media (AOM), we hypothesize that blocking MIF activity will relieve mouse AOM. A mouse AOM model was constructed by injecting lipopolysaccharide (LPS) into the middle ear of C57BL/6 mice through the tympanic membrane (TM). MIF levels were measured by real-time PCR (RT-PCR) and ELISA after LPS application. Normal or AOM mice were given PBS or ISO-1 (MIF antagonist) every day for 10 days and the hearing levels were determined by measuring auditory brainstem response (ABR) threshold. After the ABR test finished, H&E staining was conducted and the inflammation was also measured by detecting interleukin (IL)-1β, tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) levels with RT-PCR and ELISA. TLR-4 expression was determined by western blotting and NF-κB activation was determined by electrophoretic mobility shift assays. Compared with the normal control, MIF levels in the middle ear of LPS-induced AOM mice were significant increased. The ABR results showed that mean ABR thresholds in ISO-1 treated AOM mice were significantly reduced compared with PBS treated AOM mice since day 7, indicating that ISO-1 treatment potentially improved the hearing levels of AOM mice. H&E staining showed that ISO-1 treatment could reduce the mucosal thickness of AOM mice. In ISO-1 treated mice, TLR-4 expression and levels of IL-1β, TNF-α and VEGF were significantly lower compared with PBS treated AOM mice. ISO-1 treatment also significantly inhibited NF-κB activation in AOM mice compared with PBS treated AOM mice. These results suggested that blocking the activity of MIF by ISO-1 could reduce the inflammation in AOM mice in which process TLR-4 and NF-κB were involved. The reduction in MIF activity is conducive to alleviate mouse AOM, which may serve as a potential therapeutic target for the treatment of AOM. PMID:25108100

  6. MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability

    PubMed Central

    Sun, Yang; Qin, Zhen; Li, Qi; Wan, Jing-jing; Cheng, Ming-he; Wang, Peng-yuan; Su, Ding-feng; Yu, Jian-guang; Liu, Xia

    2016-01-01

    Aim: MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages. Methods: Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3′-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip). Results: LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability. Conclusion: miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases. PMID:27063215

  7. Stimulation of beta-adrenoceptors inhibits calcium-dependent potassium-channels in mouse macrophages

    SciTech Connect

    Rosati, C.; Hannaert, P.; Dausse, J.P.; Braquet, P.; Garay, R.

    1986-12-01

    K/sup +/ efflux in mouse macrophages exhibited a rate constant (k/sub k/) of 0.67 +/- 0.04 (h)/sup -1/. This was strongly stimulated by increasing concentrations of the Ca/sup 2 +/ ionophore A23187 up to a maximal value of 4.01 +/- 0.25 (h)/sup -1/ with an IC/sub 50/ of 7.6 +/- 1.9 ..mu..M. Similar results were obtained with the Ca/sup 2 +/ ionophore ionomycin. Binding experiments with /sup 3/H-dihydroalprenolol revealed a high density of beta-adrenergic receptors with apparent dissociation constant of 2.03 +/- 0.06 nM. Isoproterenol at a concentration of 10/sup -6/ -10/sup -5/ M induced a two- to threefold stimulation of endogenous levels of cyclic AMP (cAMP). A23187-stimulated K/sup +/ efflux was partially inhibited by (i) stimulation of adenylate cyclase with isoproterenol, forskolin or, PGE/sub 1/; (ii) exogenous cAMP; and (iii) inhibition of phosphodiesterase with MIX (1-methyl-3-isobutylxanthine). Maximal inhibition of K/sup +/ efflux was obtained by simultaneous addition of isoproterenol and MIX. In dose-response curves, the isoproterenol-sensitive K/sup +/ efflux was half-maximally inhibited (IC/sub 50/) with 2-5 x 10/sup -10/ M of isoproterenol concentration. Propranolol was able to completely block the effect of isoproterenol, with an IC/sub 50/ of about 1-2 x 10/sup -7/ M. Isoproterenol and MIX did not inhibit A23187-stimulated K/sup +/ efflux in an incubation medium where NaCl was replaced by sucrose (or choline), suggesting the involvement of an Na/sup +/:Ca/sup 2 +/ exchange mechanism. The results show that stimulation of beta-adrenoceptors in mouse macrophages counter balances the opening of K/sup +/ channels induced by the calcium ionophore A23187. This likely reflects a decrease in cytoslic free calcium content via a cAMP-mediated stimulation of Na/sup +/:Ca/sup 2 +/ exchange.

  8. Substrate-dependent nitric oxide synthesis by secreted endoplasmic reticulum aminopeptidase 1 in macrophages.

    PubMed

    Goto, Yoshikuni; Ogawa, Kenji; Nakamura, Takahiro J; Hattori, Akira; Tsujimoto, Masafumi

    2015-06-01

    In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)-γ and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis. PMID:25577645

  9. Inhalation of ozone produces a decrease in superoxide anion radical production in mouse alveolar macrophages

    SciTech Connect

    Ryer-Powder, J.E.; Amoruso, M.A.; Czerniecki, B.; Witz, G.; Goldstein, B.D.

    1988-11-01

    The potentiation of fatal bacterial pneumonia in mice by prior inhalation of ozone occurs at levels of this oxidant pollutant that are frequently present in ambient air. A likely mechanism for this effect is an ozone-induced inhibition in the ability of pulmonary alveolar macrophages (PAM) to produce superoxide anion radical (O2-) demonstrated in the present study. A 25% decrease in PAM O2- production, as measured by nitroblue tetrazolium reduction, occurred after exposure of Swiss-Webster mice to 0.11 ppm ozone for 3 h (p less than 0.05). After 1 ppm there was almost complete inhibition of O2- release. In contrast, the rat, which is highly resistant to the potentiation of bacterial infections by ozone, was less sensitive to inhibition of PAM O2- production, as measured by cytochrome c reduction (mouse IC50, 0.41 ppm; rat IC50, 3.0 ppm ozone for 3 h). The observed decrement in mouse PAM O2- production was not associated with any change in phagocytic ability, as measured by both latex bead ingestion and 51Cr-labeled sheep red blood cell ingestion. This decrease in O2- production in the presence of normal phagocytic activity is analogous to certain of the findings in the neutrophils of children with chronic granulomatous disease. A decrease in rat PAM membrane cytochrome b558 levels was observed after ozone exposure of 3 ppm for 3 h, preliminarily suggesting that the mechanism by which ozone interferes with PAM O2- production may be through interaction with this heme-containing electron carrier.

  10. Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

    PubMed

    Isidro, Raymond A; Isidro, Angel A; Cruz, Myrella L; Hernandez, Siomara; Appleyard, Caroline B

    2015-12-01

    The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated. PMID:26403093

  11. Comparative Proteomic Analysis of the Molecular Responses of Mouse Macrophages to Titanium Dioxide and Copper Oxide Nanoparticles Unravels Some Toxic Mechanisms for Copper Oxide Nanoparticles in Macrophages

    PubMed Central

    Triboulet, Sarah; Aude-Garcia, Catherine; Armand, Lucie; Collin-Faure, Véronique; Chevallet, Mireille; Diemer, Hélène; Gerdil, Adèle; Proamer, Fabienne; Strub, Jean-Marc; Habert, Aurélie; Herlin, Nathalie; Van Dorsselaer, Alain; Carrière, Marie; Rabilloud, Thierry

    2015-01-01

    Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions. PMID:25902355

  12. Intraperitoneal inflammation decreases endometriosis in a mouse model

    PubMed Central

    Nowak, N.M.; Fischer, O.M.; Gust, T.C.; Fuhrmann, U.; Habenicht, U.-F.; Schmidt, A.

    2008-01-01

    BACKGROUND The role of the immune system in the pathogenesis of endometriosis remains elusive. It has been shown that patients have an altered peritoneal environment with increased levels of inflammatory cytokines, activated macrophages and reduced clearance of retrogradely transported endometrial fragments. However, it is not known if this unique inflammatory situation is cause or consequence of endometriosis. This study investigates the impact of a pre-existing peritoneal inflammation on endometriosis establishment in a mouse model. METHODS Endometriosis was induced by intraperitoneal injection of enhanced green fluorescent protein (EGFP)-expressing endometrium in mice. In parallel, a peritonitis model was established via intraperitoneal injection of thioglycolate medium (TM). Finally, endometriosis was induced in the inflamed peritoneal cavity and lesion establishment as well as morphological and histological characteristics were analysed. RESULTS Induction of endometriosis in an inflamed peritoneal cavity resulted in fewer lesions and significantly lower sum of lesion surface area per mouse in the TM-treated group. Additionally, a higher amount of non-attached debris could be detected in the peritoneal cavity of TM-treated mice. CONCLUSIONS An intraperitoneal inflammation decreases endometriosis establishment in this mouse model. Thus, a pre-existing peritoneal inflammation might not be a factor favouring the development of endometriosis. PMID:18653673

  13. Regulatory role of PI3K-protein kinase B on the release of interleukin-1β in peritoneal macrophages from the ascites of cirrhotic patients

    PubMed Central

    Tapia-Abellán, A; Ruiz-Alcaraz, A J; Antón, G; Miras-López, M; Francés, R; Such, J; Martínez-Esparza, M; García-Peñarrubia, P

    2014-01-01

    Great effort has been paid to identify novel targets for pharmaceutical intervention to control inflammation associated with different diseases. We have studied the effect of signalling inhibitors in the secretion of the proinflammatory and profibrogenic cytokine interleukin (IL)-1β in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients and compared with those obtained from the blood of healthy donors. Peritoneal M-DM were isolated from non-infected ascites of cirrhotic patients and stimulated in vitro with lipopolysaccharide (LPS) and heat-killed Candida albicans in the presence or absence of inhibitors for c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 1 (MEK1), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). The IL1B and CASP1 gene expression were evaluated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). The expression of IL-1β and caspase-1 were determined by Western blot. IL-1β was also assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Results revealed that MEK1 and JNK inhibition significantly reduced the basal and stimulated IL-1β secretion, while the p38 MAPK inhibitor had no effect on IL-1β levels. On the contrary, inhibition of PI3K increased the secretion of IL-1β from stimulated M-DM. The activating effect of PI3K inhibitor on IL-1β release was mediated mainly by the enhancement of the intracellular IL-1β and caspase-1 content release to the extracellular medium and not by increasing the corresponding mRNA and protein expression levels. These data point towards the role of MEK1 and JNK inhibitors, in contrast to the PI3K-protein kinase B inhibitors, as potential therapeutic tools for pharmaceutical intervention to diminish hepatic damage by reducing the inflammatory response mediated by IL-1β associated with liver failure. PMID:25080058

  14. Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals.

    PubMed

    Pridans, Clare; Lillico, Simon; Whitelaw, Bruce; Hume, David A

    2014-01-01

    The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large "cargo" for applications in gene therapy and vaccine delivery. PMID:26015955

  15. Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages.

    PubMed

    Hamers, Anouk A J; van Dam, Laura; Teixeira Duarte, José M; Vos, Mariska; Marinković, Goran; van Tiel, Claudia M; Meijer, Sybren L; van Stalborch, Anne-Marieke; Huveneers, Stephan; Te Velde, Anje A; de Jonge, Wouter J; de Vries, Carlie J M

    2015-01-01

    Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn's disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases. PMID:26241646

  16. Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages

    PubMed Central

    Hamers, Anouk A. J.; van Dam, Laura; Teixeira Duarte, José M.; Vos, Mariska; Marinković, Goran; van Tiel, Claudia M.; Meijer, Sybren L.; van Stalborch, Anne-Marieke; Huveneers, Stephan; te Velde, Anje A.

    2015-01-01

    Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn’s disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases. PMID:26241646

  17. Effect of Chlamydia pneumoniae on Cellular ATP Content in Mouse Macrophages: Role of Toll-Like Receptor 2

    PubMed Central

    Yaraei, Kambiz; Campbell, Lee Ann; Zhu, Xiaodong; Liles, W. Conrad; Kuo, Cho-chou; Rosenfeld, Michael E.

    2005-01-01

    Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live C. pneumoniae and inactivated C. pneumoniae induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of C. pneumoniae on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated C. pneumoniae cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated C. pneumoniae increased the ATP content of the cells. In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In cells treated with inactivated C. pneumoniae, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of C. pneumoniae, while sustained induction required live organisms. The capacity of C. pneumoniae to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4. PMID:15972526

  18. Complement Component C1q Programs a Pro-Efferocytic Phenotype while Limiting TNFα Production in Primary Mouse and Human Macrophages

    PubMed Central

    Hulsebus, Holly J.; O’Conner, Sean D.; Smith, Emily M.; Jie, Chunfa; Bohlson, Suzanne S.

    2016-01-01

    Deficiency in complement component C1q is associated with an inability to clear apoptotic cells (efferocytosis) and aberrant inflammation in lupus, and identification of the pathways involved in these processes should reveal important regulatory mechanisms in lupus and other autoimmune or inflammatory diseases. In this study, C1q-dependent regulation of TNFα/IL-6 expression and efferocytosis was investigated using primary mouse bone marrow-derived macrophages and human monocyte-derived macrophages. C1q downregulated LPS-dependent TNFα production in mouse and human macrophages. While prolonged stimulation with C1q (18 h) was required to elicit a dampening of TNFα production from mouse macrophages, the human macrophages responded to C1q with immediate downregulation of TNFα. IL-6 production was unchanged in mouse and upregulated by human macrophages following prolonged stimulation with C1q. Our previous studies indicated that C1q programmed enhanced efferocytosis in mouse macrophages by enhancing expression of Mer tyrosine kinase and its ligand Gas6, a receptor–ligand pair that also inhibits proinflammatory signaling. Here, we demonstrated that C1q-dependent programming of human macrophage efferocytosis required protein synthesis; however, neither Mer nor the related receptor Axl was upregulated in human cells. In addition, while the C1q-collagen-like tails are sufficient for promoting C1q-dependent phagocytosis of antibody-coated targets, the C1q-tails failed to program enhanced efferocytosis or dampen TNFα production. These data further elucidate the mechanisms by which C1q regulates proinflammatory signaling and efferocytosis in macrophages, functions that are likely to influence the progression of autoimmunity and chronic inflammation. PMID:27379094

  19. Mesenchymal Stem Cell-Educated Macrophages Ameliorate LPS-Induced Systemic Response

    PubMed Central

    Hu, Yaoqin; Qin, Chaojin; Zheng, Guoping; Tao, Huikang; Zhang, Yan; Qiu, Guanguan; Ge, Menghua; Huang, Lanfang; Chen, Lina; Cheng, Baoli

    2016-01-01

    Both bone marrow and adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory effects. The goal of this study was to determine whether ASCs-educated macrophages could directly ameliorate LPS-induced systemic response in a mouse model. Mouse peritoneal macrophages were cocultured with ASCs in a Transwell system for 2 days to educate macrophages. Mice were divided into 5 groups: control, LPS, LPS + ASCs, LPS + untreated macrophages, and LPS + educated macrophages. Educated macrophages decreased lung inflammation, weight loss, pulmonary edema, and inflammatory cytokine response. In vitro, ASCs increased expression of M2 macrophages independent of direct cell-to-cell contact when macrophages were treated with LPS or serum from patients with acute respiratory distress syndrome (ARDS). When macrophages were cultured with serum from ARDS patients who were treated with ASCs or placebo in our previous clinical trial, there was no difference in M2 macrophage levels before and after ASCs treatment indicating a suboptimal response to the treatment protocol. ASCs also reduced the levels of LPS-induced proinflammatory cytokines in vitro which were mimicked by IL-10 and blocked by antibodies for IL-10 and IL-10 receptor supporting the notion that educated macrophages exert their anti-inflammatory effects via IL-10-dependent mechanisms. PMID:27546994

  20. Mesenchymal Stem Cell-Educated Macrophages Ameliorate LPS-Induced Systemic Response.

    PubMed

    Hu, Yaoqin; Qin, Chaojin; Zheng, Guoping; Lai, Dengming; Tao, Huikang; Zhang, Yan; Qiu, Guanguan; Ge, Menghua; Huang, Lanfang; Chen, Lina; Cheng, Baoli; Shu, Qiang; Xu, Jianguo

    2016-01-01

    Both bone marrow and adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory effects. The goal of this study was to determine whether ASCs-educated macrophages could directly ameliorate LPS-induced systemic response in a mouse model. Mouse peritoneal macrophages were cocultured with ASCs in a Transwell system for 2 days to educate macrophages. Mice were divided into 5 groups: control, LPS, LPS + ASCs, LPS + untreated macrophages, and LPS + educated macrophages. Educated macrophages decreased lung inflammation, weight loss, pulmonary edema, and inflammatory cytokine response. In vitro, ASCs increased expression of M2 macrophages independent of direct cell-to-cell contact when macrophages were treated with LPS or serum from patients with acute respiratory distress syndrome (ARDS). When macrophages were cultured with serum from ARDS patients who were treated with ASCs or placebo in our previous clinical trial, there was no difference in M2 macrophage levels before and after ASCs treatment indicating a suboptimal response to the treatment protocol. ASCs also reduced the levels of LPS-induced proinflammatory cytokines in vitro which were mimicked by IL-10 and blocked by antibodies for IL-10 and IL-10 receptor supporting the notion that educated macrophages exert their anti-inflammatory effects via IL-10-dependent mechanisms. PMID:27546994

  1. Interactions of IFN-gamma with IL-3 and IL-4 in the regulation of serotonin and arachidonate release from mouse peritoneal mast cells.

    PubMed Central

    Holliday, M R; Banks, E M; Dearman, R J; Kimber, I; Coleman, J W

    1994-01-01

    We have examined the interactions between interferon-gamma (IFN-gamma), interleukin-3 (IL-3) and interleukin-4 (IL-4) in the regulation of IgE/antigen-induced secretory responses of mouse peritoneal mast cells. The cytokines were added either alone or in various combinations to cultured mast cells sensitized passively with IgE antibody. In experiments with unfractionated peritoneal cells (containing approx. 1% mast cells), IL-3 and IL-4 enhanced in an additive manner antigen-induced release of serotonin (5-HT), while IFN-gamma inhibited release regardless of whether IL-3 and/or IL-4 were present. In experiments employing mast cells purified to > 90%, IL-3 and IL-4 retained their enhancing activities whereas the inhibitory effect of IFN-gamma was considerably diminished. Nevertheless, IFN-gamma still inhibited significantly IL-4-enhanced secretion. The effects of IL-3 and IL-4 +/- IFN-gamma on arachidonate release were identical to those seen for 5-HT release, indicating that the secretion of both preformed mediators and newly synthesized eicosanoids is regulated in a similar way by these cytokines. PMID:8045595

  2. Treatment of dextran sodium sulfate-induced experimental colitis by adoptive transfer of peritoneal cells

    PubMed Central

    Liu, Ting; Ren, Jun; Wang, Wei; Wei, Xia-wei; Shen, Guo-bo; Liu, Yan-tong; Luo, Min; Xu, Guang-chao; Shao, Bin; Deng, Sen-yi; He, Zhi-yao; Liang, Xiao; Liu, Yu; Wen, Yan-Zhu; Xiang, Rong; Yang, Li; Deng, Hong-xin; Wei, Yu-quan

    2015-01-01

    The adoptive transfer of the natural regulatory B cells and macrophages should be a useful treatment for inflammation and autoimmune disease. However, it is usually difficult to isolate these cells from the tissues and expand them. Here, we investigated the feasibility of adoptively transferring peritoneal cells (PCs) as a treatment for DSS-induced colitis. We found that peritoneal cavity can provide an easily accessible site for harvesting enough number of PCs, namely, two-dose PCs for the treatment from a mouse in one operation. Adoptive therapy of these cells from healthy mice or those with disease is effectively in reducing the disease activity score. The natural B cells and macrophages of the infused PCs can selectively migrate to lesion sites and regulate the expression of Stat3, NF−κB, Smad3 and Smad7. Additionally, PCs exert dual activity of IL-10 and TGF-β secreted spontaneously by both peritoneal B cells and macrophages, which in turn enhance the induction of regulatory B cells and Macrophages in microenvironment of inflammation. Moreover, PCs can re-establish immunological tolerance in the OVA-immunized mice. Thus, our findings provide a new strategy for colitis therapy and could be of importance in additional exploration of other inflammation and autoimmune diseases therapy. PMID:26565726

  3. Systemic and Intravitreal Delivery of Dendrimers to Activated Microglia/Macrophage in Ischemia/Reperfusion Mouse Retina

    PubMed Central

    Kambhampati, Siva P.; Clunies-Ross, Alexander J. M.; Bhutto, Imran; Mishra, Manoj K.; Edwards, Malia; McLeod, D. Scott; Kannan, Rangaramanujam M.; Lutty, Gerard

    2015-01-01

    Purpose Microglial activation and associated neuroinflammation play a key role in the pathogenesis of many diseases of the retina, including viral infection, diabetes, and retinal degeneration. Strategies to target activated microglia and macrophages and attenuate inflammation may be valuable in treating these diseases. We seek to develop dendrimer-based formulations that target retinal microglia and macrophages in a pathology-dependent manner, and deliver drugs, either intravenously or intravitreally. Methods Retinal uptake of cyanine dye (Cy5)-conjugated dendrimer (D-Cy5) was assessed in normal and ischemia/reperfusion (I/R) mouse eyes. Microglia/macrophage uptake of the dendrimer was assessed with immunofluorescence using rabbit Iba-1 antibody with Cy3-tagged secondary antibody (microglia/macrophage). Uptake in retina and other organs was quantified using fluorescence spectroscopy. Results Clearance of D-Cy5 from normal eyes was almost complete by 72 hours after intravitreal injection and 24 hours after intravenous delivery. In eyes with activated microglia after I/R injury, D-Cy5 was retained by activated microglia/macrophage (Iba1+ cells) up to 21 days after intravitreal and intravenous administration. In I/R eyes, the relative retention of intravitreal and intravenous D-Cy5 was comparable, if a 30-fold higher intravenous dose was used. Conclusions Intravitreal and systemic dendrimers target activated microglia and show qualitatively similar retinal biodistribution when administered by either route. Results provide proof-of-concept insights for developing dendrimer drug formulations as treatment options for retinal diseases associated with microglia or macrophage activation such as age-related macular degeneration, diabetic retinopathy, and retinal degenerations. PMID:26193917

  4. Comparative functional characterization of mouse bone marrow-derived mast cells and peritoneal mast cells in response to non-immunological stimuli.

    PubMed

    Singh, R; Kumar, P; Gupta, P P

    2001-04-01

    The cultured mouse mast cells that are dependent on spleen-derived factor for their proliferation and maintenance and have been shown to be similar to mucosal mast cells in terms of their T-cell dependence and histochemical staining characteristics. Mast cell heterogeneity has been confirmed by functional characterization of mouse bone marrow-derived mast cells (MBMMC) and mouse peritoneal mast cells (MPMCs). MPMCs released around 30% of histamine when stimulated with compound 48/80 whereas MBMMC were almost unresponsive to the same stimulus. Calcium Ionophore A23187 on the other hand, released histamine in dose-dependent manner from MBMMC. The study was undertaken to investigate the effect of antiallergic drug, disodium cromoglycate (DSCG), a synthetic cromone and quercetin, a plant-derived flavonoid on Ca ionophore A23187 induced histamine release from MBMMC. MBMMCs were almost unresponsive to DSCG whereas Ca Ionophore induced histamine release was blocked by Quercetin. The results indicate that response of mast cells at one anatomic site to a given stimulus does not necessarily predict the response of mast cells at a different anatomic location to the same stimulus. It shows functional heterogeneity within a single species. So, it cannot be assumed that antiallergic compounds stabilizing mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites. PMID:11491575

  5. Local Delivery of Polarized Macrophages Improves Reperfusion Recovery in a Mouse Hind Limb Ischemia Model

    PubMed Central

    Jetten, Nadine; Donners, Marjo M. P. C.; Wagenaar, Allard; Cleutjens, Jack P. M.; van Rooijen, Nico; de Winther, Menno P. J.; Post, Mark J.

    2013-01-01

    Aims Enhancement of collateral development in coronary or peripheral artery disease is a therapeutic target, but it has proven difficult to achieve. Macrophages are key players in collateral remodeling, yet the effect of different macrophage subsets on arteriogenesis has not been investigated. Methods and Results Murine macrophages were cultured from bone marrow and polarized into M1 (IFNγ), M2a (IL-4) or M2c (IL-10) subsets. C57BL/6 mice underwent femoral artery ligation followed by intramuscular injection of macrophage subsets. Using eGFP expressing macrophages, cells could be detected at least 6 days after ligation and were located in the perivascular space of collateral vessels. After 14 days, perfusion ratio was increased in animals treated with M1 as well as M2a and M2c macrophages compared to control. Depletion of circulating monocytes by clodronate liposome injections did not hamper reperfusion recovery, however, treatment with exogenous polarized macrophages improved perfusion ratio after 14 days again. We used IL10Rfl/fl/LysMCre+ mice to study the effect of inhibition of endogenous polarization towards specifically M2c macrophages on arteriogenesis. Deletion of the IL10-receptor (IL10R) in the myeloid lineage did not affect reperfusion recovery, yet the pro-arteriogenic effect of exogenously injected M2c macrophages was still present. Conclusions Local injection of polarized macrophages promotes reperfusion recovery after femoral artery ligation and is not influenced by depletion of circulatory monocytes. Preventing endogenous M2c polarization did not affect reperfusion recovery suggesting that M2c’s are not required for collateralization, but are sufficient to induce collateral formation upon exogenous administration. This is the first study using local injection of macrophage subsets showing the pro-arteriogenic effect of polarized macrophages. PMID:23894348

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

    PubMed

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

    2015-12-01

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

  7. Lysophosphatidylcholine Triggers TLR2- and TLR4-Mediated Signaling Pathways but Counteracts LPS-Induced NO Synthesis in Peritoneal Macrophages by Inhibiting NF-κB Translocation and MAPK/ERK Phosphorylation

    PubMed Central

    Carneiro, Alan Brito; Iaciura, Bruna Maria Ferreira; Nohara, Lilian Lie; Lopes, Carla Duque; Veas, Esteban Mauricio Cordero; Mariano, Vania Sammartino; Bozza, Patricia Torres; Lopes, Ulisses Gazos; Atella, Georgia Correa; Almeida, Igor Correia; Silva-Neto, Mário Alberto Cardoso

    2013-01-01

    Background Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway. Methodology/Principal Findings HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells. Conclusions/Significance The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression. PMID:24312681

  8. The Macrophage Inhibitor CNI-1493 Blocks Metastasis in a Mouse Model of Ewing Sarcoma through Inhibition of Extravasation

    PubMed Central

    Hesketh, Anthony J.; Maloney, Caroline; Behr, Christopher A.; Edelman, Morris C.; Glick, Richard D.; Al-Abed, Yousef; Symons, Marc; Soffer, Samuel Z.; Steinberg, Bettie M.

    2015-01-01

    Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma. PMID:26709919

  9. Experimental Evolution of Legionella pneumophila in Mouse Macrophages Leads to Strains with Altered Determinants of Environmental Survival

    PubMed Central

    Ensminger, Alexander W.; Yassin, Yosuf; Miron, Alexander; Isberg, Ralph R.

    2012-01-01

    The Gram-negative bacterium, Legionella pneumophila, is a protozoan parasite and accidental intracellular pathogen of humans. We propose a model in which cycling through multiple protozoan hosts in the environment holds L. pneumophila in a state of evolutionary stasis as a broad host-range pathogen. Using an experimental evolution approach, we tested this hypothesis by restricting L. pneumophila to growth within mouse macrophages for hundreds of generations. Whole-genome resequencing and high-throughput genotyping identified several parallel adaptive mutations and population dynamics that led to improved replication within macrophages. Based on these results, we provide a detailed view of the population dynamics of an experimentally evolving bacterial population, punctuated by frequent instances of transient clonal interference and selective sweeps. Non-synonymous point mutations in the flagellar regulator, fleN, resulted in increased uptake and broadly increased replication in both macrophages and amoebae. Mutations in multiple steps of the lysine biosynthesis pathway were also independently isolated, resulting in lysine auxotrophy and reduced replication in amoebae. These results demonstrate that under laboratory conditions, host restriction is sufficient to rapidly modify L. pneumophila fitness and host range. We hypothesize that, in the environment, host cycling prevents L. pneumophila host-specialization by maintaining pathways that are deleterious for growth in macrophages and other hosts. PMID:22693450

  10. The peritoneal tumour microenvironment of high-grade serous ovarian cancer.

    PubMed

    Leinster, D Andrew; Kulbe, Hagen; Everitt, Gemma; Thompson, Richard; Perretti, Mauro; Gavins, Felicity N E; Cooper, Dianne; Gould, David; Ennis, Darren P; Lockley, Michelle; McNeish, Iain A; Nourshargh, Sussan; Balkwill, Frances R

    2012-06-01

    High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3(+) T cells and CD68(+) macrophages, with occasional neutrophils. Alpha-smooth muscle actin(+) (α-SMA(+) ) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6-48 h) with an extensive CD45(+) leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3(+) T lymphocytes and CD68(+) macrophages with α-SMA(+) cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2-3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated

  11. Relationship between macrophages in mouse uteri and angiogenesis in endometrium during the peri-implantation period.

    PubMed

    Tan, Wenya; Chen, Leining; Guo, Lei; Ou, Xianghong; Xie, Duo; Quan, Song

    2014-10-15

    The objective of this study is to examine the change in macrophage numbers, inducible form of NO synthase (iNOS), and vascular endothelial growth factor (VEGF) expression both before and after embryo implantation in the uterine tissue of mice. In order to explore the mechanism of macrophages in endometrial angiogenesis, 8-week-old female mice were divided into three groups: pregnant group, pseudopregnant group (mated to male mice that had been vasectomized), and estrous group (unmated). Individuals from these three groups were sacrificed at time intervals D1.5 to D6.5. Formalin-fixed paraffin-embedded tissue was used for immunocytochemical localization of Mφ, iNOS, and VEGF utilizing standard methodology. The proportion of macrophages in the peripheral blood was determined by flow cytometry, and the relationship between macrophage, iNOS, and VEGF expression was analyzed. The proportion of peripheral blood macrophages in the pregnancy group was significantly higher than that in the other groups. The results of immunohistochemistry determined that the macrophages exhibited changes in both numbers and distribution. The number of macrophages in the endometrium of the pregnancy and pseudopregnancy groups was significantly higher than that in the control (estrous) group. In the pregnancy group, macrophage numbers dramatically decreased and gradually transferred to the perimetrium on D4.5. Immunostaining revealed strong staining in the pregnancy group and weaker staining in the pseudopregnant and control groups for both iNOS and VEGF. There was strong, dense immunostaining at the implantation site for both iNOS and VEGF, whereas light immunostaining was seen in interimplantation tissues on D5.5 to D6.5. In the pregnant group, peripheral blood and uterine macrophage proportions were negatively correlated, whereas the amount of macrophages, iNOS, and VEGF expression in the endometrium were positively correlated. The expression of iNOS and VEGF in the endometrium also

  12. Candida albicans killing by RAW 264.7 mouse macrophage cells: effects of Candida genotype, infection ratios, and gamma interferon treatment.

    PubMed

    Marcil, A; Harcus, D; Thomas, D Y; Whiteway, M

    2002-11-01

    Phagocytic cells such as neutrophils and macrophages are potential components of the immune defense that protects mammals against Candida albicans infection. We have tested the interaction between the mouse macrophage cell line RAW 264.7 and a variety of mutant strains of C. albicans. We used an end point dilution assay to monitor the killing of C. albicans at low multiplicities of infection (MOIs). Several mutants that show reduced virulence in mouse systemic-infection models show reduced colony formation in the presence of macrophage cells. To permit analysis of the macrophage-Candida interaction at higher MOIs, we introduced a luciferase reporter gene into wild-type and mutant Candida cells and used loss of the luminescence signal to quantify proliferation. This assay gave results similar to those for the end point dilution assay. Activation of the macrophages with mouse gamma interferon did not enhance anti-Candida activity. Continued coculture of the Candida and macrophage cells eventually led to death of the macrophages, but for the RAW 264.7 cell line this was not due to apoptotic pathways involving caspase-8 or -9 activation. In general Candida cells defective in the formation of hyphae were both less virulent in animal models and more sensitive to macrophage engulfment and growth inhibition. However the nonvirulent, hypha-defective cla4 mutant line was considerably more resistant to macrophage-mediated inhibition than the wild-type strain. Thus although mutants sensitive to engulfment are typically less virulent in systemic-infection models, sensitivity to phagocytic macrophage cells is not the unique determinant of C. albicans virulence. PMID:12379711

  13. Microbiological aspects of peritonitis associated with continuous ambulatory peritoneal dialysis.

    PubMed Central

    von Graevenitz, A; Amsterdam, D

    1992-01-01

    The process of continuous ambulatory peritoneal dialysis has provided a useful, relatively inexpensive, and safe alternative for patients with end-stage renal disease. Infectious peritonitis, however, has limited a more widespread acceptance of this technique. The definition of peritonitis in this patient population is not universally accepted and does not always include the laboratory support of a positive culture (or Gram stain). In part, the omission of clinical microbiological findings stems from the lack of sensitivity of earlier microbiological efforts. Peritonitis results from decreased host phagocytic efficiency with depressed phagocytosis and bactericidal capacity of peritoneal macrophages. During episodes of peritonitis, fluid movement is reversed, away from the lymphatics and peritoneal membrane and toward the cavity. As a result, bloodstream infections are rare. Most peritonitis episodes are caused by bacteria. Coagulase-negative staphylococci are the most frequently isolated organisms, usually originating from the skin flora, but a wide array of microbial species have been documented as agents of peritonitis. Clinical microbiology laboratories need to be cognizant of the diverse agents so that appropriate primary media can be used. The quantity of dialysate fluid that is prepared for culture is critical and should constitute at least 10 ml. The sensitivity of the cultural approach depends on the volume of dialysate, its pretreatment (lysis or centrifugation), the media used, and the mode of incubation. The low concentration of microorganisms in dialysate fluids accounts for negative Gram stain results. Prevention of infection in continuous ambulatory peritoneal dialysis patients is associated with the socioeconomic status of the patient, advances in equipment (catheter) technology, and, probably least important, the application of prophylactic antimicrobial agents. PMID:1735094

  14. Lysophosphatidic acid receptor 1 antagonist ki16425 blunts abdominal and systemic inflammation in a mouse model of peritoneal sepsis.

    PubMed

    Zhao, Jing; Wei, Jianxin; Weathington, Nathaniel; Jacko, Anastasia M; Huang, Hai; Tsung, Allan; Zhao, Yutong

    2015-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator of inflammation via the LPA receptors 1-6. We and others have previously described proinflammatory and profibrotic activities of LPA signaling in bleomycin- or lipopolysaccharide (LPS)-induced pulmonary fibrosis or lung injury models. In this study, we investigated if LPA signaling plays a role in the pathogenesis of systemic sepsis from an abdominal source. We report here that antagonism of the LPA receptor LPA1 with the small molecule ki16425 reduces the severity of abdominal inflammation and organ damage in the setting of peritoneal endotoxin exposure. Pretreatment of mice with intraperitoneal ki16425 eliminates LPS-induced peritoneal neutrophil chemokine and cytokine production, liver oxidative stress, liver injury, and cellular apoptosis in visceral organs. Mice pretreated with ki16425 are also protected from LPS-induced mortality. Tissue myeloperoxidase activity is not affected by LPA1 antagonism. We have shown that LPA1 is associated with LPS coreceptor CD14 and the association is suppressed by ki16425. LPS-induced phosphorylation of protein kinase C δ (PKCδ) and p38 mitogen-activated protein kinase (p38 MAPK) in liver cells and interleukin 6 production in Raw264 cells are likewise blunted by LPA1 antagonism. These studies indicate that the small molecule inhibitor of LPA1, ki16425, suppresses cytokine responses and inflammation in a peritoneal sepsis model by blunting downstream signaling through the LPA1-CD14-toll-like receptor 4 receptor complex. This anti-inflammatory effect may represent a therapeutic strategy for the treatment of systemic inflammatory responses to infection of the abdominal cavity. PMID:25701366

  15. Calcium/calmodulin-dependent protein kinase II regulates cyclooxygenase-2 expression and prostaglandin E2 production by activating cAMP-response element-binding protein in rat peritoneal macrophages

    PubMed Central

    Zhou, Xueyuan; Li, Junying; Yang, Wenxiu

    2014-01-01

    Prostaglandin E2 (PGE2) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE2 production is regulated by arachidonic acid release and cyclooxygenase-2 (COX-2) expression. In the present study, we found that COX-2 expression can be achieved by activating Ca2+/Calmodulin (CaM)-dependent protein kinase II (CaMKII) and cAMP-response element-binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca2+]i), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX-2 expression and PGE2 production in a chronological order. These results suggested that Ca2+/CaM-dependent CaMKII plays an important role in mediating COX-2 expression and PGE2 production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE2 production, which plays an essential role in inflammation and cancers. PMID:24773364

  16. Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages

    SciTech Connect

    Woods, Courtney G.; Fu Jingqi; Xue Peng; Hou Yongyong; Pluta, Linda J.; Yang Longlong; Zhang Qiang; Thomas, Russell S.; Andersen, Melvin E.; Pi Jingbo

    2009-07-01

    Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (< 0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1{beta}, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways.

  17. Elimination of Leishmania donovani amastigotes by activated macrophages.

    PubMed Central

    Haidaris, C G; Bonventre, P F

    1981-01-01

    Tissue macrophages are the obligatory host cells for Leishmania donovani, the causative agent of visceral leishmaniasis. In this study we sought to determine whether activated macrophages, as defined by the functional criterion of tumor cell cytotoxicity, were also able to exert a microbicidal effect on ingested L. donovani amastigotes. We found that mouse peritoneal macrophages activated by a variety of means exerted a cytotoxic effect on tumor cell targets but were not able to kill L. donovani amastigotes unless the infected macrophages were exposed continually to an activating stimulus. Corynebacterium parvum, Mycobacterium tuberculosis H37Ra, and lymphokine-activated peritoneal macrophages from C57BL/6J mice were cytotoxic for EMT6 tumor cell targets. However, L. donovani Sudan strain 1S amastigotes were not killed by these macrophages unless the activated state was maintained by daily addition of lymphokine to the infected monolayers for several days postinfection. The killing of amastigotes was dependent on the time of exposure to lymphokine, as well as on the concentration of lymphokine added to the culture. Images PMID:7287190

  18. Peritonitis - spontaneous

    MedlinePlus

    ... a catheter used in peritoneal dialysis. Antibiotics may control infection in cases of spontaneous peritonitis with liver or kidney disease. Intravenous therapy can treat dehydration . You may need to stay in the hospital so health care providers can rule out conditions ...

  19. The E3 ubiquitin ligase RNF144B is LPS-inducible in human, but not mouse, macrophages and promotes inducible IL-1β expression.

    PubMed

    Ariffin, Juliana K; Kapetanovic, Ronan; Schaale, Kolja; Gatica-Andrades, Marcela; Blumenthal, Antje; Schroder, Kate; Sweet, Matthew J

    2016-07-01

    Differences in human and mouse immune responses may partly reflect species-specific adaptations and can provide important insights into human immunity. In this study, we show that RNF144B, which encodes an E3 ubiquitin ligase, was lipopolysaccharide-inducible in primary human macrophages and in human macrophage-like THP-1 cells. In contrast, Rnf144b was not lipopolysaccharide-inducible in several mouse cell populations, including primary macrophages from C57BL/6 and BALB/c mice and RAW264.7 macrophages. Similarly, Rnf144b was not up-regulated by infection of C57BL/6 mice with Escherichia coli Although the human and mouse RNF144B genes have conserved transcription start sites, cap analysis of gene expression data confirmed that the RNF144B promoter directs transcription in human but not mouse macrophages. The human and mouse RNF144B genes are controlled by highly conserved TATA-containing promoters, but subtle differences in transcription factor binding sites may account for differential regulation. Using gene silencing, we showed that RNF144B is necessary for priming of inflammasome responses in primary human macrophages. Specifically, RNF144B promotes lipopolysaccharide-inducible IL-1b mRNA expression but does not regulate expression of several other lipopolysaccharide-inducible cytokines (e.g., interleukin-10, interferon-γ) or affect expression of inflammasome components or substrates (e.g., procaspase-1, pro-interleukin-18). Our findings thus revealed a species-specific regulatory mechanism for selective inflammasome priming in human macrophages. PMID:26819317

  20. The effects of oral Cardax (disodium disuccinate astaxanthin) on multiple independent oxidative stress markers in a mouse peritoneal inflammation model: influence on 5-lipoxygenase in vitro and in vivo.

    PubMed

    Lockwood, Samuel F; Penn, Marc S; Hazen, Stanley L; Bikádi, Zsolt; Zsila, Ferenc

    2006-06-01

    Disodium disuccinate astaxanthin ('rac'-dAST; Cardax) is a water-dispersible C40 carotenoid derivative under development for oral and parenteral administration for cardioprotection of the at-risk ischemic cardiovascular patient. In experimental infarction models in animals (rats, rabbits, and dogs), significant myocardial salvage has been obtained, up to 100% at the appropriate dose in dogs. The documented mechanism of action in vitro includes direct scavenging of biologically produced superoxide anion; in vivo in rabbits, modulation of the complement activity of serum has also been shown. A direct correlation between administration of the test compound in animals and reductions of multiple, independent markers of oxidative stress in serum was recently obtained in a rat experimental infarction model. For the current study, it was hypothesized that oral Cardax administration would inhibit oxidative damage of multiple relevant biological targets in a representative, well-characterized murine peritoneal inflammation model. A previously developed mass spectrometry-based (LC/ESI/MS/MS) approach was used to interrogate multiple distinct pathways of oxidation in a black mouse (C57/BL6) model system. In vivo markers of oxidant stress from peritoneal lavage samples (supernatants) were evaluated in mice on day eight (8) after treatment with either Cardax or vehicle (lipophilic emulsion without drug) orally by gavage at 500 mg/kg once per day for seven (7) days at five (5) time points: (1) baseline prior to treatment (t=0); (2) 16 h following intraperitoneal (i.p.) injection with thioglycollate to elicit a neutrophilic infiltrate; (3) 4 h following i.p. injection of yeast cell wall (zymosan; t=16 h/4 h thioglycollate+zymosan); (4) 72 h following i.p. injection with thioglycollate to elicit monocyte/macrophage infiltration; and (5) 72 h/4 h thioglycollate+zymosan. A statistically significant sparing effect on the arachidonic acid (AA) and linoleic acid (LA) substrates was

  1. Two structurally distinct {kappa}B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages

    SciTech Connect

    Ohmori, Y.; Fukumoto, S.; Hamilton, T.A.

    1995-10-01

    The mouse KC gene is an {alpha}-chemokine gene whose transcription is induced in mononuclear phagocytes by LPS. DNA sequences necessary for transcriptional control of KC by LPS were identified in the region flanking the transcription start site. Transient transfection analysis in macrophages using deletion mutants of a 1.5-kb sequence placed in front of the chloramphenicol acetyl transferase (CAT) gene identified an LPS-responsive region between residues -104 and +30. This region contained two {kappa}B sequence motifs. The first motif (position -70 to -59, {kappa}B1) is highly conserved in all three human GRO genes and in the mouse macrophage inflammatory protein-2 (MIP-2) gene. The second {kappa}B motif (position -89 to -78, {kappa}B2) was conserved only between the mouse and the rat KC genes. Consistent with previous reports, the highly conserved {kappa}B site ({kappa}B1) was essential for LPS inducibility. Surprisingly, the distal {kappa}B site ({kappa}B2) was also necessary for optimal response; mutation of either {kappa}B site markedly reduced sensitivity to LPS in RAW264.7 cells and to TNF-{alpha} in NIH 3T3 fibroblasts. Although both {kappa}B1 and {kappa}B2 sequences were able to bind members of the Rel homology family, including NF{kappa}B1 (P50), RelA (65), and c-Rel, the {kappa}B1 site bound these factors with higher affinity and functioned more effectively than the {kappa}B2 site in a heterologous promoter. These findings demonstrate that transcriptional control of the KC gene requires cooperation between two {kappa}B sites and is thus distinct from that of the three human GRO genes and the mouse MIP-2 gene. 71 refs., 8 figs.

  2. Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages.

    PubMed

    Harada, Nobuhiko; Kanayama, Masaya; Maruyama, Atsushi; Yoshida, Aruto; Tazumi, Kyoko; Hosoya, Tomonori; Mimura, Junsei; Toki, Tsutomu; Maher, Jonathan M; Yamamoto, Masayuki; Itoh, Ken

    2011-04-01

    Iron is an essential element of hemoglobin, and efficient iron recycling from senescent erythrocytes by splenic macrophages is required for erythrocyte hemoglobin synthesis during erythropoiesis. Ferroportin 1 (Fpn1) is the sole iron exporter in mammals, and it also regulates iron reutilization. In this study, we demonstrated genetically that a redox-sensitive transcription factor, Nrf2, regulates Fpn1 mRNA expression in macrophages. Nrf2 activation by several electrophilic compounds commonly resulted in the upregulation of Fpn1 mRNA in bone marrow-derived and peritoneal macrophages obtained from wild-type mice but not from Nrf2 knockout mice. Further, Nrf2 activation enhanced iron release from the J774.1 murine macrophage cell line. Previous studies showed that inflammatory stimuli, such as LPS, downregulates macrophage Fpn1 by transcriptional and hepcidin-mediated post-translational mechanisms leading to iron sequestration by macrophages. We showed that two Nrf2 activators, diethyl maleate and sulforaphane (SFN; a natural Nrf2 activator found in broccoli), restored the LPS-induced suppression of Fpn1 mRNA in human and mouse macrophages, respectively. Furthermore, SFN counteracted the LPS-induced increase of Hepcidin mRNA by an Nrf2-independent mechanism in mouse peritoneal macrophages. These results demonstrate that Nrf2 regulates iron efflux from macrophages through Fpn1 gene transcription and suggest that Nrf2 may control iron metabolism during inflammation. PMID:21303654

  3. Azithromycin protects mice against ischemic stroke injury by promoting macrophage transition towards M2 phenotype.

    PubMed

    Amantea, Diana; Certo, Michelangelo; Petrelli, Francesco; Tassorelli, Cristina; Micieli, Giuseppe; Corasaniti, Maria Tiziana; Puccetti, Paolo; Fallarino, Francesca; Bagetta, Giacinto

    2016-01-01

    To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotective effects of the macrolide antibiotic azithromycin in a mouse model system of transient middle cerebral artery occlusion. Intraperitoneal administration of azithromycin significantly reduced blood-brain barrier damage and cerebral infiltration of myeloid cells, including neutrophils and inflammatory macrophages. These effects resulted in a dose-dependent reduction of cerebral ischemic damage, and in a remarkable amelioration of neurological deficits up to 7 days after the insult. Neuroprotection was associated with increased arginase activity in peritoneal exudate cells, which was followed by the detection of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic area at 24-48 h of reperfusion. Pharmacological inhibition of peritoneal arginase activity counteracted azithromycin-induced neuroprotection, pointing to a major role for drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype. PMID:26518285

  4. Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

    PubMed Central

    Evans, Teresa A.; Barkauskas, Deborah S.; Myers, Jay T.; Huang, Alex Y.

    2014-01-01

    Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury. PMID:25489963

  5. Berteroin Present in Cruciferous Vegetables Exerts Potent Anti-Inflammatory Properties in Murine Macrophages and Mouse Skin

    PubMed Central

    Jung, Yoo Jin; Jung, Jae In; Cho, Han Jin; Choi, Myung-Sook; Sung, Mi-Kyung; Yu, Rina; Kang, Young-Hee; Park, Jung Han Yoon

    2014-01-01

    Berteroin (5-methylthiopentyl isothiocyanate) is a sulforaphane analog present in cruciferous vegetables, including Chinese cabbage, rucola salad leaves, and mustard oil. We examined whether berteroin exerts anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin inflammation models. Berteroin decreased LPS-induced release of inflammatory mediators and pro-inflammatory cytokines in Raw 264.7 macrophages. Berteroin inhibited LPS-induced degradation of inhibitor of κBα (IκBα) and nuclear factor-κB p65 translocation to the nucleus and DNA binding activity. Furthermore, berteroin suppressed degradation of IL-1 receptor-associated kinase and phosphorylation of transforming growth factor β activated kinase-1. Berteroin also inhibited LPS-induced phosphorylation of p38 MAPK, ERK1/2, and AKT. In the mouse ear, berteroin effectively suppressed TPA-induced edema formation and down-regulated iNOS and COX-2 expression as well as phosphorylation of AKT and ERK1/2. These results demonstrate that berteroin exhibits potent anti-inflammatory properties and suggest that berteroin can be developed as a skin anti-inflammatory agent. PMID:25393510

  6. Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

    PubMed Central

    Shanafelt, A B; Kastelein, R A

    1989-01-01

    Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed. Images PMID:2662186

  7. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability.

    PubMed

    Chen, Kejie; Shanmugam, Nanda Kumar N; Pazos, Michael A; Hurley, Bryan P; Cherayil, Bobby J

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  8. Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability

    PubMed Central

    Chen, Kejie; Shanmugam, Nanda Kumar N.; Pazos, Michael A.; Hurley, Bryan P.; Cherayil, Bobby J.

    2016-01-01

    Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis. PMID:27505062

  9. Leishmania infantum: infection of macrophages in vitro with promastigotes.

    PubMed

    Méndez, S; Nell, M; Alunda, J M

    1996-06-01

    Leishmania infantum promastigotes in axenic culture exhibit limited infectivity for mouse peritoneal macrophages (M phi) in vitro using standard culture conditions (37 degrees C; 95% air/5% CO2) compared to Leishmania donovani promastigotes which induce notable infections. The infectivity of logarithmic (log) and stationary (stat) phase promastigotes of L. infantum was enhanced by the addition of fresh homologous serum, but no amastigotes were observed after 4 days. Prolonged infections, including transformation and survival of intracellular amastigotes in BALB/c mouse and hamster resident peritoneal M phi and M phi cell line J774.G8 were obtained by incubating M phi for 48 h at 26 degrees C prior to standard culture. Enhanced infectivity was observed in a number of L. infantum strains subject to this transient thermal change. PMID:8875306

  10. Peritoneal Disorders

    MedlinePlus

    Your peritoneum is the tissue that lines your abdominal wall and covers most of the organs in your abdomen. ... the surface of this tissue. Disorders of the peritoneum are not common. They include Peritonitis - an inflammation ...

  11. Peritonitis - secondary

    MedlinePlus

    ... blood pressure. Tests may include: Blood culture Blood chemistry, including pancreatic enzymes Complete blood count Liver and kidney function tests X-rays or CT scan Peritoneal fluid culture Urinalysis

  12. Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

    PubMed Central

    Ufimtseva, Elena

    2016-01-01

    The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells. PMID:27066505

  13. Modifying Effects of Nanosized Diamonds on Hydrolytic Potential of Macrophages In Vitro.

    PubMed

    Neshchadim, D V; Arkhipov, S A; Shkurupy, V A; Akhramenko, E S; Troitskii, A V; Karpov, M A

    2015-07-01

    The effects of nanosized particles (4-6 nm) of synthetic diamonds on mouse macrophage expression and secretion of lysosomal cathepsins (B and D) and MMP-1 and MMP-9 were studied in vitro. Culturing of peritoneal macrophages in medium with diamond nanoparticles led to an increase in the counts of macrophages expressing the above enzymes and to stimulation of their secretion. However, the manifestations of these effects varied significantly for various enzymes. The data indicate modulation of macrophage functions by nanodiamonds. These results help better understand the possible role of the "corpuscular" xenobiotic factors in the pathogenesis of diseases associated with macrophage capturing of these factors irrespective of their chemical "activity". PMID:26212815

  14. Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages.

    PubMed

    Horton, M R; Olman, M A; Bao, C; White, K E; Choi, A M; Chin, B Y; Noble, P W; Lowenstein, C J

    2000-10-01

    Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation. PMID:11000131

  15. [Macrophages promote the migration of neural stem cells into mouse spinal cord injury site].

    PubMed

    Cheng, Zhijian; Zhu, Wen; Li, Haopeng; He, Xijing

    2016-09-01

    Objective To explore the role of macrophages in the migration of neural stem cells (NSCs) in vivo and in vitro . Methods NSCs with green fluorescent protein (GFP) were isolated from GFP transgenic mice and the immunofluorescence cytochemical staining of nestin was used to identify NSCs. After spinal cord injury was induced, the tissue level of macrophage chemotactic protein-1 (MCP-1) mRNA was detected using quantitative real time PCR. The migration of GFP-NSCs was investigated 1 week after GFP-NSCs were injected into both sides of the damaged area. The effect of macrophage on the migration of NSCs in vitro was tested by Transwell(TM) system and the content of MCP-1 was detected by ELISA. Results NSCs highly expressed nestin. Compared with the control group, the level of MCP-1 mRNA significantly increased in the spinal cord injury group. The NSCs which were injected into the spinal cord migrated into the center of the injured site where F4/80 was highly expressed. Macrophages significantly increased the number of migrating NSCs in vitro and the secretion of MCP-1. Conclusion Macrophages induce NSC migrating into the spinal cord injury site possibly through promoting the secretion of MCP-1. PMID:27609570

  16. [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

  17. Blueberries reduce pro-inflammatory cytokine TNF-alpha and IL-6 production in mouse macrophages by inhibiting NF Kappa B activation and the MAPK pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Blueberries (BB) have been reported to attenuate atherosclerosis in apoE deficient (ApoE-/-) mice. The aim of this study was to evaluate the effects of BB in reducing pro-inflammatory cytokine production in mouse macrophages. ApoE-/- mice were fed AIN-93G diet (CD) or CD formulated to contain 1% fre...

  18. Comparison of the response of primary murine peritoneal macrophages and the U937 human histiocytic cell line to challenge with in vitro generated clinically relevant UHMWPE particles.

    PubMed

    Matthews, J B; Green, T R; Stone, M H; Wroblewski, B M; Fisher, J; Ingham, E

    2000-01-01

    The response of primary murine macrophages and the U937 human histiocytic cell line to challenge with clinically relevant UHMWPE wear debris of known particle size and dose was evaluated. Particles with mean sizes of 0.24, 0.45, 1.71, 7.62 and 88 microm were co-cultured with cells for 24 hours prior to assessment of cell viability and production of the osteolytic mediators IL-1beta, IL-6, TNFalpha and, in supernatants from murine phagocytes, PGE2 and GM-CSF. All particle fractions were evaluated at particle volume (microm3) to cell number ratios of 10 : 1 and 100 : 1 (and, additionally, 0.1 : 1 and 1 : 1 for U937 cells). These ratios had previously been identified as the most stimulatory and clinically relevant. Although the results for the cell line were highly variable, stimulation with phagocytosable particles (range 0.1 to 15 microm) resulted in enhanced levels of cytokine secretion by both murine macrophages and U937 histiocytes. The most biologically active particles were sub-micrometre in size. However, U937 cells responded to wear debris at much lower particle volume to cell number ratios (>0.1 microm3 per cell) than the murine cells (> 10 microm3 per cell). No GM-CSF was produced by particle or LPS stimulated murine macrophages. Similarly, U937 histiocytes failed to secrete any IL-1beta. Neither macrophage population responded to stimulation with the largest (88 microm) particles. These results confirm earlier findings and suggest that the size of UHMWPE wear particles is a critical factor in macrophage activation. Moreover, primary murine macrophages have been demonstrated to be a suitable model for studying cell-particle interactions in vitro. PMID:11202151

  19. Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

    PubMed Central

    Tao, Lili; Zhu, Wenhan; Hu, Bi-Jie

    2013-01-01

    Legionella pneumophila, the etiological agent for Legionnaires' disease, is ubiquitous in the aqueous environment, where it replicates as an intracellular parasite of free-living protozoa. Our understanding of L. pneumophila pathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmental L. pneumophila isolates interact similarly with the model host systems, we analyzed intracellular replication of several recently isolated such strains and found that these strains cannot productively grow in bone marrow-derived macrophages of A/J mice, which are permissive for all examined laboratory strains. By focusing on one strain called LPE509, we found that its deficiency in intracellular replication in primary A/J macrophages is not caused by the lack of important pathogenic determinants because this strain replicates proficiently in two protozoan hosts and the human macrophage U937 cell. We also found that in the early phase of infection, the trafficking of this strain in A/J macrophages is similar to that of JR32, a derivative of strain Philadelphia 1. Furthermore, infection of these cells by LPE509 caused extensive cell death in a process that requires the Dot/Icm type IV secretion system. Finally, we showed that the cell death is caused neither by the activation of the NAIP5/NLRC4 inflammasome nor by the recently described caspase 11-dependent pathway. Our results revealed that some environmental L. pneumophila strains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratory L. pneumophila strains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding to L. pneumophila infection. PMID:23753633

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

    PubMed

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

    2011-01-01

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

  1. Red blood cell homeostasis: recognition of distinct types of damaged homologous red blood cells by a mouse macrophage cell line.

    PubMed

    Singer, J A; Morrison, M; Walker, W S

    1987-06-01

    The mouse macrophage (M phi) cell line IC-21 preferentially ingests a subpopulation of homologous red blood cells (MRBC) from normal mice. This subpopulation presumably bears the so-called transfusion lesion, a consequence of damage acquired during the drawing and processing of blood. To determine if all damaged MRBC were recognized by a common receptor site on IC-21 M phi, we prepared suspensions of MRBC damaged in vitro by treatment with tannic acid and compared the phagocytic uptake of these cells with those bearing the transfusion lesion. Trypsin treatment of IC-21 M phi rendered them unable to recognize MRBC bearing the transfusion lesion; but it had no effect on the uptake of tannic acid-damaged MRBC, showing that IC-21 M phi have separate recognition sites for these two populations of damaged MRBC. PMID:3474332

  2. Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein.

    PubMed

    Pahuja, Parveen; Srinivasan, Alagiri; Puri, Munish

    2014-04-01

    X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering. PMID:24385107

  3. Zinc- and oxidative property-dependent degradation of pro-caspase-1 and NLRP3 by ziram in mouse macrophages.

    PubMed

    Muroi, Masashi; Tanamoto, Ken-ichi

    2015-06-15

    The NLRP3 inflammasome, composed of caspase-1, NLRP3 and ASC, plays a critical role in the clearance of microbial pathogens. Here, we found that the treatment of mouse macrophages with the zinc-containing dithiocarbamate ziram, a widely used fungicide in agriculture, caused a decrease in pro-caspase-1 and NLRP3 levels while not affecting ASC level. Ziram did not affect levels of pro-caspase-1 and NLRP3 mRNA, and no cleavage products of pro-caspase-1 including p10 subunit, which is an autocleavage product of pro-caspase-1, were detected, indicating that the decrease was associated with degradation of these proteins. The decrease was inhibited by SH-type antioxidants, N-acetyl cysteine, dithiothreitol and 2-mercaptoethanol, or a metal chelator EDTA but not by inhibitors of proteasome, lysosomes, autophagy and matrix metalloproteases. Thiram, a comparator for ziram that does not contain zinc, showed a weaker decrease in protein levels. Furthermore, the zinc-containing dithiocarbamate, zinc diethyldithiocarbamate, efficiently decreased the levels of pro-caspase-1 and NLRP3, whereas dithiocarbamates, dimethyldithiocarbamate and diethyldithiocarbamate without zinc, were less active. The organic zinc compound [3,4-toluenedithiolato(2-)]zinc hydrate did not induce a decrease in protein levels. Ziram also inhibited IL-1β production by macrophages in response to lipopolysaccharide and bacterial clearance during Salmonella infection of macrophage cells. These results indicate that ziram causes degradation of pro-caspase-1 and NLRP3 in a zinc- and oxidative property-dependent manner and suggest that exposure to ziram may compromise the clearance of microbial pathogens. PMID:25929180

  4. Determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and BALB/c mouse macrophages.

    PubMed

    Tamari, Farshad; Tychowski, Joanna; Lorentzen, Laura

    2013-01-01

    The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections. PMID:23748896

  5. Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse.

    PubMed Central

    Wiktor-Jedrzejczak, W; Bartocci, A; Ferrante, A W; Ahmed-Ansari, A; Sell, K W; Pollard, J W; Stanley, E R

    1990-01-01

    Osteopetrotic (op/op) mutant mice suffer from congenital osteopetrosis due to a severe deficiency of osteoclasts. Furthermore, the total number of mononuclear phagocytes is extremely low in affected mice. Serum, 11 tissues, and different cell and organ conditioned media from op/op mice were shown to be devoid of biologically active colony-stimulating factor 1 (CSF-1), whereas all of these preparations from littermate control +/+ and +/op mice contained the growth factor. The deficiency was specific for CSF-1 in that serum or conditioned media from op/op mice possessed elevated levels of at least three other macrophage growth factors. Partial correction of the op/op defect was observed following intraperitoneal implantation of diffusion chambers containing L929 cells, which in culture produce CSF-1 as their sole macrophage growth factor. No rearrangement of the CSF-1 gene in op/op mice was detected by Southern analysis. However, in contrast to control lung fibroblasts, which contained 4.6- and 2.3-kilobase CSF-1 mRNAs, only the 4.6-kilobase species was detected in op/op cells. An alteration in the CSF-1 gene is strongly implicated as the primary defect in op/op mice because they do not contain detectable CSF-1, their defect is correctable by administration of CSF-1, the op locus and the CSF-1 gene map within the same region of mouse chromosome 3, their CSF-1 mRNA biosynthesis is altered, and the op/op phenotype is consistent with the phenotype expected in a CSF-1 deficient mouse. Images PMID:2191302

  6. Mediation of macrophage cytolytic and phagocytic activities by antibodies of different classes and class-specific Fc-receptors.

    PubMed

    Walker, W S

    1977-08-01

    The classes of antibodies that mediate the phagocytosis and cytolysis of 51Cr-labeled chicken erythrocytes by IC-21 macrophages, an established line of mouse peritoneal macrophages, were identified. The phagocytic activity of IC-21 macrophages, as determined by a functional inhibition assay with mouse myeloma proteins, depended mainly on IgM and IgG2a antibodies and to a lesser extent on IgG2b antibodies. Extracellular cytolysis of target cells was mediated solely by IgG2b antibodies. These results correlate with the previously documented specificities of discrete Fc-receptors for IgG2a and IgG2b immunoglobulins on IC-21 cells. Thus, phagocytosis and cytolysis appear to be mediated by antibodies of different classes operating through separate and distinct sites on the surface of IC-21 macrophages. PMID:886183

  7. Peritoneal Dialysis.

    PubMed

    Al-Natour, Mohammed; Thompson, Dustin

    2016-03-01

    Peritoneal dialysis is becoming more important in the management of patients with end-stage renal disease. Because of the efforts of the "Fistula First Breakthrough Initiative," dialysis venous access in the United States has become focused on promoting arteriovenous fistula creation and reducing the number of patients who start dialysis with a tunneled catheter. This is important because tunneled catheters can lead to infection, endocarditis, and early loss of more long-term access. When planned for, peritoneal dialysis can offer patients the opportunity to start dialysis at home without jeopardizing central access or the possibilities of eventual arteriovenous fistula creation. The purpose of this review is to highlight the indications, contraindications, and procedural methods for implanting peritoneal dialysis catheters in the interventional radiology suite. PMID:27011420

  8. Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes.

    PubMed

    Fernández-Arenas, Elena; Bleck, Christopher K E; Nombela, César; Gil, Concha; Griffiths, Gareth; Diez-Orejas, Rosalía

    2009-04-01

    The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO. PMID:19134116

  9. Molecular Responses of Mouse Macrophages to Copper and Copper Oxide Nanoparticles Inferred from Proteomic Analyses*

    PubMed Central

    Triboulet, Sarah; Aude-Garcia, Catherine; Carrière, Marie; Diemer, Hélène; Proamer, Fabienne; Habert, Aurélie; Chevallet, Mireille; Collin-Faure, Véronique; Strub, Jean-Marc; Hanau, Daniel; Van Dorsselaer, Alain; Herlin-Boime, Nathalie; Rabilloud, Thierry

    2013-01-01

    The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents. PMID:23882024

  10. Degradation of connective tissue matrices by macrophages. II. Influence of matrix composition on proteolysis of glycoproteins, elastin, and collagen by macrophages in culture

    SciTech Connect

    Jones, P.A.; Werb, Z.

    1980-12-01

    Thioglycollate-elicited mouse peritoneal macrophages were cultured in contact with the mixture of extracellular matrix proteins produced by rat smooth muscle cells in culture. Both live macrophages and their conditioned media hydrolyzed glycoproteins, elastin, and collagen. Live macrophages also degraded extracellular connective tissue proteins secreted by endothelial cells and fibroblasts. The glycoproteins in the matrix markedly inhibited the rate of digestion of the other macromolecules, particularly elastin. When plasminogen was added to the matrix, activation of plasminogen to plasmin resulted in the hydrolysis of the glycoprotein components, which then allowed the macrophage elastase easier access to its substrate, elastin. Thus, although plasmin has no direct elastinolytic activity, its presence accelerated the rate of hydrolysis of elastin and therefore the rate of matrix degradation. These findings may be important in an understanding of disease states, such as emphysema and atherosclerosis, that are characterized by the destruction of connective tissue.

  11. Troglitazone regulates peroxisome proliferator-activated receptors and inducible nitric oxide synthase in murine ovarian macrophages.

    PubMed

    Minge, Cadence E; Ryan, Natalie K; Van Der Hoek, Kylie H; Robker, Rebecca L; Norman, Robert J

    2006-01-01

    Peroxisome proliferator-activated receptor-gamma (PPARG) and PPAR-alpha (PPARA) control metabolic processes in many cell types and act as anti-inflammatory regulators in macrophages. PPAR-activating ligands include thiazolidinediones (TZDs), such as troglitazone, once frequently used to treat insulin resistance as well as symptoms of polycystic ovary syndrome (PCOS). Since macrophages within the ovary mediate optimal follicle development, TZD actions to improve PCOS symptoms are likely to be partly mediated through these specifically localized immune cells. In mouse ovary, PPARG protein was expressed in granulosa cells and in isolated cells localized to theca, stroma, and corpora lutea, consistent with EMR1+ macrophages. Isolation of immune cells (EMR1+ or H2+) showed that Pparg and Ppara were expressed in ovarian macrophages at much higher levels than in peritoneal macrophages. Ovulatory human chorionic gonadotropin downregulated expression of Pparg and Ppara in EMR1+ ovarian macrophages, but no hormonal responsiveness was observed in H2+ cells. Downstream anti-inflammatory effects of PPARG activation were analyzed by in vitro treatment of isolated macrophages with troglitazone. Interleukin-1 beta (Il1b) expression was not altered, and tumor necrosis factor-alpha (Tnf) expression was affected in peritoneal macrophages only. In ovarian macrophages, inducible nitric oxide synthase (Nos2), an important proinflammatory enzyme that regulates ovulation, was significantly reduced by troglitazone treatment, an effect that was restricted to cells from the preovulatory ovary. Thus, expression of PPARs within ovarian macrophages is hormonally regulated, reflecting the changing roles of these cells during the ovulatory cycle. Additionally, ovarian macrophages respond directly to troglitazone to downregulate expression of proinflammatory Nos2, providing mechanistic information about ovarian effects of TZD treatment. PMID:16192401

  12. Hemopexin down-regulates LPS-induced proinflammatory cytokines from macrophages

    PubMed Central

    Liang, Xueya; Lin, Tian; Sun, Guangjie; Beasley-Topliffe, Laura; Cavaillon, Jean-Marc; Warren, H. Shaw

    2009-01-01

    Detection of LPS in tissues is an integral component of innate immunity that acts to protect against invasion by Gram-negative bacteria. Plasma down-regulates LPS-induced cytokine production from macrophages, thereby limiting systemic inflammation in blood and distant tissues. To identify the protein(s) involved in this process, we used classical biochemical chromatographic techniques to identify fractions of mouse sera that suppress LPS-induced TNF from bone marrow-derived macrophages (BMDMs). Fractionation yielded microgram quantities of a protein that was identified by MS to be hemopexin (Hx). Mouse Hx purified on hemin-agarose beads and rhHx decreased the production of cytokines from BMDMs and peritoneal macrophages induced by LPS. Preincubation of LPS with Hx did not affect the activity of LPS on LAL, whereas preincubation of Hx with macrophages followed by washing resulted in decreased activity of these cells in response to LPS, suggesting that Hx acts on macrophages rather than LPS. Heme-free Hx did not stimulate HO-1 in the macrophages. Purified Hx also decreased TNF and IL-6 from macrophages induced by the synthetic TLR2 agonist Pam3Cys. Our data suggest that Hx, which is an acute-phase protein that increases during inflammation, limits TLR4 and TLR2 agonist-induced macrophage cytokine production directly through a mechanism distinct from HO-1. PMID:19395472

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

    PubMed

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

    2016-01-01

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

  14. Production of leukotrienes by macrophage cells irradiated with ultraviolet light

    SciTech Connect

    Minoui, S.

    1986-01-01

    Mouse peritoneal macrophages were cultured, labelled with /sup 14/C-arachidonic acid, and then were irradiated with UV light (254 nm). Also, some /sup 14/C-arachidonic acid labelled macrophages were treated with Ca-ionophore (A-23187). The UV-treated macrophages produced two to three times as much arachidonic acid metabolites as did the Ca-ionophore treated cells, the UV irradiated cells produced about 20 ng of LTC/sub 4/ and 5 ng of LTB/sub 4/ per million cells, whereas the Ca-ionophore treated cells produced 10 ng LTC/sub 4/ and 1 ng LTB/sub 4/ per million cells. The irradiated cultures also exhibited a high degree of aggregation of viable macrophages around the lysed cells. There was little aggregation in the Ca-ionophore treated cultures. In phagocytosis and cell aggregation leukotrienes are produced by the viable macrophage cells. Leukotrienes are arachidonic acid oxygenation products that are thought to be mediators both in the expression of the immune-based and inflammatory responses. This study shows that macrophage cells under stressful conditions produced by a trauma-causing agent (UV light) respond by producing leukotrienes and chemotactic factors. These responses of the macrophage cells are the result of multiple biochemical events that promote the production of leukotrienes in the cultures.

  15. Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury

    PubMed Central

    Xie, Jieshi; Yang, Le; Tian, Lei; Li, Weiyang; Yang, Lin; Li, Liying

    2016-01-01

    Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation. PMID:27273604

  16. Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury.

    PubMed

    Xie, Jieshi; Yang, Le; Tian, Lei; Li, Weiyang; Yang, Lin; Li, Liying

    2016-01-01

    Macrophage migration inhibitor factor (MIF), a multipotent innate immune mediator, is an upstream component of the inflammatory cascade in diseases such as liver disease. Monocyte chemoattractant protein-1 (MCP-1), a highly representative chemokine, is critical in liver disease pathogenesis. We investigated the role of MIF in regulating hepatocytic MCP-1 expression. MIF and MCP-1 expression were characterized by immunochemistry, RT-PCR, ELISA, and immunoblotting in CCl4-treated mouse liver and isolated hepatocytes. MIF was primarily distributed in hepatocytes, and its expression increased upon acute liver injury. Its expression was also increased in injured hepatocytes, induced by LPS or CCl4, which mimic liver injury in vitro. MIF was expressed earlier than MCP-1, strongly inducing hepatocytic MCP-1 expression. Moreover, the increase in MCP-1 expression induced by MIF was inhibited by CD74- or CD44-specific siRNAs and SB203580, a p38 MAPK inhibitor. Further, CD74 or CD44 deficiency effectively inhibited MIF-induced p38 activation. MIF inhibitor ISO-1 reduced MCP-1 expression and p38 phosphorylation in CCl4-treated mouse liver. Our results showed that MIF regulates MCP-1 expression in hepatocytes of injured liver via CD74, CD44, and p38 MAPK in an autocrine manner, providing compelling information on the role of MIF in liver injury, and implying a new regulatory mechanism for liver inflammation. PMID:27273604

  17. [Peritoneal echinococcosis].

    PubMed

    Vara-Thorbeck, C; Vara-Thorbeck, R

    1986-01-01

    Secondary peritoneal echinococcosis was recorded from 50 in 312 patients (16 per cent) who had been hospitalised for liver echinococcosis. Hydatido and peritoneal hydatidiosis were recorded from 34 of these patients and thus accounted for the two most common pathological forms of secondary peritoneal echinococcosis, according to Dévè. Peritoneal echinococcosis usually is not diagnosed until conspicuous symptoms grow manifest due to cyst growth or other complications. Positive responses were recorded from all the above cases to laboratory tests (eosinophilia in over five to nine per cent) and were also established on the basis of immune reactions, including the complement fixation reaction according to Weinberg, the intracutaneous test by Casoni, and latex echinococcus reaction. Surgery, at present, is the only promising therapeutic approach to the problem. Surgical intervention could not even be avoided by application of mebendazol. Postoperative lethality amounted to four per cent and morbidity to ten per cent. They were comparatively low, measured by the generally poor prognosis of the disease. PMID:3776378

  18. Dialysis - peritoneal

    MedlinePlus

    ... The number of exchanges and amount of dwell time depends on the method of PD you use and other factors. Your ... PD: Continuous ambulatory peritoneal dialysis (CAPD) . For this ... routine until it is time to drain the fluid. You are not hooked ...

  19. Counter-regulatory paracrine actions of FGF-23 and 1,25(OH)2 D in macrophages.

    PubMed

    Han, Xiaobin; Li, Linqiang; Yang, Jiancheng; King, Gwendalyn; Xiao, Zhousheng; Quarles, Leigh Darryl

    2016-01-01

    Mechanisms underlying the association between fibroblastic growth factor 23 (FGF-23) and inflammation are uncertain. We found that FGF-23 was markedly up-regulated in LPS/INF-γ-induced proinflammatory M1 macrophages and Hyp mouse-derived peritoneal macrophages, but not in IL-4-induced M2 anti-inflammatory macrophages. NF-КB and JAK/STAT1 pathways mediated the increased transcription of FGF-23 in response to M1 polarization. FGF-23 stimulated TNF-α, but not IL-6, expression in M0 macrophages and suppressed Arginase-1 expression in M2 macrophages through FGFR-mediated mechanisms. 1,25(OH)2 D stimulated Arginase-1 expression and inhibited FGF-23 stimulation of TNF-α. FGF-23 has proinflammatory paracrine functions and counter-regulatory actions to 1,25(OH)2 D on innate immune responses. PMID:26762170

  20. Synergistic interaction of catecholamine hormones and Mycobacterium avium results in the induction of interleukin-10 mRNA expression by murine peritoneal macrophages.

    PubMed

    Chen, L; Boomershine, C; Wang, T; Lafuse, W P; Zwilling, B S

    1999-01-01

    The results of this investigation provides evidence that catecholamine hormones interact with macrophages that are infected with Mycobacterium avium resulting in the induction of IL-10 mRNA and protein. The effect of catecholamine hormones was prevented by treating the cells with the beta-adrenergic receptor antagonist propranolol but not by alpha-adrenergic antagonist phentolamine. The effect of catecholamine stimulation was mimicked by the addition of beta-2 adrenergic agonists and by the addition of cAMP to the infected macrophage cultures. These observations suggest that sympathetic nervous system activation together with microbial infection results in a synergistic interaction that could result in the control of inflammatory processes. PMID:10378878

  1. Difference in the toxicity mechanism between ion and nanoparticle forms of silver in the mouse lung and in macrophages.

    PubMed

    Arai, Yuta; Miyayama, Takamitsu; Hirano, Seishiro

    2015-02-01

    The health effects of silver nanoparticles (AgNPs) have not been well investigated, despite AgNPs now being widely used in consumer products. We investigated the metabolic behavior and toxicity of AgNPs in comparison to silver nitrate (AgNO3) both in vivo and in vitro. AgNPs (20 nm diameter) suspended in 1% albumin solution or AgNO3 solution was injected into the mouse lung. Less than 1% of the initial dose of AgNPs and more than 7% of the initial dose of AgNO3 was recovered in the liver 4h after administration, suggesting that the ionic form of silver was absorbed by the lung tissue and entered the systemic circulation more efficiently than AgNPs. The pro-inflammatory cytokine, IL-1β, and neutrophils in bronchoalveolar lavage fluid (BALF) increased following intratracheal instillation of AgNPs or AgNO3. AgNO3 recruited more neutrophils in the alveolar space than did AgNPs. In the in vitro study, AgNO3 was more cytotoxic than 20, 60, or 100 nm diameter AgNPs in a mouse macrophage cell line (J774.1). To investigate the intracellular distribution of Ag in detail, J774.1 cells were exposed to AgNO3 or 20 nm AgNPs and the distribution of Ag to cytosolic proteins was investigated using HPLC-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Ag was mainly distributed to metallothioneins (MT) and to high molecular weight proteins in AgNO3- and AgNPs-exposed cells, respectively. Confocal laser microscopic examination of LysoTracker(®)-labeled cells indicated that AgNPs were colocalized with lysosomes in J774.1 cells. These results suggest that AgNPs were transported to lysosomes and only gradually dissolved in the macrophages, causing milder inflammatory stimulation in the mouse lung compared to AgNO3. PMID:25527144

  2. Induction of M2 regulatory macrophages through the β2 adrenergic receptor with protection during endotoxemia and acute lung injury

    PubMed Central

    Grailer, Jamison J.; Haggadone, Mikel D.; Sarma, J. Vidya; Zetoune, Firas S.; Ward, Peter A.

    2014-01-01

    Main drivers of acute inflammation are macrophages, which are known to have receptors for catecholamines. Based on their function, macrophages are broadly categorized as either M1 (pro-inflammatory) or M2 phenotypes (anti-inflammatory). In this study, we investigated catecholamine-induced alterations in the phenotype of activated macrophages. In the presence of lipopolysaccharide (LPS), mouse peritoneal macrophages acquired an M1 phenotype. However, the co-presence of LPS and either epinephrine or norepinephrine resulted in a strong M2 phenotype including high levels of arginase-1 and interleukin (IL) -10, and reduced expression of M1 markers. Furthermore, epinephrine enhanced macrophage phagocytosis and promoted type 2 T cell responses in vitro, which are known features of M2 macrophages. Analysis of M2 subtype-specific markers indicated that LPS and catecholamine co-treated macrophages were not alternatively activated, but of the regulatory macrophage subtype. Interestingly, catecholamines signaled through the β2 adrenergic receptor, but not the canonical cAMP/protein kinase A signaling pathway. Instead, the M2 pathway required an intact phosphoinositol 3-kinase pathway. Blockade of the β2 adrenergic receptor reduced survival and enhanced injury during mouse models of endotoxemia and LPS-induced acute lung injury, respectively. These results demonstrate a role for the β2 adrenergic receptor in promoting the M2 macrophage phenotype. PMID:24642449

  3. Use of the short-term inflammatory response in the mouse peritoneal cavity to assess the biological activity of leached vitreous fibers.

    PubMed Central

    Donaldson, K; Addison, J; Miller, B G; Cullen, R T; Davis, J M

    1994-01-01

    We used a special-purpose glass microfiber sample, Johns-Manville Code 100/475, to study the effects of various acid and alkali treatments on biological activity as assessed by inflammation in the mouse peritoneal cavity, the leaching of Si, and the phase contrast optical microscopy (PCOM) fiber number. We used mild and medium treatments with oxalic acid and Tris buffer and harsh treatment with concentrated HCl and NaOH. Mild oxalic acid and Tris treatment for 2 weeks had no effect on any of the end-points, but prolonging the mild oxalic acid treatment time to 2 months reduced the biological activity and the fiber number. Medium oxalic acid treatment reduced the biological activity and the fiber number and caused a loss of Si. Medium Tris alkali treatment reduced the PCOM-countable fibers and the biological activity but did not cause a substantial loss of Si. Harsh treatment with strong HCl did not affect the fiber number or cause leaching but the biological activity was reduced; strong NaOH reduced the fiber number and biological activity, and caused marked leaching of Si. The medium oxalic acid conditions (pH 1.4) were more acid than those found in lung cells but produced the same effects (reduction in fiber number and biological activity) as the more physiological mild treatment (pH 4.0), when prolonged. This study suggests that medium oxalic acid treatment can be used as a short-term assay to compare loss of Si, reduction in fiber number, and change in biological activity of vitreous fibers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7882922

  4. Circulating cell-free DNA indicates M1/M2 responses during septic peritonitis.

    PubMed

    Xin, Yi; Gao, Xingjuan; Wang, Wenxiao; Xu, Xiaojuan; Yu, Lijuan; Ju, Xiuli; Li, Aimin

    2016-09-01

    Circulating cell-free DNA (cfDNA) has been widely suggested as clinical indicator in diseases, including sepsis. It was thought that the cfDNA was coming from the cell lysis, necrosis and apoptosis caused by tissue damages during sepsis. M1 or M2 macrophage-type responses kill or repair in vivo, which is highly relevant with the tissue damages in sepsis. The correlation between cfDNA and M1/M2 responses during sepsis was never investigated. Here, we used bacteria injection induced septic peritonitis mouse model in both M1-dominant C57bl/6 and M2-dominant Balb/c mouse strains. We found that M2-dominant Balb/c mice showed better prognosis of septic peritonitis than C57bl/6 mice, which is corresponded with lower level of cfDNA in septic Balb/c mice compared to septic C57bl/6 mice. By assessing the M1 and M2 related cytokines in both septic Balb/c and C57bl/6 mice, we found out that Balb/c mice has lower tumor necrosis factor α (TNFα) and higher interleukin 10 (IL-10) productions than C57bl/6 mice during septic peritonitis. Especially, when monitoring the monocyte subtypes in peripheral blood of these septic mice, we found out that C57bl/6 showed higher inflammatory (Ly6C(high)) monocyte (corresponding to M1 macrophage) proportion than Balb/c mice. Interestingly, we find out that cfDNA is highly correlated with the ratio of Ly6C(high) monocytes versus Ly6C(low) monocytes, which represents M1/M2 (killing/healing) responses. Our study suggested that the cfDNA is a good indicator for evaluating M1/M2 responses in septic peritonitis. PMID:27335257

  5. Endogenous interferon production by endotoxin-responsive macrophages provides an autostimulatory differentiation signal.

    PubMed Central

    Vogel, S N; Fertsch, D

    1984-01-01

    Previous studies have demonstrated that peritoneal macrophages (resident or thioglycolate-induced) derived from mouse strains fully responsive to gram-negative endotoxins continue to differentiate in vitro, as evidenced by an increased capacity to phagocytose via the Fc receptor with time in culture. In contrast, macrophages derived from endotoxin-hyporesponsive mouse strains (e.g., C3H/HeJ or C57BL/10ScN) exhibit no such increase in phagocytic capacity, and, in fact, significantly lose the capacity to phagocytose particles opsonized with immunoglobulin G with time in culture. This defect was found to be fully correctable by the addition to the cultures of an exogenous source of alpha, beta, or gamma interferon. In this study, we compared C3H/HeN (endotoxin-responsive) and C3H/HeJ (endotoxin-responsive) and C3H/HeJ (endotoxin-hyporesponsive) macrophages in an attempt to elucidate the mechanism responsible for this difference in phagocytic (differentiative) potential. The following observations support the hypothesis that endotoxin-responsive macrophages, in contrast to endotoxin-hyporesponsive macrophages, produce significantly higher levels of an autostimulatory differentiation signal that appears to be macrophage-derived interferon. (i) Anti-alpha/beta-interferon antibody greatly reduces the ability of C3H/HeN macrophages to phagocytose opsonized erythrocytes: (ii) C3H/HeJ macrophages can be made more phagocytic by coculture with C3H/HeN macrophages or by treatment with supernatants derived from C3H/HeN macrophage cultures; and (iii) C3H/HeN macrophages spontaneously lose Mac-1 antigen with time in culture. C3H/HeJ macrophages must be interferon-treated to be equivalently down-regulated. PMID:6378797

  6. Regulation of mannose receptor synthesis and turnover in mouse J774 macrophages.

    PubMed

    Fiani, M L; Beitz, J; Turvy, D; Blum, J S; Stahl, P D

    1998-07-01

    The mannose receptor, present on the plasma membrane of macrophages, promotes the internalization of glycoproteins and glycoconjugates via both endocytic and phagocytic pathways. The expression of this receptor is tightly modulated during monocyte/Mphi differentiation and cellular activation. We isolated clonal populations from murine J774 macrophage tumor cells, which differ in their surface expression of functional mannose receptors. To examine the potential mechanisms regulating receptor function in these cell lines, the interaction of receptor with ligand as well as receptor synthesis and degradation was analyzed. J774 clones with both high and low levels of mannose receptor activity were found to synthesize significant amounts of receptor protein, suggesting that the protein may be regulated at the level of synthesis and degradation. In J774 clones expressing very low receptor activity and protein, the half-life of mannose receptor molecules was substantially decreased. The evolution of multiple mechanisms modulating mannose receptor function may be critical in fine-tuning the role of this receptor in antigen processing and in scavenger and host defense functions. PMID:9665280

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

    PubMed Central

    Lima, Djalma S.; Zamboni, Dario S.

    2010-01-01

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

  8. Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

    SciTech Connect

    Chen, Shayan; Feng, Wenli; Yang, Xiao; Yang, Wanzhu; Ru, Yongxin; Liao, Jinfeng; Wang, Lina; Lin, Yongmin; Ren, Qian; Zheng, Guoguang

    2014-04-18

    Highlights: • We study the impact of leukemic microenvironment on P2X family receptors in Mφs. • Bone marrow and spleen Mφs are studied in Notch1-induced mouse leukemia model. • Increased expression of P2X7R is found in Mφs during the development of leukemia. • Elevated P2X7R-mediated calcium response is found in Mφs at late stage of leukemia. • More apoptotic Mφs are found in bone marrow and spleen at late stage of leukemia. - Abstract: Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca{sup 2+} response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia.

  9. Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling

    SciTech Connect

    Su, Dian; Gaffrey, Matthew J.; Guo, Jia; Hatchell, Kayla E.; Chu, Rosalie K.; Clauss, Therese RW; Aldrich, Joshua T.; Wu, Si; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.; Thrall, Brian D.; Qian, Weijun

    2014-02-11

    Protein S-glutathionylation (SSG) is an important regulatory posttranslational modification of protein cysteine (Cys) thiol redox switches, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols and enrichment using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys-sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG-modification compared to controls.. This approach was extended to identify potential SSG modified Cys-sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys-sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment. These proteins covered a range of molecular types and molecular functions with free radical scavenging, and cell death and survival included as the most significantly enriched functional categories. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of S-glutathionylated proteins. The analytical strategy also provides a unique approach to determining the major pathways and cell processes most susceptible to glutathionylation at a proteome-wide scale.

  10. Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling.

    PubMed

    Su, Dian; Gaffrey, Matthew J; Guo, Jia; Hatchell, Kayla E; Chu, Rosalie K; Clauss, Therese R W; Aldrich, Joshua T; Wu, Si; Purvine, Sam; Camp, David G; Smith, Richard D; Thrall, Brian D; Qian, Wei-Jun

    2014-02-01

    S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible

  11. Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages

    PubMed Central

    Eichenfield, Dawn Z; Troutman, Ty Dale; Link, Verena M; Lam, Michael T; Cho, Han; Gosselin, David; Spann, Nathanael J; Lesch, Hanna P; Tao, Jenhan; Muto, Jun; Gallo, Richard L; Evans, Ronald M; Glass, Christopher K

    2016-01-01

    Although macrophages can be polarized to distinct phenotypes in vitro with individual ligands, in vivo they encounter multiple signals that control their varied functions in homeostasis, immunity, and disease. Here, we identify roles of Rev-erb nuclear receptors in regulating responses of mouse macrophages to complex tissue damage signals and wound repair. Rather than reinforcing a specific program of macrophage polarization, Rev-erbs repress subsets of genes that are activated by TLR ligands, IL4, TGFβ, and damage-associated molecular patterns (DAMPS). Unexpectedly, a complex damage signal promotes co-localization of NF-κB, Smad3, and Nrf2 at Rev-erb-sensitive enhancers and drives expression of genes characteristic of multiple polarization states in the same cells. Rev-erb-sensitive enhancers thereby integrate multiple damage-activated signaling pathways to promote a wound repair phenotype. DOI: http://dx.doi.org/10.7554/eLife.13024.001 PMID:27462873

  12. Apocynin suppresses the progression of atherosclerosis in apoE-deficient mice by inactivation of macrophages

    SciTech Connect

    Kinoshita, Hiroyuki; Matsumura, Takeshi; Ishii, Norio; Fukuda, Kazuki; Senokuchi, Takafumi; Motoshima, Hiroyuki; Kondo, Tatsuya; Taketa, Kayo; Kawasaki, Shuji; Hanatani, Satoko; Takeya, Motohiro; Nishikawa, Takeshi; Araki, Eiichi

    2013-02-08

    Highlights: ► We examined the anti-athrogenic effect of apocynin in atherosclerotic model mice. ► Apocynin prevented atherosclerotic lesion formation. ► Apocynin suppressed ROS production in aorta and in macrophages. ► Apocynin suppressed cytokine expression and cell proliferation in macrophages. ► Apocynin may be beneficial compound for the prevention of atherosclerosis. -- Abstract: Production of reactive oxygen species (ROS) and other proinflammatory substances by macrophages plays an important role in atherogenesis. Apocynin (4-hydroxy-3-methoxy-acetophenone), which is well known as a NADPH oxidase inhibitor, has anti-inflammatory effects including suppression of the generation of ROS. However, the suppressive effects of apocynin on the progression of atherosclerosis are not clearly understood. Thus, we investigated anti-atherosclerotic effects of apocynin using apolipoprotein E-deficient (apoE{sup –/–}) mice in vivo and in mouse peritoneal macrophages in vitro. In atherosclerosis-prone apoE{sup –/–} mice, apocynin suppressed the progression of atherosclerosis, decreased 4-hydroxynonenal-positive area in atherosclerotic lesions, and mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in aorta. In mouse peritoneal macrophages, apocynin suppressed the Ox-LDL-induced ROS generation, mRNA expression of MCP-1, IL-6 and granulocyte/macrophage colony-stimulating factor, and cell proliferation. Moreover, immunohistochemical studies revealed that apocynin decreased the number of proliferating cell nuclear antigen-positive macrophages in atherosclerotic lesions of apoE{sup –/–} mice. These results suggested that apocynin suppressed the formation of atherosclerotic lesions, at least in part, by inactivation of macrophages. Therefore, apocynin may be a potential therapeutic material to prevent the progression of atherosclerosis.

  13. Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1.

    PubMed

    Köröskényi, Krisztina; Kiss, Beáta; Szondy, Zsuzsa

    2016-07-01

    Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway. PMID:27066978

  14. Anti-inflammatory effects of vina-ginsenoside R2 and majonoside R2 isolated from Panax vietnamensis and their metabolites in lipopolysaccharide-stimulated macrophages.

    PubMed

    Jeong, Jin-Ju; Van Le, Thi Hong; Lee, Sang-Yun; Eun, Su-Hyeon; Nguyen, Minh Duc; Park, Jeong Hill; Kim, Dong-Hyun

    2015-09-01

    Panax vietnamensis Ha et Grushv., with its main constituents vina-ginsenoside R2 (VR2) and majonoside R2 (MR2), is used in traditional folk medicine in the hill tribes of Vietnam for anti-fatigue, anti-inflammatory, and life-saving purposes. In a preliminary study, VR2 and MR2 were shown to be metabolized to pseudoginsenoside RT4 (PRT4) and ocotillol by human gut microbiota. Therefore, we measured the anti-inflammatory effects of VR2, MR2, and their metabolites in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. Among these ginsenosides, only VR2 exhibited cytotoxicity against peritoneal macrophages. MR2, PRT4, and ocotillol inhibited LPS-stimulated transcription factor (NF)-κB activation, and expression of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-1. However, these ginsenosides did not inhibit peptidoglycan-induced NF-κB activation in the macrophages. These three ginsenosides also inhibited LPS-stimulated cyclooxygenase-2 and inducible NO synthase expression, and phosphorylation of NF-κB signal molecules IL-1 receptor-associated kinase 1 and tumor growth factor-β-activated kinase 1 in peritoneal macrophages. Treatment with either PRT4 or ocotillol inhibited the Alexa Fluor 488-conjugated LPS-mediated shift of macrophages, as observed by flow cytometry. They also potently inhibited the binding of LPS to TLR4 on peritoneal macrophages, both with and without transfected MyD88 siRNA. Among the tested ginsenosides, ocotillol exhibited the strongest inhibitory effect on inflammation in LPS-stimulated macrophages via the NF-κB signaling pathway. Based on these findings, orally administered VR2 and MR2 of P. vietnamensis may be metabolized to ocotillol via PRT4, and the metabolites, particularly ocotillol, may inhibit inflammation by inhibiting the binding of LPS to TLR4 on macrophages. PMID:26256699

  15. Further characterization of macrophage adsorption of suppressor cell activity from tumor-allosensitized spleen

    SciTech Connect

    Zografos-Miller, L.E.; Argyris, B.F.

    1983-06-01

    Suppressor cell activity from P815-allosensitized C57BL/6 spleen can be decreased by incubating the tumor-allosensitized spleen cells on monolayers of thioglycollate-stimulated BDF1 peritoneal macrophages for 2 or 4 hr. The adsorption response appears to be specific for macrophages, because adsorption of suppressor cell activity does not occur following incubation of P815-allosensitized spleen cells on confluent monolayers of mouse spleen cells or mouse embryonic fibroblasts. Pretreatment of macrophage monolayers with X irradiation (2,000 rads) or anti-Thy 1.2 serum (and complement) does not affect their ability to bind suppressor cell activity. Adsorption of suppressor cell activity from P815-allosensitized spleen can also be carried out by proteose peptone-stimulated or Corynebacterium parvum-stimulated macrophages. Blockage of macrophage Fc receptors decreases the ability of thioglycollate-stimulated macrophages to adsorb suppressor cell activity. Monolayers of P815 or P388 cells, two cell types positive for Fc receptors, are unable to adsorb suppressor cell activity from the tumor-allosensitized spleen. The significance of our findings is discussed in terms of the relationship between macrophages and suppressor cells in the immune response to normal or tumor allografts.

  16. Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

    PubMed Central

    Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

    1998-01-01

    The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

  17. Zinc Insufficiency Mediates Ethanol-Induced Alveolar Macrophage Dysfunction in the Pregnant Female Mouse

    PubMed Central

    Konomi, Juna V.; Harris, Frank L.; Ping, Xiao-Du; Gauthier, Theresa W.; Brown, Lou Ann S.

    2015-01-01

    Aims: (a) Establish the minimum number of weeks of chronic ethanol ingestion needed to perturb zinc homeostasis, (b) Examine intracellular zinc status in the alveolar macrophages (AMs) when ethanol ingestion is combined with pregnancy, (c) Investigate whether in vitro zinc treatment reverses the effects of ethanol ingestion on the AM. Methods: C57BL/6 female mice were fed a liquid diet (±25% ethanol-derived calories) during preconception and pregnancy. The control group was pair-fed to the ethanol group. In the isolated AMs, we measured intracellular AM zinc levels, zinc transporter expression, alternative activation and phagocytic index. Zinc acetate was added to some cells prior to analysis. Results: Intracellular zinc levels in the AM decreased within 3 weeks of ethanol ingestion. After ethanol ingestion prior to and during pregnancy, zinc transporter expression and intracellular zinc levels were decreased in the AMs when compared with controls. Bacterial clearance was decreased because the AMs were alternatively activated. In vitro additions of zinc reversed these effects of ethanol. Conclusion: Ethanol ingestion prior to and during pregnancy perturbed AM zinc balance resulting in impaired bacterial clearance, but these effects were ameliorated by in vitro zinc treatments. PMID:25371044

  18. Genome-wide analysis of TIAR RNA ligands in mouse macrophages before and after LPS stimulation

    PubMed Central

    Kharraz, Yacine; Lefort, Anne; Libert, Frédérick; Mann, Christopher J.; Gueydan, Cyril; Kruys, Véronique

    2016-01-01

    TIA-1 related protein (TIAR) is a RNA-binding protein involved in several steps of gene expression such as RNA splicing Aznarez et al. (2008) [1] and translation Piecyk et al. (2000) [2]. TIAR contains three RNA recognition motifs (RRMs) allowing its interaction with specific sequences localized in the untranslated regions (UTRs) of several mRNAs. In myeloid cells, TIAR has been shown to bind and regulate the translation and stability of various mRNA-encoding proteins important for the inflammatory response, such as TNFα Piecyk et al. (2000), Gueydan et al. (1999) [2], [3], Cox-2 Cok et al. (2003) [4] or IL-8 Suswam et al. (2005) [5]. Here, we generated two macrophage-like RAW 264.7 cell lines expressing either a tagged full-length TIAR protein or a RRM2-truncated mutant unable to bind RNA with high affinity Dember et al. (1996), Kim et al. (2013) . By a combination of RNA-IP and microarray analysis (RIP-chip), we identified mRNAs specifically bound by the full-length protein both in basal conditions and in response to LPS (GSE77577). PMID:26981431

  19. Baicalin ameliorates experimental inflammatory bowel disease through polarization of macrophages to an M2 phenotype.

    PubMed

    Zhu, Wei; Jin, Zaishun; Yu, Jianbo; Liang, Jun; Yang, Qingdong; Li, Fujuan; Shi, Xuekui; Zhu, Xiaodong; Zhang, Xiaoli

    2016-06-01

    Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract. Baicalin, originally isolated from the root of the Chinese herb Huangqin (Scutellaria baicalensis Georgi) and its main active ingredient, has a protective effect against inflammatory responses in several diseases. The present study investigated the effects of baicalin on macrophage polarization and its therapeutic role in IBD. Murine peritoneal macrophages and mice with colitis were treated with baicalin. Macrophage subset distribution, M1 and M2 macrophage-associated mRNA expression, and interferon regulatory factor 4 and 5 (IRF4 and IRF5) expression were analyzed. siRNA transfection into mouse peritoneal macrophages was utilized to suppress IRF4. Fluorescence-activated cell sorting, western blot, and real-time PCR analyses were performed. Baicalin (50μM) limited lipopolysaccharide (LPS)-induced M1 macrophage polarization; decreased LPS-induced tumor necrosis factor α, interleukin (IL)-23, and IRF5 expression; and increased IL-10, arginase-1 (Arg-1), and IRF4 expression. siRNA-mediated IRF4 silencing significantly impaired baicalin activity. Furthermore, pretreatment with baicalin (100mg/kg) in mice with dextran sodium sulfate (DSS)-induced colitis ameliorated the severity of colitis and significantly decreased the disease activity index (baicalin group, 3.33±0.52 vs. DSS group, 5.67±1.03). Baicalin (100mg/kg) also repressed IRF5 protein expression and promoted IRF4 protein expression in the lamina propria mononuclear cells, and induced macrophage polarization to the M2 phenotype. In summary, our results showed that baicalin upregulates IRF4 protein expression and reverses LPS-induced macrophage subset redistribution. Thus, baicalin alleviates DSS-induced colitis by modulating macrophage polarization to the M2 phenotype. PMID:27039210

  20. 9-cis β-Carotene Increased Cholesterol Efflux to HDL in Macrophages.

    PubMed

    Bechor, Sapir; Zolberg Relevy, Noa; Harari, Ayelet; Almog, Tal; Kamari, Yehuda; Ben-Amotz, Ami; Harats, Dror; Shaish, Aviv

    2016-01-01

    Cholesterol efflux from macrophages is a key process in reverse cholesterol transport and, therefore, might inhibit atherogenesis. 9-cis-β-carotene (9-cis-βc) is a precursor for 9-cis-retinoic-acid (9-cis-RA), which regulates macrophage cholesterol efflux. Our objective was to assess whether 9-cis-βc increases macrophage cholesterol efflux and induces the expression of cholesterol transporters. Enrichment of a mouse diet with βc from the alga Dunaliella led to βc accumulation in peritoneal macrophages. 9-cis-βc increased the mRNA levels of CYP26B1, an enzyme that regulates RA cellular levels, indicating the formation of RA from βc in RAW264.7 macrophages. Furthermore, 9-cis-βc, as well as all-trans-βc, significantly increased cholesterol efflux to high-density lipoprotein (HDL) by 50% in RAW264.7 macrophages. Likewise, food fortification with 9-cis-βc augmented cholesterol efflux from macrophages ex vivo. 9-cis-βc increased both the mRNA and protein levels of ABCA1 and apolipoprotein E (APOE) and the mRNA level of ABCG1. Our study shows, for the first time, that 9-cis-βc from the diet accumulates in peritoneal macrophages and increases cholesterol efflux to HDL. These effects might be ascribed to transcriptional induction of ABCA1, ABCG1, and APOE. These results highlight the beneficial effect of βc in inhibition of atherosclerosis by improving cholesterol efflux from macrophages. PMID:27447665

  1. 9-cis β-Carotene Increased Cholesterol Efflux to HDL in Macrophages

    PubMed Central

    Bechor, Sapir; Zolberg Relevy, Noa; Harari, Ayelet; Almog, Tal; Kamari, Yehuda; Ben-Amotz, Ami; Harats, Dror; Shaish, Aviv

    2016-01-01

    Cholesterol efflux from macrophages is a key process in reverse cholesterol transport and, therefore, might inhibit atherogenesis. 9-cis-β-carotene (9-cis-βc) is a precursor for 9-cis-retinoic-acid (9-cis-RA), which regulates macrophage cholesterol efflux. Our objective was to assess whether 9-cis-βc increases macrophage cholesterol efflux and induces the expression of cholesterol transporters. Enrichment of a mouse diet with βc from the alga Dunaliella led to βc accumulation in peritoneal macrophages. 9-cis-βc increased the mRNA levels of CYP26B1, an enzyme that regulates RA cellular levels, indicating the formation of RA from βc in RAW264.7 macrophages. Furthermore, 9-cis-βc, as well as all-trans-βc, significantly increased cholesterol efflux to high-density lipoprotein (HDL) by 50% in RAW264.7 macrophages. Likewise, food fortification with 9-cis-βc augmented cholesterol efflux from macrophages ex vivo. 9-cis-βc increased both the mRNA and protein levels of ABCA1 and apolipoprotein E (APOE) and the mRNA level of ABCG1. Our study shows, for the first time, that 9-cis-βc from the diet accumulates in peritoneal macrophages and increases cholesterol efflux to HDL. These effects might be ascribed to transcriptional induction of ABCA1, ABCG1, and APOE. These results highlight the beneficial effect of βc in inhibition of atherosclerosis by improving cholesterol efflux from macrophages. PMID:27447665

  2. C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

    NASA Astrophysics Data System (ADS)

    Russ, K. A.; Elvati, P.; Parsonage, T. L.; Dews, A.; Jarvis, J. A.; Ray, M.; Schneider, B.; Smith, P. J. S.; Williamson, P. T. F.; Violi, A.; Philbert, M. A.

    2016-02-01

    There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern

  3. Lipocalin-2 promotes m1 macrophages polarization in a mouse cardiac ischaemia-reperfusion injury model.

    PubMed

    Cheng, L; Xing, H; Mao, X; Li, L; Li, X; Li, Q

    2015-01-01

    Ischaemia-reperfusion (IR) injury is a major issue in cardiac transplantation. Inflammatory processes play a major role in myocardial IR injury. Lipocalin-2 (Lcn2), which is also known as neutrophil gelatinase-associated lipocalin, has multiple functions that include the regulation of cell death/survival, cell migration/invasion, cell differentiation and iron delivery. In our study, the hearts of C57BL/6 mice were flushed with and stored in cold Bretschneider solution for 8 h and then transplanted into a syngeneic recipient. We found that Lcn2 neutralization decreased the recruitment of neutrophils and macrophages. Troponin T (TnT) production, 24 h after myocardial IR injury, was reduced through anti-Lcn2 antibody administration. The cardiac output at 60 mmHg of afterload pressure was significantly increased in hearts administrated with anti-Lcn2 antibody administration (anti-Lcn-2: 58.9 ± 5.62 ml/min; control: 25.8 ± 4.1 ml/min; P < 0.05). Anti-Lcn2 antibody treatment suppressed M1 marker (IL-12, IL-23 and iNOS) expression but increased M2 marker (IL-10, Arg1 and Mrc1) expression. Furthermore, in our vitro and vivo experiments, we found that anti-Lcn2 antibody treatment failed to induce M1-related gene expression in response to LPS and that Lcn2 neutralization enhanced the expression of M2-related genes following IL-4 treatment. In conclusion, Lcn2 promotes M1 polarization, and Lcn2 neutralization attenuates cardiac IR injury. PMID:25359467

  4. Toxicity and oxidative stress induced by semiconducting polymer dots in RAW264.7 mouse macrophages

    NASA Astrophysics Data System (ADS)

    Ye, Fangmao; White, Collin C.; Jin, Yuhui; Hu, Xiaoge; Hayden, Sarah; Zhang, Xuanjun; Gao, Xiaohu; Kavanagh, Terrance J.; Chiu, Daniel T.

    2015-05-01

    The rapid development and acceptance of PDots for biological applications depends on an in depth understanding of their cytotoxicity. In this paper, we performed a comprehensive study of PDot cytotoxicity at both the gross cell effect level (such as cell viability, proliferation and necrosis) and more subtle effects (such as redox stress) on RAW264.7 cells, a murine macrophage cell line with high relevance to in vivo nanoparticle disposition. The redox stress measurements assessed were inner mitochondrial membrane lipid peroxidation (nonyl-acridine orange, NAO), total thiol level (monobromobimane, MBB), and pyridine nucleotide redox status (NAD(P)H autofluorescence). Because of the extensive work already performed with QDots on nanotoxicity and also because of their comparable size, QDots were chosen as a comparison/reference nanoparticle for this study. The results showed that PDots exhibit cytotoxic effects to a much lesser degree than their inorganic analogue (QDots) and are much brighter, allowing for much lower concentrations to be used in various biological applications. In addition, at lower dose levels (2.5 nM to 10 nM) PDot treatment resulted in higher total thiol level than those found with QDots. At higher dose levels (20 nM to 40 nM) QDots caused significantly higher thiol levels in RAW264.7 cells, than was seen with PDots, suggesting that QDots elicit compensation to oxidative stress by upregulating GSH synthesis. At the higher concentrations of QDots, NAD(P)H levels showed an initial depletion, then repletion to a level that was greater than vehicle controls. PDots showed a similar trend but this was not statistically significant. Because PDots elicit less oxidative stress and cytotoxicity at low concentrations than QDots, and because they exhibit superior fluorescence at these low concentrations, PDots are predicted to have enhanced utility in biomedical applications.

  5. C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages.

    PubMed

    Russ, K A; Elvati, P; Parsonage, T L; Dews, A; Jarvis, J A; Ray, M; Schneider, B; Smith, P J S; Williamson, P T F; Violi, A; Philbert, M A

    2016-02-21

    There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis. PMID:26866469

  6. Immunostimulatory early phenotype of tumor-associated macrophages does not predict tumor growth outcome in an HLA-DR mouse model of prostate cancer.

    PubMed

    Riabov, Vladimir; Kim, David; Chhina, Surmeet; Alexander, Richard B; Klyushnenkova, Elena N

    2015-07-01

    Tumor-associated macrophages (TAM) were shown to support the progression of many solid tumors. However, anti-tumor properties of TAM were also reported in several types of cancer. Here, we investigated the phenotype and functions of TAM in two transgenic mouse models of prostate cancer that display striking differences in tumor growth outcome. Mice expressing prostate-specific antigen (PSA) as a self-antigen specifically in prostate (PSAtg mice) rejected PSA-expressing transgenic adenocarcinoma of mouse prostate (TRAMP) tumors. However, the introduction of HLA-DRB1*1501 (DR2b) transgene presenting PSA-derived peptides in a MHC class II-restricted manner exacerbated the growth of TRAMP-PSA tumors in DR2bxPSA F 1 mice. Despite the difference in tumor growth outcome, tumors in both strains were equally and intensively infiltrated by macrophages on the first week after tumor challenge. TAM exhibited mixed M1/M2 polarization and simultaneously produced pro-inflammatory (TNFα, IL1β) and anti-inflammatory (IL10) cytokines. TAM from both mouse strains demonstrated antigen-presenting potential and pronounced immunostimulatory activity. Moreover, they equally induced apoptosis of tumor cells. In vivo depletion of macrophages in DR2bxPSA F 1 but not PSAtg mice aggravated tumor growth suggesting that macrophages more strongly contribute to anti-tumor immunity when specific presentation of PSA to CD4+ T cells is possible. In summary, we conclude that in the early stages of tumor progression, the phenotype and functional properties of TAM did not predict tumor growth outcome in two transgenic prostate cancer models. Furthermore, we demonstrated that during the initial stage of prostate cancer development, TAM have the potential to activate T cell immunity and mediate anti-tumor effects. PMID:25893810

  7. Mouse macrophage polarity and ROCK1 activity depend on RhoA and non-apoptotic Caspase 3.

    PubMed

    Liu, Yianzhu; Minze, Laurie J; Mumma, Lindsay; Li, Xian C; Ghobrial, Rafik M; Kloc, Malgorzata

    2016-02-15

    The macrophages have different subtypes with different functions in immune response and disease. It has been generally accepted that M1 macrophages are responsible for stimulation of immune system and inflammation while M2 macrophages play a role in tissue repair. Irrespective of the type, macrophage functions depend on actin cytoskeleton, which is under the control of small GTPase RhoA pathway and its downstream effector ROCK1. We generated RhoA-deleted macrophages and compared the effect of RhoA deletion on M0, M1 and M2 macrophage phenotype. Our studies showed that, unexpectedly, the RhoA deletion did not eliminate macrophage ROCK1 expression and increased ROCK1 activity. The RhoA deletion effect on macrophage phenotype, structure and polarity was different for each subtype. Moreover, our study indicates that the up-regulation of ROCK1 activity in RhoA-deleted macrophages and macrophage phenotype/polarity are dependent on non-apoptotic Caspase-3 and are sensitive to Caspase-3 inhibition. These novel findings will revise/complement our understanding of RhoA pathway regulation of cell structure and polarity. PMID:26875770

  8. Diagnostic Magnetic Resonance Imaging of Atherosclerosis in Apolipoprotein E Knockout Mouse Model Using Macrophage-Targeted Gadolinium-Containing Synthetic Lipopeptide Nanoparticles

    PubMed Central

    Shen, Zu T.; Zheng, Shaokuan; Gounis, Matthew J.; Sigalov, Alexander B.

    2015-01-01

    Cardiovascular disease is the leading cause of death in Western cultures. The vast majority of cardiovascular events, including stroke and myocardial infarction, result from the rupture of vulnerable atherosclerotic plaques, which are characterized by high and active macrophage content. Current imaging modalities including magnetic resonance imaging (MRI) aim to characterize anatomic and structural features of plaques rather than their content. Previously, we reported that macrophage-targeted delivery of gadolinium (Gd)-based contrast agent (GBCA-HDL) using high density lipoproteins (HDL)-like particles significantly enhances the detection of plaques in an apolipoprotein (apo) E knockout (KO) mouse model, with an atherosclerotic wall/muscle normalized enhancement ratio (NER) of 120% achieved. These particles are comprised of lipids and synthetic peptide fragments of the major protein of HDL, apo A-I, that contain a naturally occurring modification which targets the particles to macrophages. Targeted delivery minimizes the Gd dose and thus reduces the adverse effects of Gd. The aims of the current study were to test whether varying the GBCA-HDL particle shape and composition can further enhance atherosclerotic plaque MRI and control organ clearance of these agents. We show that the optimized GBCA-HDL particles are efficiently delivered intracellularly to and uptaken by both J774 macrophages in vitro and more importantly, by intraplaque macrophages in vivo, as evidenced by NER up to 160% and higher. This suggests high diagnostic power of our GBCA-HDL particles in the detection of vulnerable atherosclerotic plaques. Further, in contrast to discoidal, spherical GBCA-HDL exhibit hepatic clearance, which could further diminish adverse renal effects of Gd. Finally, activated macrophages are reliable indicators of any inflamed tissues and are implicated in other areas of unmet clinical need such as rheumatoid arthritis, sepsis and cancer, suggesting the expanded diagnostic

  9. THE EFFECT OF TUNGSTATE NANOPARTICLES ON REACTIVE OXYGEN SPECIES AND CYTOTOXICITY IN RAW 264.7 MOUSE MONOCYTE MACROPHAGE CELLS

    PubMed Central

    Dunnick, Katherine M.; Badding, Melissa A.; Schwegler-Berry, Diane; Patete, Jonathan M.; Koenigsmann, Christopher; Wong, Stanislaus S.; Leonard, Stephen S.

    2015-01-01

    Due to their unique size, surface area, and chemical characteristics, nanoparticles’ use in consumer products has increased. However, the toxicity of nanoparticle (NP) exposure during the manufacturing process has not been fully assessed. Tungstate NP are used in numerous products, including but not limited to scintillator detectors and fluorescent lighting. As with many NP, no apparent toxicity studies have been completed with tungstate NP. The hypothesis that tungstate NP in vitro exposure results in reactive oxygen species (ROS) formation and cytotoxicity was examined. Differences in toxicity based on tungstate NP size, shape (sphere vs. wire), and chemical characteristics were determined. RAW 264.7 mouse monocyte macrophages were exposed to tungstate NP, and ROS formation was assessed via electron spin resonance (ESR), and several assays including hydrogen peroxide, intracellular ROS, and Comet. Results showed ROS production induced by tungstate nanowire exposure, but this exposure did not result in oxidative DNA damage. Nanospheres showed neither ROS nor DNA damage following cellular exposure. Cells were exposed over 72 h to assess cytotoxicity using an MTT (tetrazolium compound) assay. Results showed that differences in cell death between wires and spheres occurred at 24 h but were minimal at both 48 and 72 h. The present results indicate that tungstate nanowires are more reactive and produce cell death within 24 h of exposure, whereas nanospheres are less reactive and did not produce cell death. Results suggest that differences in shape may affect reactivity. However, regardless of the differences in reactivity, in general both shapes produced mild ROS and resulted in minimal cell death at 48 and 72 h in RAW 264.7 cells. PMID:25208664

  10. Dynamic analysis of phospholipid metabolism of mouse macrophages treated with common non-steroidal anti-inflammatory drugs.

    PubMed

    Peng, Haibo; Wu, Xia; Zhao, Lifang; Feng, Yifan

    2016-01-01

    Through studying the changes of the total phospholipid components in mouse macrophages under the inflammatory status and the drug intervention status, we found the targets of non-steroidal anti-inflammatory drugs on phospholipids, thus providing the basis for the targets of in vitro anti-inflammatory effects of non-steroidal anti-inflammatory drugs. After RAW264.7 cells were pretreated with common non-steroidal anti-inflammatory drugs (aspirin and ibuprofen) and, respectively, stimulated with KLA for various periods (0.5, 4, 12, 16, and 24 h), the phospholipids were extracted. The dynamic changes of phospholipids in cells under various stimulations were analyzed with UPLC-Q-TOF-MS technique. Through the statistical analysis of Simca-P, we explored the potential targets of non-steroidal anti-inflammatory drugs on phospholipids. Through the dynamic analysis of phospholipids, we found two biomarkers (PC(17:1/18:1), PA(18:0/18:4)) which might be in vitro intervention inflammatory response targets of non-steroidal anti-inflammatory drugs. The analysis results show that in anti-inflammatory effects, non-steroidal anti-inflammatory drugs can inhibit COX, induce the cellular fatty acid desaturation and the changes of phospholipid components, stimulate free fatty acids, activate calcium ion channels of endoplasmic reticulum, and promote cell endocytosis, thus controlling inflammation and activating cells. Non-steroidal anti-inflammatory drugs can promote endocytosis, alter cell inflammatory response, and activate the process cells, thus realizing the anti-inflammatory effects. PMID:26441061

  11. T Cells and Macrophages Responding to Oxidative Damage Cooperate in Pathogenesis of a Mouse Model of Age-Related Macular Degeneration

    PubMed Central

    Cruz-Guilloty, Fernando; Saeed, Ali M.; Duffort, Stephanie; Cano, Marisol; Ebrahimi, Katayoon B.; Ballmick, Asha; Tan, Yaohong; Wang, Hua; Laird, James M.; Salomon, Robert G.; Handa, James T.; Perez, Victor L.

    2014-01-01

    Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis. PMID:24586307

  12. Detection of disseminated peritoneal tumors by fluorescein diacrylate in mice

    NASA Astrophysics Data System (ADS)

    Harada, Yoshinori; Furuta, Hirokazu; Murayama, Yasutoshi; Dai, Ping; Fujikawa, Yuta; Urano, Yasuteru; Nagano, Tetsuo; Morishita, Koki; Hasegawa, Akira; Takamatsu, Tetsuro

    2009-02-01

    Tumor invasion to the peritoneum is a poor prognostic factor in cancer patients. Accurate diagnosis of disseminated peritoneal tumors is essential to accurate cancer staging. To date, peritoneal washing cytology during laparotomy has been used for diagnosis of peritoneal dissemination of gastrointestinal cancer, but its sensitivity has not been satisfactory. Thus, a more direct approach is indispensable to detect peritoneal dissemination in vivo. Fluorescein diacrylate (FDAcr) is an esterase-sensitive fluorescent probe derived from fluorescein. In cancer cells, fluorescent fluorescein generated by exogenous application of FDAcr selectively deposits owing to its stronger hydrolytic enzyme activity and its lower leakage rate. We examined whether FDAcr can specifically detect disseminated peritoneal tumors in athymic nude mouse models. Intraperitoneally administered FDAcr revealed disseminated peritoneal microscopic tumors not readily recognized on white-light imaging. These results suggest that FDAcr is a useful probe for detecting disseminated peritoneal tumors.

  13. Physiological function and inflamed-brain migration of mouse monocyte-derived macrophages following cellular uptake of superparamagnetic iron oxide nanoparticles-Implication of macrophage-based drug delivery into the central nervous system.

    PubMed

    Tong, Hsin-I; Kang, Wen; Shi, Yingli; Zhou, Guangzhou; Lu, Yuanan

    2016-05-30

    This study was designed to use superparamagnetic iron oxide nanoparticles (SPIONs) as evaluating tools to study monocyte-derived macrophages (MDM)-mediated delivery of small molecular agents into the diseased brains. MDM were tested with different-configured SPIONs at selected concentrations for their impacts on carrier cells' physiological and migratory properties, which were found to depend largely on particle size, coating, and treatment concentrations. SHP30, a SPION of 30-nm core size with oleic acids plus amphiphilic polymer coating, was identified to have high cellular uptake efficiency and cause little cytotoxic effects on MDM. At lower incubation dose (25μg/mL), few alteration was observed in carrier cells' physiological and in vivo migratory functions, as tested in a lipopolysaccharide-induced acute neuroinflammation mouse model. Nevertheless, significant increase in monocyte-to-macrophage differentiation, and decrease in in vivo carrier MDM inflamed-brain homing ability were found in groups treated with a higher dose of SHP30at 100μg/mL. Overall, our results have identified MDM treatment at 25μg/mL SHP30 resulted in little functional changes, provided valuable parameters for using SPIONs as evaluating tools to study MDM-mediated therapeutics carriage and delivery, and supported the concepts of using monocytes-macrophages as cellular vehicles to transport small molecular agents to the brain. PMID:27001531

  14. Amphiphilic Polymer-coated CdSe/ZnS Quantum Dots Induce Pro-inflammatory Cytokine Expression in Mouse Lung Epithelial Cells and Macrophages

    PubMed Central

    Lee, Vivian; McMahan, Ryan S.; Hu, Xiaoge; Gao, Xiaohu; Faustman, Elaine M.; Griffith, William C.; Kavanagh, Terrance J.; Eaton, David L.; McGuire, John K.; Parks, William C.

    2015-01-01

    Quantum dots (Qdots) are semiconductor nanoparticles with size-tunable fluorescence capabilities with diverse applications. Qdots typically contain cadmium or other heavy metals, hence raising concerns of their potential toxicity, especially in occupational settings where inhalation of nanomaterials may increase the risk of lung disease. Accordingly, we assessed the effects of tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coated CdSe/ZnS Qdots on mouse lung epithelial cells and macrophages. Mouse tracheal epithelial cells (MTEC), grown as organotypic cultures, bone marrow-derived macrophages (BMDM), and primary alveolar macrophages (AM) were derived from C57BL/6J or A/J mice and treated with TOPO-PMAT CdSe/ZnS Qdots (10–160 nM) for up to 24 h. Cadmium analysis showed that Qdots remained in the apical compartment of MTEC cultures, whereas they were avidly internalized by AM and BMDM, which did not differ between strains. In MTEC, Qdots selectively induced expression (mRNA and protein) of neutrophil chemokines CXCL1 and CXCL2 but only low to no detectable levels of other factors assessed. In contrast, 4 h exposure to Qdots markedly increased expression of CXCL1, IL6, IL12, and other pro-inflammatory factors in BMDM. Higher inflammatory response was seen in C57BL/6J than in A/J BMDM. Similar expression responses were observed in AM, although overall levels were less robust than in BMDM. MTEC from A/J mice were more sensitive to Qdot pro-inflammatory effects while macrophages from C57BL/6J mice were more sensitive. These findings suggest that patterns of Qdot-induced pulmonary inflammation are likely to be cell type specific and genetic background dependent. PMID:24983898

  15. Amphiphilic polymer-coated CdSe/ZnS quantum dots induce pro-inflammatory cytokine expression in mouse lung epithelial cells and macrophages.

    PubMed

    Lee, Vivian; McMahan, Ryan S; Hu, Xiaoge; Gao, Xiaohu; Faustman, Elaine M; Griffith, William C; Kavanagh, Terrance J; Eaton, David L; McGuire, John K; Parks, William C

    2015-05-01

    Quantum dots (Qdots) are semiconductor nanoparticles with size-tunable fluorescence capabilities with diverse applications. Qdots typically contain cadmium or other heavy metals, hence raising concerns of their potential toxicity, especially in occupational settings where inhalation of nanomaterials may increase the risk of lung disease. Accordingly, we assessed the effects of tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coated CdSe/ZnS Qdots on mouse lung epithelial cells and macrophages. Mouse tracheal epithelial cells (MTEC), grown as organotypic cultures, bone marrow-derived macrophages (BMDM), and primary alveolar macrophages (AM) were derived from C57BL/6J or A/J mice and treated with TOPO-PMAT CdSe/ZnS Qdots (10-160 nM) for up to 24 h. Cadmium analysis showed that Qdots remained in the apical compartment of MTEC cultures, whereas they were avidly internalized by AM and BMDM, which did not differ between strains. In MTEC, Qdots selectively induced expression (mRNA and protein) of neutrophil chemokines CXCL1 and CXCL2 but only low to no detectable levels of other factors assessed. In contrast, 4 h exposure to Qdots markedly increased expression of CXCL1, IL6, IL12, and other pro-inflammatory factors in BMDM. Higher inflammatory response was seen in C57BL/6J than in A/J BMDM. Similar expression responses were observed in AM, although overall levels were less robust than in BMDM. MTEC from A/J mice were more sensitive to Qdot pro-inflammatory effects while macrophages from C57BL/6J mice were more sensitive. These findings suggest that patterns of Qdot-induced pulmonary inflammation are likely to be cell-type specific and genetic background dependent. PMID:24983898

  16. Effect of low power laser irradiation on macrophage phagocytic capacity

    NASA Astrophysics Data System (ADS)

    Lu, Cuixia; Song, Sheng; Tang, Yu; Zhou, Feifan

    2011-03-01

    Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immunity in mammals. Laser irradiation has been found to produce photobiological effects with evidence of interference with immunological functions. However, the effects of laser on the immune response have not been extensively characterized. In this study, we focused our attention on the effects of He-Ne laser on the phagocytic activity of macrophages by using flow cytometry (FCM). After irradiating at fluence of 0, 1, 2 J/cm2 with He-Ne laser (632.8 nm, 3mw), the cells were incubated with microsphere and then subjected to FACS analysis. The results showed that Low-power laser irradiation (LPLI) leads to an increase in phagocytosis on both mouse peritoneal macrophages and the murine macrophage-like cell line RAW264.7. In addition, we demonstrated that LPLI increased phagocytosis of microsphere in a dose-dependent manner, reaching a maximum at fluence of 2 J/cm2. Taken together, our results indicated that Low-power laser irradiation with appropriate dosage can enhance the phagocytosis of macrophage, and provided a theoretical base for the clinical use of the He-Ne laser.

  17. Mobilization of stored triglycerides from macrophages as free fatty acids.

    PubMed

    von Hodenberg, E; Khoo, J C; Jensen, D; Witztum, J L; Steinberg, D

    1984-01-01

    Because many or most lipid-laden foam cells in atheromas and in xanthomas derive from macrophages, it is important to understand how they accumulate lipids and how they can divest themselves of lipids. The mobilization of stored triglycerides from macrophages was studied in cell cultures. Mouse resident peritoneal macrophages and J774 macrophages increased their triglyceride content six- to tenfold during a 24-hour incubation with free fatty acids complexed to albumin. Subsequent incubation in fresh medium containing free fatty acid-poor albumin was accompanied by a fall in cell triglyceride content (50% in 20 hours) and a corresponding increase in medium-free fatty acid. Release of free fatty acid was linear as a function of time, provided fresh medium was added hourly. When medium was not changed, release rates fell off rapidly, probably due to re-uptake of released free fatty acid. Chloroquine did not affect the rate of free fatty acid release. The results suggest that macrophages-foam cells can reduce their triglyceride stores via the action of a nonlysosomal (presumably cytoplasmic) neutral triglyceride lipase. PMID:6508637

  18. Disruption of Lipid Rafts Interferes with the Interaction of Toxoplasma gondii with Macrophages and Epithelial Cells

    PubMed Central

    Cruz, Karla Dias; Cruz, Thayana Araújo; Veras de Moraes, Gabriela; Paredes-Santos, Tatiana Christina; Attias, Marcia; de Souza, Wanderley

    2014-01-01

    The intracellular parasite Toxoplasma gondii can penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization of T. gondii in both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the LLC-MK2 cell lineage originated from kidney epithelial cells. PMID:24734239

  19. Fms-like tyrosine kinase 3 (Flt3) ligand depletes erythroid island macrophages and blocks medullar erythropoiesis in the mouse.

    PubMed

    Jacobsen, Rebecca N; Nowlan, Bianca; Brunck, Marion E; Barbier, Valerie; Winkler, Ingrid G; Levesque, Jean-Pierre

    2016-03-01

    The cytokines granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (Flt3-L) mobilize hematopoietic stem and progenitor cells into the peripheral blood of primates, humans, and mice. We recently reported that G-CSF administration causes a transient blockade of medullar erythropoiesis by suppressing erythroblastic island (EI) macrophages in the bone marrow. In the study described here, we investigated the effect of mobilizing doses of Flt3-L on erythropoiesis in mice in vivo. Similar to G-CSF, Flt3-L caused whitening of the bone marrow with significant reduction in the numbers of EI macrophages and erythroblasts. This was compensated by an increase in the numbers of EI macrophages and erythroblasts in the spleen. However, unlike G-CSF, Flt3-L had an indirect effect on EI macrophages, as it was not detected at the surface of EI macrophages or erythroid progenitors. PMID:26607596

  20. The M2 macrophages induce autophagic vascular disorder and promote mouse sensitivity to urethane-related lung carcinogenesis.

    PubMed

    Li, G-G; Guo, Z-Z; Ma, X-F; Cao, N; Geng, S-N; Zheng, Y-Q; Meng, M-J; Lin, H-H; Han, G; Du, G-J

    2016-06-01

    Tumor vessels are known to be abnormal, with typically aberrant, leaky and disordered vessels. Here, we investigated whether polarized macrophage phenotypes are involved in tumor abnormal angiogenesis and what is its mechanism. We found that there was no difference in chemotaxis of polarized M1 and M2 macrophages to lewis lung carcinoma (LLC) cells and that either M1 or M2 macrophage-conditioned media had no effect on LLC cell proliferation. Unexpectedly, the M2 but not M1 macrophage-conditioned media promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and simultaneously increased endothelial cell permeability in vitro and angiogenic index in the chick embryo chorioallantoic membrane (CAM). The treatment with M2 but not M1 macrophage-conditioned media increased autophagosomes as well as microtubule-associated protein light chain 3B (LC3-B) expression (a robust marker of autophagosomes) but decreased p62 protein expression (a selective autophagy substrate) in HUVECs, the treatment with chloroquine that blocked autophagy abrogated the abnormal angiogenic efficacy of M2 macrophage-conditioned media. These results were confirmed in urethane-induced lung carcinogenic progression. Urethane-induced lung carcinogenesis led to more M2 macrophage phenotype and increased abnormal angiogenesis concomitant with the upregulation of LC3-B and the downregulation of p62. Clodronate liposome-induced macrophage depletion, chloroquine-induced autophagic prevention or salvianolic acid B-induced vascular protection decreased abnormal angiogenesis and lung carcinogenesis. In addition, we found that the tendency of age-related M2 macrophage polarization also promoted vascular permeability and carcinogenesis in urethane carcinogenic progression. These findings indicate that the M2 macrophages induce autophagic vascular disorder to promote lung cancer progression, and the autophagy improvement represents an efficacious strategy for abnormal angiogenesis and cancer

  1. Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

    SciTech Connect

    Kanakura, Y.; Thompson, H.; Nakano, T.; Yamamura, T.; Asai, H.; Kitamura, Y.; Metcalfe, D.D.; Galli, S.J.

    1988-09-01

    Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of (35S) sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate (35S) proteoglycans. When ''MMC-like'' cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1-W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these ''second generation PMC'' formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.

  2. Silencing CCR2 in Macrophages Alleviates Adipose Tissue Inflammation and the Associated Metabolic Syndrome in Dietary Obese Mice.

    PubMed

    Kim, Jongkil; Chung, Kunho; Choi, Changseon; Beloor, Jagadish; Ullah, Irfan; Kim, Nahyeon; Lee, Kuen Yong; Lee, Sang-Kyung; Kumar, Priti

    2016-01-01

    Adipose tissue macrophage (ATM)-mediated inflammation is a key feature contributing to the adverse metabolic outcomes of dietary obesity. Recruitment of macrophages to obese adipose tissues (AT) can occur through the engagement of CCR2, the receptor for MCP-1 (monocyte chemoattractant protein-1), which is expressed on peripheral monocytes/macrophages. Here, we show that i.p. administration of a rabies virus glycoprotein-derived acetylcholine receptor-binding peptide effectively delivers complexed siRNA into peritoneal macrophages and ATMs in a mouse model of high-fat diet-induced obesity. Treatment with siRNA against CCR2 inhibited macrophage infiltration and accumulation in AT and, therefore, proinflammatory cytokines produced by macrophages. Consequently, the treatment significantly improved glucose tolerance and insulin sensitivity profiles, and also alleviated the associated symptoms of hepatic steatosis and reduced hepatic triglyceride production. These results demonstrate that disruption of macrophage chemotaxis to the AT through cell-targeted gene knockdown strategies can provide a therapeutic intervention for obesity-related metabolic diseases. The study also highlights a siRNA delivery approach for targeting specific monocyte subsets that contribute to obesity-associated inflammation without affecting the function of other tissue-resident macrophages that are essential for host homeostasis and survival. PMID:26812653

  3. Establishment of a novel method to evaluate peritoneal microdissemination and therapeutic effect using luciferase assay.

    PubMed

    Takahashi, Ryo; Yokobori, Takehiko; Osone, Katsuya; Tatsuki, Hironori; Takada, Takahiro; Suto, Toshinaga; Yajima, Reina; Kato, Toshihide; Fujii, Takaaki; Tsutsumi, Souichi; Kuwano, Hiroyuki; Asao, Takayuki

    2016-03-01

    Peritoneal dissemination is a major cause of recurrence in patients with malignant tumors in the peritoneal cavity. Effective anticancer agents and treatment protocols are necessary to improve outcomes in these patients. However, previous studies using mouse models of peritoneal dissemination have not detected any drug effect against peritoneal micrometastasis. Here we used the luciferase assay to evaluate peritoneal micrometastasis in living animals and established an accurate mouse model of early peritoneal microdissemination to evaluate tumorigenesis and drug efficacy. There was a positive correlation between luminescence intensity in in vivo luciferase assay and the extent of tumor dissemination evaluated by ex vivo luciferase assay and mesenteric weight. This model has advantages over previous models because optimal luciferin concentration without cell damage was validated and peritoneal microdissemination could be quantitatively evaluated. Therefore, it is a useful model to validate peritoneal micrometastasis formation and to evaluate drug efficacy without killing mice. PMID:26716425

  4. Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein.

    PubMed

    Rayamajhi, Manira; Zak, Daniel E; Chavarria-Smith, Joseph; Vance, Russell E; Miao, Edward A

    2013-10-15

    The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SSs). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol is detected through murine NAIP2 and NAIP5/6, respectively. In this study, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages; however, priming with polyinosinic-polycytidylic acid induces it and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized bone marrow-derived macrophages by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly, independent of priming. Human macrophages are known to express only one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP. PMID:24043898

  5. Cystathionine-γ-lyase gene silencing with siRNA in monocytes/ macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse.

    PubMed

    Badiei, A; Chambers, S T; Gaddam, R R; Bhatia, M

    2016-03-01

    Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition. PMID:26949091

  6. The co‐transcriptome of uropathogenic E scherichia coli‐infected mouse macrophages reveals new insights into host–pathogen interactions

    PubMed Central

    Mavromatis, Charalampos (Harris); Bokil, Nilesh J.; Totsika, Makrina; Kakkanat, Asha; Schaale, Kolja; Cannistraci, Carlo V.; Ryu, Taewoo; Beatson, Scott A.; Ulett, Glen C.; Schembri, Mark A.; Sweet, Matthew J.

    2015-01-01

    Summary Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic E scherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host–pathogen co‐transcriptome analyses using RNA sequencing. Mouse bone marrow‐derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow‐derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up‐regulated at 24 h post‐infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co‐cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment. PMID:25410299

  7. Activation of adenosine A(3) receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells.

    PubMed

    Hofer, M; Vacek, A; Pospísil, M; Holá, J; Streitová, D; Znojil, V

    2009-01-01

    Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells. PMID:18380545

  8. Peritoneal fluid culture

    MedlinePlus

    Culture - peritoneal fluid ... sent to the laboratory for Gram stain and culture. The sample is checked to see if bacteria ... based on more than just the peritoneal fluid culture (which may be negative even if you have ...

  9. Peritoneal fluid analysis

    MedlinePlus

    ... at fluid that has built up in the space in the abdomen around the internal organs. This area is called the peritoneal space. ... sample of fluid is removed from the peritoneal space using a needle and syringe. Your health care ...

  10. Peritoneal Fluid Analysis

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Peritoneal Fluid Analysis Share this page: Was this page helpful? Formal name: Peritoneal Fluid Analysis Related tests: Pleural Fluid Analysis , Pericardial Fluid ...

  11. Intranasal delivery of bone marrow-derived mesenchymal stem cells, macrophages, and microglia to the brain in mouse models of Alzheimer's and Parkinson's disease.

    PubMed

    Danielyan, Lusine; Beer-Hammer, Sandra; Stolzing, Alexandra; Schäfer, Richard; Siegel, Georg; Fabian, Claire; Kahle, Philipp; Biedermann, Tilo; Lourhmati, Ali; Buadze, Marine; Novakovic, Ana; Proksch, Barbara; Gleiter, Christoph H; Frey, William H; Schwab, Matthias

    2014-01-01

    In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinson's disease (PD). The present study examined delivery of bone marrow-derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD [(Thy1)-h[A30P] αS] and an APP/PS1 model of Alzheimer's disease (AD) via intranasal application (INA). INA of microglia in naive BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1 × 10(4)) after INA of 1 × 10(6) cells, while the total amount of cells detected in peripheral organs did not exceed 3.4 × 10(3). Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] αS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13-month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular amyloid β (macrophages in APP/PS1 model) or α-synuclein [MSCs in (Thy1)-h[A30P] αS model] immunoreactivity. Here, we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated

  12. Prostaglandin E specifically upregulates the expression of the mannose-receptor on mouse bone marrow-derived macrophages.

    PubMed Central

    Schreiber, S; Blum, J S; Chappel, J C; Stenson, W F; Stahl, P D; Teitelbaum, S L; Perkins, S L

    1990-01-01

    The macrophage mannose receptor (MMR) facilitates the binding and internalization of microorganisms and glycoproteins with terminal mannose residues. The receptor is progressively upregulated as bone marrow precursor cells mature into macrophages and thus may serve as a marker of differentiation. Prostaglandins of the E series (PGE) are known inhibitors of monocyte and macrophage precursor proliferation, an effect often associated with cellular maturation. MMR expression was therefore assessed after exposure of bone marrow macrophage precursor (BMMP) cells to these prostanoids. Receptor expression was determined by ligand binding and via immunoprecipitation of newly synthesized receptor molecules. PGE1 and PGE2 at 10(-9)-10(-6) M upregulated MMR surface expression and biosynthesis four- to sixfold in a dose-dependent manner. BMMPs responsive to prostaglandins were characterized by plastic adherence, F4/80 antigen expression, and nonspecific esterase activity. Prostaglandins accelerated the expression of the MMR in cells by 48-72h, with maximal levels of receptor expression being identical in control or treated cells. Thus, prostaglandins enhanced mannose receptor expression in adherent but not fully differentiated macrophage precursors. This effect is specific for PGE and is mimicked by dibutyrl cyclic AMP. These results indicate that prostaglandins accelerate MMR expression and hence the differentiation of macrophage precursor cells. Cells resident in the bone marrow secrete abundant prostaglandins, suggesting that a paracrine mechanism may exist to regulate MMR expression and function. Images PMID:1965946

  13. Lysis of herpesvirus-infected cells by macrophages activated with free or liposome-encapsulated lymphokine produced by a murine T cell hybridoma.

    PubMed Central

    Koff, W C; Showalter, S D; Seniff, D A; Hampar, B

    1983-01-01

    Thioglycolate-induced mouse peritoneal macrophages were activated in vitro by the lymphokine designated macrophage-activating factor (MAF) produced by a murine T cell hybridoma to lyse herpes simplex virus type 2 (HSV-2)-infected murine target cells. Comparison of uninfected BALB/c 10E2 cells with HSV-2-infected 10E2 cells showed that macrophages activated with MAF selectively destroyed HSV-2-infected cells and left uninfected cells unharmed, as measured by an 18-h 51Cr-release assay. In contrast, macrophages treated with medium were as efficient as MAF-activated macrophages in suppressing the production of HSV-2 from virus-infected cells. These findings suggest that macrophages must attain an activated state to lyse HSV-2-infected cells. Finally, incubation of macrophages with liposomes containing MAF was shown to be a highly efficient method for activation of macrophages against HSV-2 infected cells. The ability to selectively destroy herpesvirus-infected cells in vitro by macrophages activated with liposome-encapsulated MAF suggests that the therapeutic efficacy of this treatment in vivo should be evaluated. PMID:6358037

  14. Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide

    PubMed Central

    Raza, Haider; John, Annie

    2015-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs), including acetaminophen (APAP), have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP) causes liver injury in humans and animals. Hepatic glutathione (GSH) depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP—induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST) and multidrug resistance (MDR1) proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM), a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h) exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems. PMID:26714183

  15. Docosahexaenoic acid consumption inhibits deoxynivalenol-induced CREB/ATF1 activation and IL-6 gene transcription in mouse macrophages.

    PubMed

    Jia, Qunshan; Zhou, Hui-Ren; Shi, Yuhui; Pestka, James J

    2006-02-01

    The mycotoxin deoxynivalenol (DON) induces IgA nephropathy in mice by upregulating IL-6 expression, which is suppressed by (n-3) PUFA consumption. The purpose of this study was to test the hypothesis that consumption of the (n-3) PUFA docosahexaenoic acid (DHA) interferes with DON-induced transcriptional and post-transcriptional upregulation of IL-6 mRNA in murine macrophages. DON evoked expression of IL-6 mRNA and IL-6 heterogenous nuclear RNA (hnRNA), an indicator of ongoing IL-6 transcription, in macrophages elicited from mice fed control AIN-93G diet for 4 wk, whereas expression of both RNA species was suppressed in macrophages from mice fed AIN-93G modified to contain 30 g DHA/kg diet for the same time period. DON enhanced IL-6 mRNA stability similarly in macrophages from control and DHA-fed mice suggesting that (n-3) PUFA effects were not post-transcriptional. DON upregulated binding activity of cAMP response element binding protein (CREB) and activator protein (AP-1) to their respective consensus sequences in nuclear extracts from control-fed mice, whereas both activities were suppressed in nuclear extracts from DHA-fed mice. DON induced phosphorylation of CREB at Ser-133 and ATF1 at Ser-63 as well as intranuclear binding of phospho-CREB/ATF1 to the cis element of the IL-6 promoter in control macrophages, whereas both activities were inhibited in macrophages from DHA-fed mice. DHA consumption blocked DON-induced phosphorylation of the CREB kinase AKT. Inhibition of AKT suppressed both CREB/ATF1 phosphorylation and IL-6 transcription. These data suggest that DHA consumption suppresses DON-induced IL-6 transcription in macrophages in part by interfering with AKT-dependent phosphorylation and subsequent binding of CREB/ATF1 to the IL-6 promoter. PMID:16424113

  16. Macrophage recognition of toxic advanced glycosylation end products through the macrophage surface-receptor nucleolin.

    PubMed

    Miki, Yuichi; Dambara, Hikaru; Tachibana, Yoshihiro; Hirano, Kazuya; Konishi, Mio; Beppu, Masatoshi

    2014-01-01

    Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs. PMID:24818254

  17. Glutamine Modulates Macrophage Lipotoxicity

    PubMed Central

    He, Li; Weber, Kassandra J.; Schilling, Joel D.

    2016-01-01

    Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli. PMID:27077881

  18. Anti-inflammatory effects of silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles in mouse macrophages infected with live Chlamydia trachomatis

    PubMed Central

    Yilma, Abebayehu N; Singh, Shree R; Dixit, Saurabh; Dennis, Vida A

    2013-01-01

    Chlamydia trachomatis is a very common sexually transmissible infection in both developing and developed countries. A hallmark of C. trachomatis infection is the induction of severe inflammatory responses which play critical roles in its pathogenesis. Antibiotics are the only treatment option currently available for controlling C. trachomatis infection; however, they are efficacious only when administered early after an infection. The objectives of this study are to explore alternative strategies in the control and regulation of inflammatory responses triggered by a C. trachomatis infection. We employed silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles, which have been shown to possess anti-inflammatory properties, as our target and the in vitro mouse J774 macrophage model of C. trachomatis infection. Our hypothesis is that small sizes of Ag-PVP nanoparticles will control inflammatory mediators triggered by a C. trachomatis infection. Cytotoxicity studies using Ag-PVP nanoparticles of 10, 20, and 80 nm sizes revealed >80% macrophage viability up to a concentration of 6.25 μg/mL, with the 10 nm size being the least toxic. All sizes of Ag-PVP nanoparticles, especially the 10 nm size, reduced the levels of the prototypic cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, as elicited from C. trachomatis infected macrophages. Additionally, Ag-PVP nanoparticles (10 nm) selectively inhibited a broad spectrum of other cytokines and chemokines produced by infected macrophages. Of significance, Ag-PVP nanoparticles (10 nm) caused perturbations in a variety of upstream (toll like receptor 2 [TLR2], nucleotide-binding oligomerization-protein 2 [NOD2], cluster of differentiation [CD]40, CD80, and CD86) and downstream (IL-1 receptor-associated kinase 3 [IRAK3] and matrix metallopeptidase 9 [MMP9]) inflammatory signaling pathways by downregulating their messenger ribonucleic acid (mRNA) gene transcript expressions as induced by C. trachomatis in macrophages

  19. Transcriptome Analysis of Genes Regulated by Cholesterol Loading in Two Strains of Mouse Macrophages Associates Lysosome Pathway and ER Stress Response with Atherosclerosis Susceptibility

    PubMed Central

    Robinet, Peggy; Smith, Jonathan D.

    2013-01-01

    Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2 ApoE−/− mice have aortic root lesions 10-fold larger than AKR ApoE−/−mice. The current study analyzes the response to cholesterol loading of macrophages from these two strains. Macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total and esterified cholesterol compared to atherosclerosis resistant AKR macrophages, while free cholesterol levels were higher in AKR cells. Gene expression profiles were obtained and data were analyzed for strain, cholesterol loading, and strain-cholesterol loading interaction effects by a fitted linear model. Pathway and transcriptional motif enrichment were identified by gene set enrichment analysis. In addition to observed strain differences in basal gene expression, we identified many transcripts whose expression was significantly altered in response to cholesterol loading, including P2ry13 and P2ry14, Trib3, Hyal1, Vegfa, Ccr5, Ly6a, and Ifit3. Eight pathways were significantly enriched in transcripts regulated by cholesterol loading, among which the lysosome and cytokine-cytokine receptor interaction pathways had the highest number of significantly regulated transcripts. Of the differentially regulated transcripts with a strain-cholesterol loading interaction effect, we identified three genes known to participate in the endoplasmic reticulum (ER) stress response, Ddit3, Trib3 and Atf4. These three transcripts were highly up-regulated by cholesterol in AKR and either down-regulated or unchanged in loaded DBA/2 macrophages, thus associating a robust ER stress response with atherosclerosis resistance. We identified significant transcripts with strain, loading, or strain-loading interaction effect that

  20. Transcriptome analysis of genes regulated by cholesterol loading in two strains of mouse macrophages associates lysosome pathway and ER stress response with atherosclerosis susceptibility.

    PubMed

    Berisha, Stela Z; Hsu, Jeffrey; Robinet, Peggy; Smith, Jonathan D

    2013-01-01

    Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2 ApoE(-/-) mice have aortic root lesions 10-fold larger than AKR ApoE(-/-) mice. The current study analyzes the response to cholesterol loading of macrophages from these two strains. Macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total and esterified cholesterol compared to atherosclerosis resistant AKR macrophages, while free cholesterol levels were higher in AKR cells. Gene expression profiles were obtained and data were analyzed for strain, cholesterol loading, and strain-cholesterol loading interaction effects by a fitted linear model. Pathway and transcriptional motif enrichment were identified by gene set enrichment analysis. In addition to observed strain differences in basal gene expression, we identified many transcripts whose expression was significantly altered in response to cholesterol loading, including P2ry13 and P2ry14, Trib3, Hyal1, Vegfa, Ccr5, Ly6a, and Ifit3. Eight pathways were significantly enriched in transcripts regulated by cholesterol loading, among which the lysosome and cytokine-cytokine receptor interaction pathways had the highest number of significantly regulated transcripts. Of the differentially regulated transcripts with a strain-cholesterol loading interaction effect, we identified three genes known to participate in the endoplasmic reticulum (ER) stress response, Ddit3, Trib3 and Atf4. These three transcripts were highly up-regulated by cholesterol in AKR and either down-regulated or unchanged in loaded DBA/2 macrophages, thus associating a robust ER stress response with atherosclerosis resistance. We identified significant transcripts with strain, loading, or strain-loading interaction effect that

  1. Cinnamon polyphenol extract affects immune responses by regulating anti- and proinflammatory and glucose transporter gene expression in mouse macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tristetraprolin (TTP/TIS11/ZFP36) family proteins have anti-inflammatory effects by polyphenoldestabilizing pro-inflammatory mRNAs. TTP expression is induced by insulin and cinnamon extract (CPE) in adipocytes, by lipopolysaccharide (LPS) in macrophages, and by green tea extract in rats. This study ...

  2. Reactive oxygen species in the tumor niche triggers altered activation of macrophages and immunosuppression: Role of fluoxetine.

    PubMed

    Ghosh, Sayan; Mukherjee, Sudeshna; Choudhury, Sreetama; Gupta, Payal; Adhikary, Arghya; Baral, Rathindranath; Chattopadhyay, Sreya

    2015-07-01

    Macrophages are projected as one of the key players responsible for the progression of cancer. Classically activated (M1) macrophages are pro-inflammatory and have a central role in host defense, while alternatively activated (M2) macrophages are associated with immunosuppression. Macrophages residing at the site of neoplastic growth are alternately activated and are referred to as tumor-associated macrophages (TAMs). These "cooperate" with tumor tissue, promoting increased proliferation and immune escape. Selective serotonin reuptake inhibitors like fluoxetine have recently been reported to possess anti-inflammatory activity. We used fluoxetine to target tumor-associated inflammation and consequent alternate polarization of macrophages. We established that murine peritoneal macrophages progressed towards an altered activation state when exposed to cell-free tumor fluid, as evidenced by increased IL-6, IL-4 and IL-10 levels. These polarized macrophages showed significant pro-oxidant bias and increased p65 nuclear localization. It was further observed that these altered macrophages could induce oxidative insult and apoptosis in cultured mouse CD3(+) T cells. To validate these findings, we replicated key experiments in vivo, and observed that there was increased serum IL-6, IL-4 and IL-10 in tumor-bearing animals, with increased % CD206(+) cells within the tumor niche. TAMs showed increased nuclear localization of p65 with decreased Nrf2 expression in the nucleus. These results were associated with increase in apoptosis of CD3(+) T cells co-cultured with TAM-spent media. We could establish that fluoxetine treatment could specifically re-educate the macrophages both in vitro and in vivo by skewing their phenotype such that immune suppression mediated by tumor-dictated macrophages was successfully mitigated. PMID:25819340

  3. Selective Imaging of Malignant Ascites in a Mouse Model of Peritoneal Metastasis Using in Vivo Dynamic Nuclear Polarization-Magnetic Resonance Imaging.

    PubMed

    Eto, Hinako; Hyodo, Fuminori; Nakano, Kenji; Utsumi, Hideo

    2016-02-16

    The presence of malignant ascites in advanced cancer patients is associated with both a poor prognosis and quality of life with a risk of abdominal infection and sepsis. Contemporary noninvasive visualization methods such as ultrasound, computed tomography, and magnetic resonance imaging (MRI) often struggle to differentiate malignant ascites from surrounding tissues. This study aimed to determine the utility of selective H2O imaging in the abdominal cavity with a free radical probe and deuterium oxide (D2O) contrast agent using in vivo dynamic nuclear polarization-MRI (DNP-MRI). Phantom imaging experiments established a linear relationship between H2O volume and image intensity using in vivo DNP-MRI. Similar results were obtained when the radical-D2O probe was used to determine selective and spatial information on H2O in vivo, modeled by the injection of saline into the abdominal cavity of mice. To demonstrate the utility of this method for disease, malignant ascites in peritoneal metastasis animal model was selected as one of the typical examples. In vivo DNP-MRI of peritoneal metastasis animal model was performed 7-21 days after intraperitoneal injection of luciferase, stably expressing the human pancreatic carcinoma (SUIT-2). The image intensity with increasing malignant ascites was significantly increased at days 7, 16, and 21. This increase corresponded to in vivo tumor progression, as measured by bioluminescent imaging. These results suggest that H2O signal enhancement in DNP-MRI using radical-D2O contrast is positively associated with the progression of dissemination and could be a useful biomarker for malignant ascites with cancer metastasis. PMID:26796949

  4. Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo.

    PubMed

    Komutarin, T; Azadi, S; Butterworth, L; Keil, D; Chitsomboon, B; Suttajit, M; Meade, B J

    2004-04-01

    The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2-200 microg/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a concentration-dependent manner. In vivo administration of T. indica extract (100-500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-gamma induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity. PMID:15019190

  5. Mast cell–macrophage dynamics in modulation of dengue virus infection in skin

    PubMed Central

    Chu, Ya-Ting; Wan, Shu-Wen; Anderson, Robert; Lin, Yee-Shin

    2015-01-01

    Dengue virus (DENV) infection causes dengue fever, dengue haemorrhagic fever, or dengue shock syndrome. Mast cells have been speculated to play a role in DENV disease although their precise roles are unclear. In this study, we used mast cell-deficient KitW-sh/W-sh mice to investigate the involvement of mast cells after intradermal DENV infection. An approximately two- to three-fold higher level of DENV NS3 antigen was detected at the skin inoculation site in DENV-infected KitW-sh/W-sh mice than in DENV-infected wild-type (WT) mice (using a dose of 1 × 109 plaque-forming units/mouse). Moreover, as an indicator of heightened pathogenesis, a more prolonged bleeding time was observed in DENV-infected KitW-sh/W-sh mice than in WT mice. Monocytes/macrophages are considered to be important targets for DENV infection, so we investigated the susceptibility and chemokine response of DENV-infected peritoneal macrophages from KitW-sh/W-sh and WT mice both ex vivo and in vivo. There was a tendency for higher DENV infection and higher secretion of CCL2 (MCP-1) from peritoneal macrophages isolated from KitW-sh/W-sh mice than those from WT mice. In vivo studies using intradermal inoculation of DENV showed about twofold higher levels of infiltrating macrophages and CCL2 (MCP-1) at the inoculation site in both mock control and DENV-inoculated KitW-sh/W-sh mice than in corresponding WT mice. In summary, compared with WT mice, KitW-sh/W-sh mice show enhanced DENV infection and macrophage infiltration at the skin inoculation site as well as increased DENV-associated bleeding time. The results indicate an intriguing interplay between mast cells and tissue macrophages to restrict DENV replication in the skin. PMID:26059780

  6. Kinsenoside isolated from Anoectochilus formosanus suppresses LPS-stimulated inflammatory reactions in macrophages and endotoxin shock in mice.

    PubMed

    Hsiao, Hung-Bo; Wu, Jin-Bin; Lin, Ho; Lin, Wen-Chuan

    2011-02-01

    In the present study, we reported that kinsenoside, a major component of Anoectochilus formosanus, inhibited inflammatory reactions in mouse peritoneal lavage macrophages and protects mice from endotoxin shock. In LPS-stimulated mouse peritoneal lavage macrophages, kinsenoside inhibited the inflammatory mediators, such as nitric oxide, TNF-[alpha], IL-1[beta], monocyte chemoattractant protein 1, and macrophage migration inhibitory factor production. Furthermore, kinsenoside decreased the formation of a nuclear factor [kappa]B-DNA complex and nuclear p65 and p50 protein levels. Kinsenoside inhibited nuclear factor [kappa]B translocation through both I[kappa]B[alpha]-dependent and -independent pathway. In contrast, it stimulated anti-inflammatory cytokine IL-10 generation and enhanced the mRNA expression of IL-10 and suppressor of cytokine signaling 3 in the same cells induced by LPS. In an animal model, both pretreatment and posttreatment of kinsenoside increased the survival rate of ICR mice challenged by LPS (80 mg/kg, i.p.). Pretreatment with kinsenoside decreased serum levels of TNF-[alpha], IL-1[beta], IL-10, monocyte chemoattractant protein 1, and migration inhibitory factor at 1 h after sublethal dose of LPS (40 mg/kg, i.p.) in mice. In contrast, kinsenoside enhanced serum IL-10 level at 24 h after LPS injection in mice. In conclusion, kinsenoside inhibited the production of inflammatory mediators and enhanced anti-inflammatory cytokine generation. Therefore, kinsenoside can alleviate acute inflammatory hazards. PMID:20661184

  7. Helicobacter pylori protein HP0986 (TieA) interacts with mouse TNFR1 and triggers proinflammatory and proapoptotic signaling pathways in cultured macrophage cells (RAW 264.7).

    PubMed

    Ansari, Suhail A; Devi, Savita; Tenguria, Shivendra; Kumar, Ashutosh; Ahmed, Niyaz

    2014-08-01

    HP0986 protein of Helicobacter pylori has been shown to trigger induction of proinflammatory cytokines (IL-8 and TNF-α) through the activation of NF-κB and also to induce Fas mediated apoptosis of human macrophage cells (THP-1). In this study, we unravel mechanistic details of the biological effects of this protein in a murine macrophage environment. Up regulation of MCP-1 and TNF-α in HP0986-induced RAW 264.7 cells occurred subsequent to the activation and translocation of NF-κB to the cell nucleus. Further, HP0986 induced apoptosis of RAW 264.7 cells through Fas activation and this was in agreement with previous observations made with THP-1 cells. Our studies indicated activation of TNFR1 through interaction with HP0986 and this elicited the aforementioned responses independent of TLR2, TLR4 or TNFR2. We found that mouse TNFR1 activation by HP0986 facilitates formation of a complex comprising of TNFR1, TRADD and TRAF2, and this occurs upstream of NF-κB activation. Furthermore, FADD also forms a second complex, at a later stage, together with TNFR1 and TRADD, resulting in caspase-8 activation and thereby the apoptosis of RAW 264.7 cells. In summary, our observations reveal finer details of the functional activity of HP0986 protein in relation to its behavior in a murine macrophage cell environment. These findings reconfirm the proinflammatory and apoptotic role of HP0986 signifying it to be an important trigger of innate responses. These observations form much needed baseline data entailing future in vivo studies of the functions of HP0986 in a murine model. PMID:24767863

  8. Sulindac, a nonsteroidal anti-inflammatory drug, selectively inhibits interferon-{gamma}-induced expression of the chemokine CXCL9 gene in mouse macrophages

    SciTech Connect

    Sakaeda, Yoshiichi; Hiroi, Miki; Shimojima, Takahiro; Iguchi, Mayumi; Kanegae, Haruhide; Ohmori, Yoshihiro . E-mail: ohmori@dent.meikai.ac.jp

    2006-11-17

    Sulindac, a non-steroidal anti-inflammatory drug, has been shown to exert an anti-tumor effect on several types of cancer. To determine the effect of sulindac on intracellular signaling pathways in host immune cells such as macrophages, we investigated the effect of the drug on interferon gamma (IFN{gamma})-induced expression of signal transducer and activator of transcription 1 (STAT1) and other genes in mouse macrophage-like cell line RAW264.7 cells. Sulindac, but not aspirin or sodium salicylate, inhibited IFN{gamma}-induced expression of the CXC ligand 9 (CXCL9) mRNA, a chemokine for activated T cells, whereas the interferon-induced expression of CXCL10 or IFN regulatory factor-1 was not affected by sulindac. Luciferase reporter assay demonstrated that sulindac inhibited IFN{gamma}-induced promoter activity of the CXCL9 gene. Surprisingly, sulindac had no inhibitory effect on IFN{gamma}-induced STAT1 activation; however, constitutive nuclear factor {kappa}B activity was suppressed by the drug. These results indicate that sulindac selectively inhibited IFN{gamma}-inducible gene expression without inhibiting STAT1 activation.

  9. Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages

    PubMed Central

    Namatame, Ichiji; Tomoda, Hiroshi; Ishibashi, Shun; Ōmura, Satoshi

    2004-01-01

    Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC50 values of 0.78 and 0.41 μM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC50 values of 6.0 and 5.5 μM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents. PMID:14718664

  10. Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages.

    PubMed

    Namatame, Ichiji; Tomoda, Hiroshi; Ishibashi, Shun; Omura, Satoshi

    2004-01-20

    Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC(50) values of 0.78 and 0.41 microM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC(50) values of 6.0 and 5.5 microM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents. PMID:14718664

  11. Peritonitis caused by Rothia mucilaginosa in a peritoneal dialysis patient.

    PubMed

    Gosmanova, Elvira O; Garrett, Tiffani R; Wall, Barry M

    2013-12-01

    Peritonitis is an important cause of morbidity in patients undergoing peritoneal dialysis. Rothia mucilaginosa has been reported as an unusual cause of peritoneal dialysis associated peritonitis. Difficulty in the management of this microorganism lies in the absence of uniform recommendations for anti-microbial therapy directed against this pathogen. The current report describes the clinical course of an episode of peritoneal dialysis associated peritonitis caused by Rothia mucilaginosa. Treatment options for this organism are summarized. PMID:24263080

  12. Linagliptin Ameliorates Methylglyoxal-Induced Peritoneal Fibrosis in Mice

    PubMed Central

    Nagai, Takuo; Doi, Shigehiro; Nakashima, Ayumu; Irifuku, Taisuke; Sasaki, Kensuke; Ueno, Toshinori; Masaki, Takao

    2016-01-01

    Recent studies have reported increases of methylglyoxal (MGO) in peritoneal dialysis patients, and that MGO-mediated inflammation plays an important role in the development of peritoneal fibrosis through production of transforming growth factor-β1 (TGF-β1). Linagliptin, a dipeptidyl peptidase-4 inhibitor, exerts anti-inflammatory effects independent of blood glucose levels. In this study, we examined whether linagliptin suppresses MGO-induced peritoneal fibrosis in mice. Male C57/BL6 mice were divided into three groups: control, MGO injection plus saline, and MGO injection plus linagliptin (n = 6 per group). Peritoneal fibrosis was induced by daily intraperitoneal injection of saline containing 40 mmol/L MGO for 21 days. Saline was administered intraperitoneally to the control group. Linagliptin (10 mg/kg) or saline were administrated by once-daily oral gavage from 3 weeks before starting MGO injections. Immunohistochemical staining revealed that linagliptin suppressed expression of α-smooth muscle actin and fibroblast-specific protein-1, deposition of type I and III collagen, and macrophage (F4/80) infiltration. Peritoneal equilibration testing showed improved peritoneal functions in mice treated with linagliptin. Peritoneal injection of MGO increased plasma levels of glucagon-like peptide-1 (GLP-1) in mice, and a further increase was observed in linagliptin-treated mice. Although MGO increased plasma glucose levels, linagliptin did not decrease plasma glucose levels. Moreover, linagliptin reduced the TGF-β1 concentration in the peritoneal fluid of MGO-treated mice. GLP-1 receptor (GLP-1R) was expressed in monocytes/macrophages and linagliptin suppressed GLP-1R expression in MGO-injected mice. These results suggest that oral administration of linagliptin ameliorates MGO-induced peritoneal fibrosis. PMID:27513960

  13. The Impact of Serum Amyloid P-Component on Gene Expression in RAW264.7 Mouse Macrophages

    PubMed Central

    Xi, Dan; Zhao, Jinzhen; Liu, Jichen; Xiong, Haowei; He, Wenshuai; Hu, Jing; Lai, Wenyan; Guo, Zhigang

    2016-01-01

    Serum amyloid P-component (SAP) contributes to host defense and prevents fibrosis. Macrophages are the most abundant inflammatory cell type in atherosclerotic plaques. In the present study, using 3H-cholesterol-labeled counting radioactivity assay, we demonstrated that the apoAI-mediated cholesterol efflux in RAW264.7 macrophages was increased by SAP treatment in a time- and dose-dependent manner. We analyzed global gene expression changes upon SAP treatment using RNA sequencing. As a result, a total of 175 differentially expressed genes were identified, of which 134 genes were downregulated and 41 genes were upregulated in SAP treated cells compared to control cells. Quantitative RT-PCR analysis confirmed decreased expression of 5 genes and an increase in expression of 1 gene upon SAP treatment. Gene ontology analysis showed that genes involved in response to stimulus were significantly enriched in differentially expressed genes. Beyond protein-coding genes, we also identified 8 differentially expressed long noncoding RNAs. Our study may provide new insights into mechanisms underlying the functional role of SAP in macrophages. PMID:27239478

  14. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  15. In vivo administration of the frog skin peptide frenatin 2.1S induces immunostimulatory phenotypes of mouse mononuclear cells.

    PubMed

    Pantic, Jelena M; Radosavljevic, Gordana D; Jovanovic, Ivan P; Arsenijevic, Nebojsa N; Conlon, J Michael; Lukic, Miodrag L

    2015-09-01

    Host-defense peptides secreted by epithelial cells exhibit cytotoxic and immunoregulatory effects in order to protect the organism against invading microorganisms. Antimicrobial peptides derived from frog skin display both immunostimulatory and immunosuppressive actions as demonstrated by in vitro cytokine production by macrophages. Frenatin 2.1S, first isolated from skin secretions of the frog, Sphaenorhynchus lacteus (Hylidae), enhances the in vitro production of pro-inflammatory IL-1β, TNF-α and IL-23 by mouse peritoneal cells. In order to test whether the immunostimulatory action of frenatin 2.1S may be reproduced in vivo, effects of intraperitoneal injections of this peptide on mononuclear cells in the peritoneum and spleen were determined 24h after administration. The data indicate that frenatin 2.1S enhances the activation state and homing capacity of Th1 type lymphocytes and NKT cells in the mouse peritoneal cavity, as evaluated by increased expression of early activation marker CD69 among T and NKT cells and chemokine receptor CXCR3 among T cells. Frenatin 2.1S significantly increases the percentage of (F4/80(+)CD11c(+)CD206(+)) pro-inflammatory M1 macrophages and enhances the expression of MHC class II molecules on F4/80(+)CD11c(+) macrophages in the mouse peritoneal cavity. Additionally, injection of frenatin 2.1S, in the presence or absence of lipopolysaccharide, increases the percentage of peritoneal B cells of the (CD19(+)CD11b(+)CD5(+)) B1a phenotype thus contributing to an inflammatory milieu. We suggest that the immunostimulatory effect of frenatin 2.1S may have therapeutic relevance in disease states, such as certain types of cancer, in which an enhanced inflammatory response may be beneficial. PMID:25861850

  16. Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules.

    PubMed Central

    Ito, N; Li, Y; Suzuki, T; Stechschulte, D J; Dileepan, K N

    1998-01-01

    The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression. PMID:9927232

  17. In vitro studies of interaction of rickettsia and macrophages: effect of ultraviolet light on Coxiella burnetti inactivation and macrophage enzymes

    SciTech Connect

    Little, J.S.; Kishimoto, R.A.; Canonico, P.G.

    1980-03-01

    The inactivation of Coxiella burnetii in suspension or in cultures of guinea pig peritoneal macrophages by ultraviolet (uv) light was studied. The effect of uv treatment on the activity of macrophage organelle marker enzymes and their subsequent equilibration in linear sucrose gradients was also determined. It was shown that uv treatment for 15 s at a distance of 10 cm inactivated C. burnetti, either in suspension or within guinea pig peritoneal macrophages. Similar uv treatment had little effect on the activity or equilibration of macrophage organelle marker enzymes in linear sucrose gradients.

  18. microRNA-155 deficiency results in decreased macrophage inflammation and attenuated atherogenesis in apoE−/− mice

    PubMed Central

    Du, Fen; Yu, Fang; Wang, Yuzhen; Hui, Yvonne; Carnevale, Kevin; Fu, Mingui; Lu, Hong; Fan, Daping

    2014-01-01

    Objective microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. Approach and Results Quantitative real-time PCR showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was positively correlated with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to LPS through targeting SOCS-1, and impaired cholesterol efflux from acetylated LDL-loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses, and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. We next examined the atherogenesis in apoE−/− and miR155−/−/apoE−/− (DKO) mice fed a western diet. Compared with apoE−/− mice, the DKO mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells, and reduced numbers of Th17 cells and CD11b+/Ly6Chigh cells in the spleen of DKO mice. Peritoneal macrophages from the DKO mice had significantly reduced pro-inflammatory cytokine expression and secretion both in the absence and presence of LPS stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed bone marrow transplantation study. Deficiency of miR155 in bone marrow-derived cells suppressed atherogenesis in apoE−/− mice, demonstrating that hematopoietic cell-derived miR155 plays a critical role. Conclusion miR155 deficiency attenuates atherogenesis in apoE−/− mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an anti-atherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis. PMID:24504735

  19. Response to stimulation with recombinant cytokines and synthesis of cytokines by murine intestinal macrophages infected with the Mycobacterium avium complex.

    PubMed Central

    Hsu, N; Young, L S; Bermudez, L E

    1995-01-01

    Current evidence suggests that the gut is the chief portal of entry for organisms of the Mycobacterium avium complex (MAC) in AIDS patients. Bacterial invasion of intestinal mucosa presumably occurs through epithelial cells, and M cells in the Peyer's patches, where the bacteria have contact with immunocompetent cells such as macrophages and T and B lymphocytes. As mucosal macrophages are probably the first line of defense against MAC, we examined their ability to inhibit intracellular growth of MAC when properly stimulated. Mouse intestinal macrophages were purified, infected with MAC 101, serovar 1, and MAC 86-2686, serovar 16, and subsequently stimulated with recombinant tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF). Viable intracellular bacteria were quantitated at 24 h after infection and again after 4 days of infection. Stimulation with TNF-alpha, IFN-gamma, and GM-CSF, but not M-CSF, was associated with mycobacteriostatic and/or mycobactericidal activity in macrophages. Treatment with 10(3) U of TNF-alpha, GM-CSF, and IFN-gamma per ml at 24 h prior to infection with MAC resulted in a significant enhancement in killing of MAC at 4 days after infection, compared with that observed for macrophages exposed to cytokines after infection. When stimulated with lipopolysaccharide or live MAC, intestinal macrophages had produced significantly less TNF-alpha and transforming growth factor beta than had splenic and peritoneal macrophages, although the levels of production of interleukin 6 and interleukin 10 among the three populations of cells were similar. Intestinal macrophages can be stimulated with cytokines to inhibit the intracellular growth of MAC, but they have differentiated abilities to produce cytokines which can modulate the anti-MAC immune response. PMID:7822018

  20. Alveolar macrophages. II. Inhibition of lymphocyte proliferation by purified macrophages from rat lung.

    PubMed Central

    Holt, P G

    1979-01-01

    Macrophages were prepared from the lung, peritoneal cavity and blood of normal, unstimulated rats from a number of strains. The macrophages were purified by adherence, and characterized via surface markers, enzyme activity and phagocytic capacity, and subsequently tested for activity in cultures of mitogen-stimulated syngeneic lymphocytes. Peritoneal macrophages and blood monocytes were mildly stimulatory, or ineffective in modulating mitogen-induced DNA synthesis; peritoneal macrophages reconstituted the blastogenic responses of macrophage-depleted lymph node cell cultures to normal limits. In contrast, alveolar macrophages were markedly inhibitory to lymphocyte proliferation; in some instances inhibitory activity was demonstrable when added alveolar macrophages comprised only 0.04% of the total cells in culture. Lymphocyte proliferation induced by T-cell mitogens was more susceptible to this inhibition than was proliferation induced by the B-cell mitogen LPS. Alveolar macrophages recovered from SPF rats, while less in number, exhibited comparable inhibitory activity. These results form part of an emerging picture picture of the normal alveolar macrophage as a potential 'suppressor' of T-cell activity in the lung. PMID:468308

  1. Assessments of Thioridazine as a Helper Compound to Dicloxacillin against Methicillin-Resistant Staphylococcus aureus: In Vivo Trials in a Mouse Peritonitis Model

    PubMed Central

    Stenger, Michael; Hendel, Kristoffer; Bollen, Peter; Licht, Peter B.; Kolmos, Hans Jørn; Klitgaard, Janne K.

    2015-01-01

    Introduction The rise in antimicrobial resistance is a major global concern and requires new treatment strategies. The use of helper compounds, such as thioridazine (TDZ), an antipsychotic drug, in combination with traditional antibiotics must be investigated. Objectives The aim of this study was to investigate the efficacy of TDZ as a helper compound for dicloxacillin (DCX) against methicillin-resistant Staphylococcus aureus (MRSA) in vivo, and compare the combination treatment of DCX+TDZ with vancomycin (VAN). Methods Mice were inoculated with an intraperitoneal (IP) injection of MRSA (108 CFU) and treated in a 12-hour cycle for 48 hours. By termination, bacterial quantities in a peritoneal flush, spleen and kidneys were obtained. In the main trial the drugs were administered subcutaneously in five treatment groups: 1) DCX, 2) TDZ, 3) DCX+TDZ, 4) VAN, 5) SALINE. Additional smaller studies with IP administration and higher subcutaneous dosages (×1.5 and ×4) of the drugs were subsequently performed. Results In the main trial no significant differences were found between DCX+TDZ and DCX or TDZ alone (p≥0.121–0.999). VAN performed significantly better than DCX+TDZ on all bacteriological endpoints (p<0.001). Higher subcutaneous dosages of DCX and TDZ improved the antibacterial efficacy, but the combination treatment was still not significantly better than monotherapy. IP drug administration of DCX+TDZ revealed a significantly better antibacterial effect than DCX or TDZ alone (p<0.001) but not significantly different from VAN (p>0.999). Conclusion In conclusion, TDZ did not prove to be a viable helper compound for dicloxacillin against MRSA in subcutaneous systemic treatment. However, IP-administration of DCX+TDZ, directly at the infection site resulted in a synergetic effect, with efficacy comparable to that of VAN. PMID:26267376

  2. Glycocalyx-Mimicking Nanoparticles for Stimulation and Polarization of Macrophages via Specific Interactions.

    PubMed

    Su, Lu; Zhang, Weiyi; Wu, Xiulong; Zhang, Yufei; Chen, Xi; Liu, Guangwei; Chen, Guosong; Jiang, Ming

    2015-09-01

    Malignant tumors develop multiple mechanisms to impair and escape from antitumor immune responses, of which tumor-associated macrophages that often show immunosuppressive phenotype (M2), play a critical role in tumor-induced immunosuppression. Therefore, strategies that can reverse M2 phenotype and even enhance immune-stimulation function of macrophage would benefit tumor immunotherapy. In this paper, self-assembled glyco-nanoparticles (glyco-NPs), as artificial glycocalyx, have been found to be able to successfully induce the polarization of mouse primary peritoneal macrophages from M2 to inflammatory type (M1). The polarization change was evidenced by the decreased expression of cell surface signaling molecules CD206 and CD23, and the increased expression of CD86. Meanwhile, secretion of cytokines supported this polarization change as well. More importantly, this phenomenon is observed not only in vitro, but also in vivo. As far as we known, this is the first report about macrophage polarization being induced by synthetic nanomaterials. Moreover, preparation, characterization of these glyco-NPs and their interaction with the macrophages are also demonstrated. PMID:25994111

  3. A biocompatibility study on peritoneal dialysis solution bags for CAPD.

    PubMed

    Carozzi, S; Nasini, M G; Schelotto, C; Caviglia, P M; Santoni, O; Pietrucci, A

    1993-01-01

    Numerous factors related to the composition of peritoneal dialysis solutions (PDS) contribute to the pathogenesis of peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). They include high osmolarity, low pH, and the presence of lactate, which may be responsible for stimulating the proliferation of peritoneal fibroblasts (PF) and for the toxicity on the peritoneal mesothelial cells (PMC). Similar effects could be hypothesized for the plasticizers released from the PDS bags, usually made of polyvinyl chloride (PVC), such as the acid esters of phthalic acid, particularly bis-(2-ethylhexyl) phthalate (BEHP). Recently, however, new BEHP-free bags (Clear-Flex, Bieffe, Italy) made of three layers (polyethylene, nylon, and polypropylene) have been introduced. The aim of this work is to evaluate in vitro the effects of samples of PDS contained in PVC bags (Bieffe) and in Clear-Flex bags on the proliferative capacity of peritoneal fibroblasts and peritoneal mesothelial cells, and the release of interferon gamma (IFN gamma), interleukin-1 (IL-1) and prostaglandin E2 (PGE2) from peritoneal T lymphocytes (PTLs) and macrophages (PM phi s). Results have shown that in the presence of PDS samples contained in PVC bags, the proliferative capacity of peritoneal fibroblasts was higher than in Clear-Flexbags. There was also an increased release of IFN-gamma and IL-1 from PTLs and PM phi s (cytokines that stimulate the collagen synthesis) and a decreased release of PGE2 (cytokines which inhibit the collagen synthesis). An inhibiting action on peritoneal mesothelial cells was also seen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8105908

  4. Resistance of C58 mice to primary systemic herpes simplex virus infection: macrophage dependence and T-cell independence.

    PubMed Central

    Schlabach, A J; Martinez, D; Field, A K; Tytell, A A

    1979-01-01

    The relative contribution of thymus-derived lymphocytes (T-cells) and of macrophages to resistance to primary infection with herpes simplex virus type 1 (HSV-1) was studied in C58 mice. Resistance was dependent on macrophage competence, but was relatively independent of T-lymphocyte competence. Although aging mice became progressively more deficient in functional T-cells, as demonstrated by a decreasing resistance to transplanted line Ib leukemia and by declining responses to T-cell nitogens (concanavalin A and phytohemagglutinin), their resistance to HSV-1 increased with increasing age. Moreover, in mice that were made selectively deficient in T-cells by the combination of adult thymectomy and treatment with anti-thymocyte serum, resistance to HSV-1 did not correlate spleen and mesenteric lymph nodes. However, selective reduction of macrophages by intraperitoneal injection of silica resulted in enhanced susceptibility to HSV-1. Furthermore, in vitro suppression of HSV-1 plaque formation in mouse embryo fibroblast cells was obtained by cocultivation of infected fibroblast monolayers with peritoneal macrophages, but not with splenic lymphocytes, from adult mice. Macrophages from weanling mice failed to suppress the development of plaques, indicating that the increase in resistance to HSV-1 with age is a result of increased macrophage competence. PMID:232692

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

    PubMed

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

    2012-06-01

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

  6. IL4/PGE{sub 2} induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

    SciTech Connect

    Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D. . E-mail: pstahl@cellbiology.wustl.edu; Barbieri, M. Alejandro

    2006-07-15

    The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E{sub 2} (PGE{sub 2}) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.

  7. Effect of methacrylic acid beads on the sonic hedgehog signaling pathway and macrophage polarization in a subcutaneous injection mouse model.

    PubMed

    Lisovsky, Alexandra; Zhang, David K Y; Sefton, Michael V

    2016-08-01

    Poly(methacrylic acid-co-methyl methacrylate) (MAA) beads promote a vascular regenerative response when used in diabetic wound healing. Previous studies reported that MAA beads modulated the expression of sonic hedgehog (Shh) and inflammation related genes in diabetic wounds. The aim of this work was to follow up on these observations in a subcutaneous injection model to study the host response in the absence of the confounding factors of diabetic wound healing. In this model, MAA beads improved vascularization in healthy mice of both sexes compared to control poly(methyl methacrylate) (MM) beads, with a stronger effect seen in males than females. MAA-induced vessels were perfusable, as evidenced from the CLARITY-processed images. In Shh-Cre-eGFP/Ptch1-LacZ non-diabetic transgenic mice, the increased vessel formation was accompanied by a higher density of cells expressing GFP (Shh) and β-Gal (patched 1, Ptch1) suggesting MAA enhanced the activation of the Shh pathway. Ptch1 is the Shh receptor and a target of the pathway. MAA beads also modulated the inflammatory cell infiltrate in CD1 mice: more neutrophils and more macrophages were noted with MAA relative to MM beads at days 1 and 7, respectively. In addition, MAA beads biased macrophages towards a MHCII-CD206+ ("M2") polarization state. This study suggests that the Shh pathway and an altered inflammatory response are two elements of the complex mechanism whereby MAA-based biomaterials effect vascular regeneration. PMID:27264502

  8. Immortalized MH-S cells lack defining features of primary alveolar macrophages and do not support mouse pneumovirus replication.

    PubMed

    Brenner, Todd A; Rice, Tyler A; Anderson, Erik D; Percopo, Caroline M; Rosenberg, Helene F

    2016-04-01

    The SV-40-transformed MH-S cell line maintains some, but not all, features of primary alveolar macrophages (AMs) from BALB/c mice. We show here that MH-S cells produce inflammatory cytokines IL-6 and CXCL10 in response to challenge with Gram-positive Lactobacillus reuteri, and to TLR2 and NOD2 ligands Pam3CSK4 and MDP, respectively. In contrast, although wild-type AMs are infected in vivo by pneumonia virus of mice (PVM), no virus replication was detected in MH-S cells. Interestingly, the surface immunophenotype of MH-S cells (CD11c(+)Siglec F(-)) differs from that of wild-type AMs (CD11c(+) Siglec F(+)) and is similar to that of immature AMs isolated from granulocyte macrophage-colony stimulating factor (GM-CSF) gene-deleted mice; AMs from GM-CSF(-/-) mice also support PVM replication. However, MH-S cells do not express the GM-CSF receptor alpha chain (CD116) and do not respond to GM-CSF. Due to these unusual features, MH-S cells should be used with caution as experimental models of AMs. PMID:26916143

  9. Ribonucleic acid synthesis in normal and immune macrophages after antigenic stimulus.

    PubMed

    Soderberg, L S; Tewari, R P; Solotorovsky, M

    1976-06-01

    Macrophage ribonucleic acid (RNA) synthesis is an important metabolic process intimately related to the function of these cells. Mouse peritoneal macrophage RNA was extracted with phenol in the presence of bentonite and electrophoresed on composite agarose-polyacrylamide gels. The pulse-chase technique was used to follow the precursor relationships in macrophage ribosomal RNA (rRNA) maturation. The rRNA species at 18S and 28S appeared at 15 and 45 min, respectively, after RNA synthesis was halted. Their appearance corresponded closely to decreases in the rRNA precursors at 45S, 36S, and 34S. Studies of RNA methylation aided in confirming the identity of these ribosomal species. Unmethylated RNA species appeared as messenger RNA between 5S and 15S, and at about 55S probably represented heterodisperse nuclear RNA. When normal macrophages were incubated with heat-killed Salmonella enteritidis, an acceleration in the maturation of RNA was observed. The accelerated maturation was indicated by the earlier appearance of 28S rRNA and the more rapid development of an equilibrium state, where further labeling did not change the RNA profile. In macrophage RNA from mice immunized with S. enteritidis, rRNA species appeared rapidly but did not accumulate to the same extent as observed for normal macrophages. Precursor rRNA and other RNA species developed as usual, suggesting specific degradation of mature rRNA. Such rRNA wastage could indicate a mechanism controlling ribosome assembly in the non-proliferating activated macrophage. The pattern of RNA synthesis in immune macrophages was essentially unchanged by the presence of heat-killed S. enteritidis in vitro. PMID:971940

  10. Characterization of oak and birch dust-induced expression of cytokines and chemokines in mouse macrophage RAW 264.7 cells.

    PubMed

    Määttä, Juha; Majuri, Marja-Leena; Luukkonen, Ritva; Lauerma, Antti; Husgafvel-Pursiainen, Kirsti; Alenius, Harri; Savolainen, Kai

    2005-11-01

    Occupational exposure to wood dust is related to several respiratory diseases, such as allergic rhinitis, chronic bronchitis, and asthma. However, virtually nothing is known about molecular mechanisms behind wood dust-induced pulmonary inflammation. To elucidate the effects of wood dust exposure on cytokine and chemokine expression in murine macrophage cell line cells, mouse RAW 264.7 cells were exposed to two selected hardwood dusts, oak and birch. TiO2 and LPS were used as controls. Expression patterns of several cytokines, chemokines, and chemokine receptors were assessed by real-time quantitative PCR system and by ELISA. Exposure to birch dust caused a major increase in TNF-alpha and IL-6 protein levels whereas a weaker induction of TNF-alpha protein was found after exposure to oak dust. Inorganic TiO2 dust did not induce significant cytokine expression. With respect to the chemokines, a dose-dependent, about 10-fold induction of CCL2 mRNA and protein was found after exposure to birch dust. Oak dust induced weakly CCL2 protein. Similarly, birch dust induced a strong expression of CCL3, CCL4, and CXCL2/3 mRNA whereas only moderate levels of these chemokine mRNAs were detected after oak dust exposure. In contrast, expression of CCL24 mRNA was inhibited by more than 40-fold by both oak and birch dusts. TiO2 dust induced about five-fold expression of CCL3 and CCL4 mRNA but did not affect significantly other chemokines. These results suggest that exposure to birch or oak dusts may influence the development of the inflammatory process in the airways by modulating the expression of macrophage-derived cytokines and chemokines. PMID:16122864

  11. Chlamydia pneumoniae Augments the Oxidized Low-Density Lipoprotein-Induced Death of Mouse Macrophages by a Caspase-Independent Pathway

    PubMed Central

    Yaraei, Kambiz; Campbell, Lee Ann; Zhu, Xiaodong; Liles, W. Conrad; Kuo, Cho-chou; Rosenfeld, Michael E.

    2005-01-01

    Chlamydia pneumoniae is a common respiratory pathogen that is associated with an increased risk of cardiovascular disease. However, the mechanisms by which C. pneumoniae contributes to cardiovascular disease have not been determined yet. C. pneumoniae infection may accelerate the death of cells within atherosclerotic lesions and contribute to the formation of unstable lesions. To test this hypothesis, the impact of C. pneumoniae infection on the death of lipid-loaded mouse macrophages was investigated. It was observed that RAW 264.7 cells are highly susceptible to the toxic effects of oxidized low-density lipoprotein (LDL) and exhibit markers of cell death within 24 h of treatment with as little as 5 μg/ml oxidized LDL. Subsequent infection with either live C. pneumoniae or heat-killed or UV-inactivated C. pneumoniae at a low multiplicity of infection for 24 to 72 h stimulated both additional binding of annexin V and the uptake of propidium iodide. Thus, C. pneumoniae augments the effects of oxidized LDL on cell death independent of a sustained infection. However, unlike oxidized LDL, C. pneumoniae infection does not activate caspase 3 or induce formation of the mitochondrial transition pore or the fragmentation of DNA, all of which are classical markers of apoptosis. Furthermore, primary bone marrow macrophages isolated from mice deficient in Toll-like receptor 2 (TLR-2) but not TLR-4 are resistant to C. pneumoniae-induced death. These data suggest that C. pneumoniae kills cells by a caspase-independent pathway and that the process is potentially mediated by activation of TLR-2. PMID:15972525

  12. In vitro evaluation of inhibitory effect of nuclear factor-kappaB activity by small interfering RNA on pro-tumor characteristics of M2-like macrophages.

    PubMed

    Kono, Yusuke; Kawakami, Shigeru; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2014-01-01

    Tumor-associated macrophages (TAMs) have an alternatively activated macrophage phenotype (M2) and promote cancer cell proliferation, angiogenesis and metastasis. Nuclear factor-kappaB (NF-κB) is one of the master regulators of macrophage polarization. Here, we investigated the effect of inhibition of NF-κB activity by small interfering RNA (siRNA) on the pro-tumor response of macrophages located in the tumor microenvironment in vitro. We used mouse peritoneal macrophages cultured in conditioned medium from colon-26 cancer cells as an in vitro TAM model (M2-like macrophages). Transfection of NF-κB (p50) siRNA into M2-like macrophages resulted in a significant decrease in the secretion of interleukin (IL)-10 (a T helper 2 (Th2) cytokine) and a significant increase of T helper 1 (Th1) cytokine production (IL-12, tumor necrosis factor-α, and IL-6). Furthermore, vascular endothelial growth factor production and matrix metalloproteinase-9 mRNA expression in M2-like macrophages were suppressed by inhibition of NF-κB expression with NF-κB (p50) siRNA. In addition, there was a reduction of arginase mRNA expression and increase in nitric oxide production. The cytokine secretion profiles of macrophages cultured in conditioned medium from either B16BL6 or PAN-02 cancer cells were also converted from M2 to classically activated (M1) macrophages by transfection of NF-κB (p50) siRNA. These results suggest that inhibition of NF-κB activity in M2-like macrophages alters their phenotype toward M1. PMID:24141263

  13. Intracellular growth inhibition of Histoplasma capsulatum induced in murine macrophages by recombinant gamma interferon is not due to a limitation of the supply of methionine or cysteine to the fungus.

    PubMed Central

    Wu-Hsieh, B A; Howard, D H

    1992-01-01

    Recombinant murine gamma interferon (rMuIFN-gamma) stimulates mouse peritoneal macrophages to inhibit the intracellular growth of the zoopathogenic fungus Histoplasma capsulatum. In some systems, the inhibition of growth of an intracellular parasite by rIFN-gamma has been related to nutritional constraints induced in the host cells by the lymphokine. Such an explanation might apply to H. capsulatum because the fungus is a functional methionine-cysteine (Met-Cys) auxotroph at 37 degrees C; its sulfite reductase is repressed at that temperature. For this reason, we set about to examine whether or not the antihistoplasma state induced in rMuIFN-gamma is due to a restriction in the availability of Met-Cys. Omission of Met-Cys from the medium in which macrophages were cultivated prevented H. capsulatum from growing within them. Addition of Met or Cys to the macrophage cultures did not antagonize the inhibitory effect induced in the cells by rMuIFN-gamma. Thus, there was no evidence from our work that rMuIFN-gamma evokes the antihistoplasma effect in mouse peritoneal macrophages by limiting the supply of Met-Cys to the fungus. PMID:1730506

  14. Insufficient Generation of Mycobactericidal Mediators and Inadequate Level of Phagosomal Maturation Are Related with Susceptibility to Virulent Mycobacterium tuberculosis Infection in Mouse Macrophages.

    PubMed

    Lee, Hyo-Ji; Ko, Hyun-Jeong; Jung, Yu-Jin

    2016-01-01

    Tuberculosis is caused by Mycobacterium tuberculosis infection, and it remains major life-threatening infectious diseases worldwide. Although, M. tuberculosis has infected one-third of the present human population, only 5-10% of immunocompetent individuals are genetically susceptible to tuberculosis. All inbred strains of mice are susceptible to tuberculosis; however, some mouse strains are much more susceptible than others. In a previous report, we showed that Th1-mediated immunity was not responsible for the differential susceptibility between mouse models. To examine whether these susceptibility differences between inbred mouse strains are due to the insufficient production of effector molecules in the early stage of innate immunity, we investigated mycobacteriostatic function of bone marrow-derived macrophages (BMDMs) in resistant (BALB/c and C57BL/6) and susceptible strains (DBA/2) that were infected with virulent M. tuberculosis (H37Rv) or attenuated M. tuberculosis (H37Ra). The growth rate of virulent M. tuberculosis in infected cells was significantly higher in DBA/2 BMDMs, whereas the growth of the attenuated strain was similar in the three inbred mouse BMDM strains. In addition, the death rate of M. tuberculosis-infected cells increased with the infectious dose when DBA/2 BMDMs were infected with H37Rv. The intracellular reactive oxygen species level was lower in DBA/2 BMDMs that were infected with virulent M. tuberculosis at an early post-infection time point. The expression levels of phagosomal maturation markers, including early endosomal antigen-1 (EEA1) and lysosome-associated membrane protein-1 (LAMP-1), were significantly decreased in DBA/2 BMDM that were infected with virulent M. tuberculosis, whereas IFNγ-treatment restored the phagosomal maturation activity. The nitric oxide (NO) production levels were also significantly lower in DBA/2 BMDMs that were infected with virulent H37Rv at late post-infection points; however, this was not observed

  15. Insufficient Generation of Mycobactericidal Mediators and Inadequate Level of Phagosomal Maturation Are Related with Susceptibility to Virulent Mycobacterium tuberculosis Infection in Mouse Macrophages

    PubMed Central

    Lee, Hyo-Ji; Ko, Hyun-Jeong; Jung, Yu-Jin

    2016-01-01

    Tuberculosis is caused by Mycobacterium tuberculosis infection, and it remains major life-threatening infectious diseases worldwide. Although, M. tuberculosis has infected one-third of the present human population, only 5–10% of immunocompetent individuals are genetically susceptible to tuberculosis. All inbred strains of mice are susceptible to tuberculosis; however, some mouse strains are much more susceptible than others. In a previous report, we showed that Th1-mediated immunity was not responsible for the differential susceptibility between mouse models. To examine whether these susceptibility differences between inbred mouse strains are due to the insufficient production of effector molecules in the early stage of innate immunity, we investigated mycobacteriostatic function of bone marrow-derived macrophages (BMDMs) in resistant (BALB/c and C57BL/6) and susceptible strains (DBA/2) that were infected with virulent M. tuberculosis (H37Rv) or attenuated M. tuberculosis (H37Ra). The growth rate of virulent M. tuberculosis in infected cells was significantly higher in DBA/2 BMDMs, whereas the growth of the attenuated strain was similar in the three inbred mouse BMDM strains. In addition, the death rate of M. tuberculosis-infected cells increased with the infectious dose when DBA/2 BMDMs were infected with H37Rv. The intracellular reactive oxygen species level was lower in DBA/2 BMDMs that were infected with virulent M. tuberculosis at an early post-infection time point. The expression levels of phagosomal maturation markers, including early endosomal antigen-1 (EEA1) and lysosome-associated membrane protein-1 (LAMP-1), were significantly decreased in DBA/2 BMDM that were infected with virulent M. tuberculosis, whereas IFNγ-treatment restored the phagosomal maturation activity. The nitric oxide (NO) production levels were also significantly lower in DBA/2 BMDMs that were infected with virulent H37Rv at late post-infection points; however, this was not observed

  16. Type I Interferon Signals in Macrophages and Dendritic Cells Control Dengue Virus Infection: Implications for a New Mouse Model To Test Dengue Vaccines

    PubMed Central

    Toh, Ying-Xiu; Valdés, Iris; Cerny, Daniela; Heinrich, Julia; Hermida, Lisset; Marcos, Ernesto; Guillén, Gerardo; Kalinke, Ulrich; Shi, Pei-Yong; Fink, Katja

    2014-01-01

    ABSTRACT Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortality. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturally not susceptible to DENV and only become infected if highly immunocompromised. Mouse models lacking both type I and type II interferon (IFN) receptors (AG129 mice) or the type I IFN receptor (IFNAR−/− mice) are susceptible to infection with mouse-adapted DENV strains but are severely impaired in mounting functional immune responses to the virus and thus are of limited use for study. Here we used conditional deletion of the type I IFN receptor (IFNAR) on individual immune cell subtypes to generate a minimally manipulated mouse model that is susceptible to DENV while retaining global immune competence. Mice lacking IFNAR expression on CD11c+ dendritic cells and LysM+ macrophages succumbed completely to DENV infection, while mice deficient in the receptor on either CD11c+ or LysM+ cells were susceptible to infection but often resolved viremia and recovered fully from infection. Conditional IFNAR mice responded with a swift and strong CD8+ T-cell response to viral infection, compared to a weak response in IFNAR−/− mice. Furthermore, mice lacking IFNAR on either CD11c+ or LysM+ cells were also sufficiently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These data demonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study of DENV immunology and screening of vaccine candidates. IMPORTANCE Dengue virus infects 400 million people every year worldwide, causing 100 million clinically apparent infections, which can be fatal if untreated. Despite many years of research, there are no effective vaccine and no antiviral treatment available for dengue. Development of vaccines has been hampered in particular by

  17. Down-regulated expression of monocyte/macrophage major histocompatibility complex receptors in human and mouse monocytes by expression of their ligands

    PubMed Central

    Yamana, H; Tashiro-Yamaji, J; Hayashi, M; Maeda, S; Shimizu, T; Tanigawa, N; Uchiyama, K; Kubota, T; Yoshida, R

    2014-01-01

    Mouse monocyte/macrophage major histocompatibility complex (MHC) receptor 1 (MMR1; or MMR2) specific for H-2Dd (or H-2Kd) molecules is expressed on monocytes from non-H-2Dd (or non-H-2Kd), but not those from H-2Dd (or H-2Kd), inbred mice. The MMR1 and/or MMR2 is essential for the rejection of H-2Dd- and/or H-2Kd-transgenic mouse skin onto C57BL/6 (H-2DbKb) mice. Recently, we found that human leucocyte antigen (HLA)-B44 was the sole ligand of human MMR1 using microbeads that had been conjugated with 80 types of HLA class I molecules covering 94·2% (or 99·4%) and 92·4% (or 96·2%) of HLA-A and B molecules of Native Americans (or Japanese), respectively. In the present study, we also explored the ligand specificity of human MMR2 using microbeads. Microbeads coated with HLA-A32, HLA-B13 or HLA-B62 antigens bound specifically to human embryonic kidney (HEK)293T or EL-4 cells expressing human MMR2 and to the solubilized MMR2-green fluorescent protein (GFP) fusion protein; and MMR2+ monocytes from a volunteer bound HLA-B62 molecules with a Kd of 8·7 × 10−9 M, implying a three times down-regulation of MMR2 expression by the ligand expression. H-2Kd (or H-2Dd) transgene into C57BL/6 mice down-regulated not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression, leading to further down-regulation of MMR expression. In fact, monocytes from two (i.e. MMR1+/MMR2+ and MMR1–/MMR2–) volunteers bound seven to nine types of microbeads among 80, indicating ≤ 10 types of MMR expression on monocytes. The physiological role of constitutive MMRs on monocytes possibly towards allogeneic (e.g. fetal) cells in the blood appears to be distinct from that of inducible MMRs on macrophages toward allografts in tissue. PMID:24842626

  18. Multicystic peritoneal mesothelioma

    PubMed Central

    Tentes, Antonios-Apostolos; Zorbas, Georgios; Pallas, Nicolaos; Fiska, Aliki

    2012-01-01

    Summary Background: Multicystic peritoneal mesothelioma is a rare disease. It is not certain if it is a benign or a borderline tumor. Although many therapeutic approaches have been used, complete cytoreductive surgery in combination with hyperthermic intraoperative intraperitoneal chemotherapy has gained acceptance. Case Report: A case of multicystic peritoneal mesothelioma in a 16-year old patient is reported. The patient underwent complete cytoreduction and received intraoperative hyperthermic intraperitoneal chemotherapy. The patient is disease-free one year after surgery. Conclusions: Complete cytoreductive surgery in combination with hyperthermic intraoperative intraperitoneal chemotherapy appears to be a rational therapeutic approach in multicystic peritoneal mesothelioma. PMID:23569544

  19. Effect of enterohaemorrhagic Escherichia coli O157:H7-specific enterohaemolysin on interleukin-1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation.

    PubMed

    Cheng, Yu-Li; Song, Li-Qiong; Huang, Yuan-Ming; Xiong, Yan-Wen; Zhang, Xiao-Ai; Sun, Hui; Zhu, Xin-Ping; Meng, Guang-Xun; Xu, Jian-Guo; Ren, Zhi-Hong

    2015-06-01

    Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7-mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin-1β (IL-1β) production in human macrophages. Here, we investigated the disparity in Ehx-induced IL-1β production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes. In contrast to the effects on human differentiated THP-1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL-1β production in mouse macrophages and splenocytes because of a disparity in pro-IL-1β cleavage into mature IL-1β upon caspase-1 activation. Additionally, Ehx significantly contributed to O157:H7-induced ATP release from THP-1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP-1 cells but not in mouse IC-21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7-induced IL-1β production was largely dependent upon caspase-1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K(+) efflux drastically diminished O157:H7-induced IL-1β production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection. PMID:25580516

  20. Effect of enterohaemorrhagic Escherichia coli O157:H7-specific enterohaemolysin on interleukin-1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation

    PubMed Central

    Cheng, Yu-Li; Song, Li-qiong; Huang, Yuan-Ming; Xiong, Yan-Wen; Zhang, Xiao-Ai; Sun, Hui; Zhu, Xin-Ping; Meng, Guang-Xun; Xu, Jian-Guo; Ren, Zhi-Hong

    2015-01-01

    Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7-mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin-1β (IL-1β) production in human macrophages. Here, we investigated the disparity in Ehx-induced IL-1β production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes. In contrast to the effects on human differentiated THP-1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL-1β production in mouse macrophages and splenocytes because of a disparity in pro-IL-1β cleavage into mature IL-1β upon caspase-1 activation. Additionally, Ehx significantly contributed to O157:H7-induced ATP release from THP-1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP-1 cells but not in mouse IC-21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7-induced IL-1β production was largely dependent upon caspase-1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K+ efflux drastically diminished O157:H7-induced IL-1β production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection. PMID:25580516

  1. Transcriptional Regulation and Macrophage Differentiation.

    PubMed

    Hume, David A; Summers, Kim M; Rehli, Michael

    2016-06-01

    Monocytes and macrophages are professional phagocytes that occupy specific niches in every tissue of the body. Their survival, proliferation, and differentiation are controlled by signals from the macrophage colony-stimulating factor receptor (CSF-1R) and its two ligands, CSF-1 and interleukin-34. In this review, we address the developmental and transcriptional relationships between hematopoietic progenitor cells, blood monocytes, and tissue macrophages as well as the distinctions from dendritic cells. A huge repertoire of receptors allows monocytes, tissue-resident macrophages, or pathology-associated macrophages to adapt to specific microenvironments. These processes create a broad spectrum of macrophages with different functions and individual effector capacities. The production of large transcriptomic data sets in mouse, human, and other species provides new insights into the mechanisms that underlie macrophage functional plasticity. PMID:27337479

  2. Anti-Inflammatory and Analgesic Effects of Pyeongwisan on LPS-Stimulated Murine Macrophages and Mouse Models of Acetic Acid-Induced Writhing Response and Xylene-Induced Ear Edema

    PubMed Central

    Oh, You-Chang; Jeong, Yun Hee; Cho, Won-Kyung; Ha, Jeong-Ho; Gu, Min Jung; Ma, Jin Yeul

    2015-01-01

    Pyeongwisan (PW) is an herbal medication used in traditional East Asian medicine to treat anorexia, abdominal distension, borborygmus and diarrhea caused by gastric catarrh, atony and dilatation. However, its effects on inflammation-related diseases are unknown. In this study, we investigated the biological effects of PW on lipopolysaccharide (LPS)-mediated inflammation in macrophages and on local inflammation in vivo. We investigated the biological effects of PW on the production of inflammatory mediators, pro-inflammatory cytokines and related products as well as the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated macrophages. Additionally, we evaluated the analgesic effect on the acetic acid-induced writhing response and the inhibitory activity on xylene-induced ear edema in mice. PW showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and interleukin-1β (IL-1β). In addition, PW strongly suppressed inducible nitric oxide synthase (iNOS), a NO synthesis enzyme, induced heme oxygenase-1 (HO-1) expression and inhibited NF-κB activation and MAPK phosphorylation. Also, PW suppressed TNF-α, IL-6 and IL-1β cytokine production in LPS-stimulated peritoneal macrophage cells. Furthermore, PW showed an analgesic effect on the writhing response and an inhibitory effect on mice ear edema. We demonstrated the anti-inflammatory effects and inhibitory mechanism in macrophages as well as inhibitory activity of PW in vivo for the first time. Our results suggest the potential value of PW as an inflammatory therapeutic agent developed from a natural substance. PMID:25569097

  3. Adsorption of Surfactant Lipids by Single-Walled Carbon Nanotubes in Mouse Lung upon Pharyngeal Aspiration: Role in Uptake by Macrophages

    PubMed Central

    Kapralov, Alexander A.; Feng, Wei Hong; Amoscato, Andrew A.; Yanamala, Naveena; Balasubramanian, Krishnakumar; Winnica, Daniel E.; Kisin, Elena R.; Kotchey, Gregg P.; Gou, Pingping; Sparvero, Louis J.; Ray, Prabir; Mallampalli, Rama K.; Klein-Seetharaman, Judith; Fadeel, Bengt; Star, Alexander; Shvedova, Anna A.; Kagan, Valerian E.

    2012-01-01

    The pulmonary route represents one of the most important portals of entry for nanoparticles into the body. However, the in vivo interactions of nanoparticles with biomolecules of the lung have not been sufficiently studied. Here, using an established mouse model of pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs), we recovered SWCNTs from the bronchoalveolar lavage fluid (BALf), purified them from possible contamination with lung cells and examined the composition of phospholipids adsorbed on SWCNTs by liquid chromatography mass spectrometry (LC-MS) analysis. We found that SWCNTs selectively adsorbed two types of the most abundant surfactant phospholipids – phosphatidylcholines (PC) and phosphatidylglycerols (PG). Molecular speciation of these phospholipids was also consistent with pulmonary surfactant. Quantitation of adsorbed lipids by LC-MS along with the structural assessments of phospholipid binding by atomic force microscopy and molecular modeling indicated that the phospholipids (~108 molecules per SWCNT) formed an uninterrupted “coating” whereby the hydrophobic alkyl chains of the phospholipids were adsorbed onto the SWCNT with the polar head groups pointed away from the SWCNT into the aqueous phase. In addition, the presence of surfactant proteins A, B and D on SWCNTs was determined by LC-MS. Finally, we demonstrated that the presence of this surfactant coating markedly enhanced the in vitro uptake of SWCNTs by macrophages. Taken together, this is the first demonstration of the in vivo adsorption of the surfactant lipids and proteins on SWCNTs in a physiologically relevant animal model. PMID:22463369

  4. Mouse pre-immunocytes as non-proliferating multipotent precursors of macrophages, interferon-producing cells, CD8alpha(+) and CD8alpha(-) dendritic cells.

    PubMed

    Bruno, L; Seidl, T; Lanzavecchia, A

    2001-11-01

    In this study we characterize in mouse bone marrow and peripheral blood a homogeneous cell subset expressing Ly6C, CD31 and CD11c, that can give rise to multiple cell types involved in the immune response. Under the aegis of M-CSF or GM-CSF these cells rapidly differentiate without division to either macrophages or immature dendritic cells, which can be further induced to mature by LPS stimulation. In fetal thymic organ cultures the same cells generate both CD8alpha(+) and CD8alpha(-) dendritic cells in comparable proportion as found in normal thymus. The Ly6C(+), CD31(+) and CD11c(+) cells express not only TLR2 and TLR4, which are characteristic of myeloid dendritic cells, but also TLR7 and TLR9, which conversely are characteristic of human interferon-producing cells. Moreover, following stimulation with influenza virus, they rapidly express high levels of IFN-alpha mRNA. Finally these precursors are increased in bone marrow and peripheral blood during systemic inflammation. These cells are defined as "pre-immunocytes" to underline the fact that they serve in a flexible fashion multiple, and often divergent, functions required for the immune response to pathogens. PMID:11745359

  5. Erucin exerts anti-inflammatory properties in murine macrophages and mouse skin: possible mediation through the inhibition of NFκB signaling.

    PubMed

    Cho, Han Jin; Lee, Ki Won; Park, Jung Han Yoon

    2013-01-01

    Erucin, an isothiocyanate, is a hydrolysis product of glucoerucin found in arugula and has recently been reported to have anti-cancer properties in various cancer cells. In this study, we assessed the anti-inflammatory effects of erucin and the underlying mechanisms, using lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages and 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin. In RAW 264.7 cells, erucin (2.5, 5 μmol/L) inhibited LPS-induced production of nitric oxide and prostaglandin E2. Erucin inhibited LPS-induced degradation of the inhibitor of κBα and translocation of p65 to the nucleus and, subsequently, reduced LPS-induced nuclear factor κB (NFκB) DNA binding activities, as well as the transcriptional activity of NFκB, leading to the decreased expression of NFκB-target genes, including tumor necrosis factor-α, interleukin (IL)-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, as well as transcriptional activity of iNOS and COX-2. In mice, erucin (100, 300 nmoles) treatment significantly inhibited phorbol ester-induced formation of ear edema and expression of iNOS and COX-2 proteins. These results indicate that erucin exerts a potent anti-inflammatory activity by inhibiting the pro-inflammatory enzymes and cytokines, which may be mediated, at least in part, via the inhibition of NFκB signaling. PMID:24132147

  6. Erucin Exerts Anti-Inflammatory Properties in Murine Macrophages and Mouse Skin: Possible Mediation through the Inhibition of NFκB Signaling

    PubMed Central

    Cho, Han Jin; Lee, Ki Won; Park, Jung Han Yoon

    2013-01-01

    Erucin, an isothiocyanate, is a hydrolysis product of glucoerucin found in arugula and has recently been reported to have anti-cancer properties in various cancer cells. In this study, we assessed the anti-inflammatory effects of erucin and the underlying mechanisms, using lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages and 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin. In RAW 264.7 cells, erucin (2.5, 5 μmol/L) inhibited LPS-induced production of nitric oxide and prostaglandin E2. Erucin inhibited LPS-induced degradation of the inhibitor of κBα and translocation of p65 to the nucleus and, subsequently, reduced LPS-induced nuclear factor κB (NFκB) DNA binding activities, as well as the transcriptional activity of NFκB, leading to the decreased expression of NFκB-target genes, including tumor necrosis factor-α, interleukin (IL)-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, as well as transcriptional activity of iNOS and COX-2. In mice, erucin (100, 300 nmoles) treatment significantly inhibited phorbol ester-induced formation of ear edema and expression of iNOS and COX-2 proteins. These results indicate that erucin exerts a potent anti-inflammatory activity by inhibiting the pro-inflammatory enzymes and cytokines, which may be mediated, at least in part, via the inhibition of NFκB signaling. PMID:24132147

  7. Lentiviral Delivery of RNAi for In Vivo Lineage-Specific Modulation of Gene Expression in Mouse Lung Macrophages

    PubMed Central

    Wilson, Andrew A; Kwok, Letty W; Porter, Emily L; Payne, Julie G; McElroy, Gregory S; Ohle, Sarah J; Greenhill, Sara R; Blahna, Matthew T; Yamamoto, Kazuko; Jean, Jyh C; Mizgerd, Joseph P; Kotton, Darrell N

    2013-01-01

    Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings. PMID:23403494

  8. Interaction of mouse splenocytes and macrophages with bacterial strains in vitro: the effect of age in the immune response.

    PubMed

    Van Beek, A A; Hoogerland, J A; Belzer, C; De Vos, P; De Vos, W M; Savelkoul, H F J; Leenen, P J M

    2016-01-01

    Probiotics influence the immune system, both at the local and systemic level. Recent findings suggest the relation between microbiota and the immune system alters with age. Our objective was to address direct effects of six bacterial strains on immune cells from young and aged mice: Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, Lactococcus lactis MG1363, Bifidobacterium breve ATCC15700, Bifidobacterium infantis ATCC15697, and Akkermansia muciniphila ATCC BAA-835. We used splenocytes and naïve or interferon-γ-stimulated bone marrow-derived macrophages (BMDM) as responder populations. All tested bacterial strains induced phenotypic and cytokine responses in splenocytes and BMDM. Based on magnitude of the cellular inflammatory response and cytokine profiles, two subgroups of bacteria were identified, i.e. L. plantarum and L. casei versus B. breve, B. infantis, and A. muciniphila. The latter group of bacteria induced high levels of cytokines produced under inflammatory conditions, including tumour necrosis factor (TNF), interleukin (IL)-6 and IL-10. Responses to L. lactis showed features of both subgroups. In addition, we compared responses by splenocytes and BMDM derived from young mice to those of aged mice, and found that splenocytes and BMDM derived from aged mice had an increased IL-10 production and dysregulated IL-6 and TNF production compared to young immune cells. Overall, our study shows differential inflammatory responses to distinct bacterial strains, and profound age-dependent effects. These findings, moreover, support the view that immune environment importantly influences bacterial immune effects. PMID:26689225

  9. Macrophage Migration Inhibitory Factor is essential for osteoclastogenic mechanisms in vitro and in vivo mouse model of arthritis

    PubMed Central

    Gu, Ran; Santos, Leilani L.; Ngo, Devi; Fan, HuaPeng; Singh, Preetinder P.; Fingerle-Rowson, Gunter; Bucala, Richard; Xu, Jiake; Quinn, Julian M. W.; Morand, Eric F.

    2015-01-01

    Macrophage migration inhibitory factor (MIF) enhances activation of leukocytes, endothelial cells and fibroblast-like synoviocytes (FLS), thereby contributing to the pathogenesis of rheumatoid arthritis (RA). A MIF promoter polymorphism in RA patients resulted in higher serum MIF concentration and worsens bone erosion; controversially current literature reported an inhibitory role of MIF in osteoclast formation. The controversial suggested that the precise role of MIF and its putative receptor CD74 in osteoclastogenesis and RA bone erosion, mediated by locally formed osteoclasts in response to receptor activator of NF-κB ligand (RANKL), is unclear. We reported that in an in vivo K/BxN serum transfer arthritis, reduced clinical and histological arthritis in MIF-/- and CD74-/- mice were accompanied by a virtual absence of osteoclasts at the synovium-bone interface and reduced osteoclast-related gene expression. Furthermore, in vitro osteoclast formation and osteoclast-related gene expression were significantly reduced in MIF-/- cells via decreasing RANKL-induced phosphorylation of NF-κB-p65 and ERK1/2. This was supported by a similar reduction of osteoclastogenesis observed in CD74-/- cells. Furthermore, a MIF blockade reduced RANKL-induced osteoclastogenesis via deregulating RANKL-mediated NF-κB and NFATc1 transcription factor activation. These data indicate that MIF and CD74 facilitate RANKL-induced osteoclastogenesis, and suggest that MIF contributes directly to bone erosion, as well as inflammation, in RA. PMID:25647268

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

    PubMed

    Yamamoto, N

    1996-10-01

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

  11. Regulation of macrophage migration by a novel plasminogen receptor Plg-RKT

    PubMed Central

    Lighvani, Shahrzad; Baik, Nagyung; Diggs, Jenna E.; Khaldoyanidi, Sophia; Parmer, Robert J.

    2011-01-01

    Localization of plasmin on macrophages and activation of pro–MMP-9 play key roles in macrophage recruitment in the inflammatory response. These functions are promoted by plasminogen receptors exposing C-terminal basic residues on the macrophage surface. Recently, we identified a novel transmembrane plasminogen receptor, Plg-RKT, which exposes a C-terminal lysine on the cell surface. In the present study, we investigated the role of Plg-RKT in macrophage invasion, chemotactic migration, and recruitment. Plg-RKT was prominently expressed in membranes of human peripheral blood monocytes and monocytoid cells. Plasminogen activation by urokinase-type plasminogen activator (uPA) was markedly inhibited (by 39%) by treatment with anti–Plg-RKT mAb. Treatment of monocytes with anti–Plg-RKT mAb substantially inhibited invasion through the representative matrix, Matrigel, in response to MCP-1 (by 54% compared with isotype control). Furthermore, chemotactic migration was also inhibited by treatment with anti–Plg-RKT mAb (by 64%). In a mouse model of thioglycollate-induced peritonitis, anti–Plg-RKT mAb markedly inhibited macrophage recruitment (by 58%), concomitant with a reduction in pro–MMP-9 activation in the inflamed peritoneum. Treatment with anti–Plg-RKT mAb did not further reduce the low level of macrophage recruitment in plasminogen-null mice. We conclude that Plg-RKT plays a key role in the plasminogen-dependent regulation of macrophage invasion, chemotactic migration, and recruitment in the inflammatory response. PMID:21940822

  12. Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

    PubMed Central

    Vázquez, Alicia; de Dios Ruiz-Rosado, Juan; Terrazas, Luis I.; Juárez, Imelda; Gomez-Garcia, Lorena; Calleja, Elsa; Camacho, Griselda; Chávez, Ana; Romero, Miriam; Rodriguez, Tonathiu; Espinoza, Bertha; Rodriguez-Sosa, Miriam

    2014-01-01

    The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 104 T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection. PMID:25170304

  13. Endogenous macrophage migration inhibitory factor reduces the accumulation and toxicity of misfolded SOD1 in a mouse model of ALS.

    PubMed

    Leyton-Jaimes, Marcel F; Benaim, Clara; Abu-Hamad, Salah; Kahn, Joy; Guetta, Amos; Bucala, Richard; Israelson, Adrian

    2016-09-01

    Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons in the brain and spinal cord. It has been suggested that the toxicity of mutant SOD1 results from its misfolding and accumulation on the cytoplasmic faces of intracellular organelles, including the mitochondria and endoplasmic reticulum (ER) of ALS-affected tissues. Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit the accumulation of misfolded SOD1 and its binding to intracellular membranes, but the role of endogenous MIF in modulating SOD1 misfolding in vivo remains unknown. To elucidate this role, we bred MIF-deficient mice with SOD1(G85R) mice, which express a dismutase-inactive mutant of SOD1 and are considered a model of familial ALS. We found that the accumulation of misfolded SOD1, its association with mitochondrial and ER membranes, and the levels of sedimentable insoluble SOD1 aggregates were significantly higher in the spinal cords of SOD1(G85R)-MIF(-/-) mice than in their SOD1(G85R)-MIF(+/+) littermates. Moreover, increasing MIF expression in neuronal cultures inhibited the accumulation of misfolded SOD1 and rescued from mutant SOD1-induced cell death. In contrast, the complete elimination of endogenous MIF accelerated disease onset and late disease progression and shortened the lifespan of the SOD1(G85R) mutant mice. These findings indicate that MIF plays a significant role in the folding and misfolding of SOD1 in vivo, and they have implications for the potential therapeutic role of up-regulating MIF within the nervous system to modulate the selective accumulation of misfolded SOD1. PMID:27551074

  14. Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism.

    PubMed

    Zhou, Xiaoye; He, Wei; Huang, Zhiping; Gotto, Antonio M; Hajjar, David P; Han, Jihong

    2008-01-25

    Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions

  15. Treatment Methods for Kidney Failure: Peritoneal Dialysis

    MedlinePlus

    ... 3.70 MB) MedlinePlus Alternate Language URL Peritoneal Dialysis Page Content On this page: What is peritoneal ... Points to Remember Clinical Trials What is peritoneal dialysis and how does it work? Peritoneal dialysis is ...

  16. Isoliquiritigenin, a flavonoid from licorice, blocks M2 macrophage polarization in colitis-associated tumorigenesis through downregulating PGE{sub 2} and IL-6

    SciTech Connect

    Zhao, Haixia; Zhang, Xinhua; Chen, Xuewei; Li, Ying; Ke, Zunqiong; Tang, Tian; Chai, Hongyan; Guo, Austin M.; Chen, Honglei; Yang, Jing

    2014-09-15

    M2 macrophage polarization is implicated in colorectal cancer development. Isoliquiritigenin (ISL), a flavonoid from licorice, has been reported to prevent azoxymethane (AOM) induced colon carcinogenesis in animal models. Here, in a mouse model of colitis-associated tumorigenesis induced by AOM/dextran sodium sulfate (DSS), we investigated the chemopreventive effects of ISL and its mechanisms of action. Mice were treated with AOM/DSS and randomized to receive either vehicle or ISL (3, 15 and 75 mg/kg). Tumor load, histology, immunohistochemistry, and gene and protein expressions were determined. Intragastric administration of ISL for 12 weeks significantly decreased colon cancer incidence, multiplicity and tumor size by 60%, 55.4% and 42.6%, respectively. Moreover, ISL inhibited M2 macrophage polarization. Such changes were accompanied by downregulation of PGE{sub 2} and IL-6 signaling. Importantly, depletion of macrophages by clodronate (Clod) or zoledronic acid (ZA) reversed the effects of ISL. In parallel, in vitro studies also demonstrated that ISL limited the M2 polarization of RAW264.7 cells and mouse peritoneal macrophages with concomitant inactivation of PGE{sub 2}/PPARδ and IL-6/STAT3 signaling. Conversely, exogenous addition of PGE{sub 2} or IL-6, or overexpression of constitutively active STAT3 reversed ISL-mediated inhibition of M2 macrophage polarization. In summary, dietary flavonoid ISL effectively inhibits colitis-associated tumorigenesis through hampering M2 macrophage polarization mediated by the interplay between PGE{sub 2} and IL-6. Thus, inhibition of M2 macrophage polarization is likely to represent a promising strategy for chemoprevention of colorectal cancer. - Highlights: • Isoliquiritigenin (ISL) prevents colitis-associated tumorigenesis. • ISL inhibits M2 macrophage polarization in vivo and in vitro. • ISL inhibits PGE{sub 2} and IL-6 signaling in colitis-associated tumorigenesis. • ISL may be an attractive candidate agent for

  17. Macrophages activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression

    PubMed Central

    Blich, Miry; Golan, Amnon; Arvatz, Gil; Sebbag, Anat; Shafat, Itay; Sabo, Edmond; Cohen-Kaplan, Victoria; Petcherski, Sirouch; Avniel-Polak, Shani; Eitan, Amnon; Hammerman, Haim; Aronson, Doron; Axelman, Elena; Ilan, Neta; Nussbaum, Gabriel; Vlodavsky, Israel

    2012-01-01

    Objective Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Results/Methods Highly purified heparanase was added to mouse peritoneal macrophages (MPM) and macrophage-like J774 cells and the levels of TNFα, MMP-9, IL-1, and MCP-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll like receptor (TLR)-2 and -4 knockout mice (KO) were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction (MI), stable angina (SA), and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with SA or acute MI. Addition or over expression of heparanase variants resulted in marked increase in TNFα, MMP-9, IL-1 and MCP-1 levels. MPM harvested from TLR-2 or TLR-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute MI, compared to patients with SA and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared to specimens of stable plaque and controls. Conclusion Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression towards vulnerability. PMID:23162016

  18. Modulation of macrophage functionality induced in vitro by chlorpyrifos and carbendazim pesticides.

    PubMed

    Helali, Imen; Ferchichi, Saiida; Maaouia, Amal; Aouni, Mahjoub; Harizi, Hedi

    2016-09-01

    The immune response is the first defense against pathogens; however, it is very sensitive and can be impacted on by agrochemicals such as carbamate and organophosphate pesticides widely present in the environment. To understand how pesticides can affect immune cell function in vitro, this study investigated the effects of chlorpyrifos (CPF) and carbendazim (CBZ), the most commonly used pesticides worldwide, on murine immune cell (i.e. macrophage) functions, including lysosomal enzyme activity and pro-inflammatory cytokines (IL-1β and TNFα) and nitric oxide (NO) production by isolated mouse peritoneal macrophages. This study showed for the first time that CPF and CBZ dose-relatedly reduced macrophage lysosomal enzyme activity and LPS-induced production of IL-1β, TNFα and NO. In general, the effects caused by CPF appeared more pronounced than those by CBZ. Collectively, these results demonstrated that CPF and CBZ exhibited marked immunomodulatory effects and could act as potent immunosuppressive factors in vitro. This inhibition of macrophage pro-inflammatory function may be an integral part of the underlying mode of action related to pesticide-induced immunosuppression. PMID:27429139

  19. Novel technique for generating macrophage foam cells for in vitro reverse cholesterol transport studies.

    PubMed

    Sengupta, Bhaswati; Narasimhulu, Chandrakala Aluganti; Parthasarathy, Sampath

    2013-12-01

    Generation of foam cells, an essential step for reverse cholesterol transport studies, uses the technique of receptor-dependent macrophage loading with radiolabeled acetylated LDL. In this study, we used the ability of a biologically relevant detergent molecule, lysophosphatidylcholine (lyso-PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabeled cholesterol/lyso-PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month of storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled, or radiolabeled)/lyso-PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by TLC. Such foam cells unloaded cholesterol when incubated with HDL but not with oxidized HDL. We propose that stable cholesterol or CE/lyso-PtdCho micelles would offer advantages over existing methods. PMID:24115226

  20. Novel technique for generating macrophage foam cells for in vitro reverse cholesterol transport studies[S

    PubMed Central

    Sengupta, Bhaswati; Narasimhulu, Chandrakala Aluganti; Parthasarathy, Sampath

    2013-01-01

    Generation of foam cells, an essential step for reverse cholesterol transport studies, uses the technique of receptor-dependent macrophage loading with radiolabeled acetylated LDL. In this study, we used the ability of a biologically relevant detergent molecule, lysophosphatidylcholine (lyso-PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabeled cholesterol/lyso-PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month of storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled, or radiolabeled)/lyso-PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by TLC. Such foam cells unloaded cholesterol when incubated with HDL but not with oxidized HDL. We propose that stable cholesterol or CE/lyso-PtdCho micelles would offer advantages over existing methods. PMID:24115226

  1. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    SciTech Connect

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-04-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approx. = 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4/sup 0/C or in medium containing 5 ..mu..M colchicine or nocodazole at 37/sup 0/C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37/sup 0/C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.

  2. Hamster bite peritonitis: Pasteurella pneumotropica peritonitis in a dialysis patient.

    PubMed

    Campos, A; Taylor, J H; Campbell, M

    2000-11-01

    We report the first case of Pasteurella pneumotropica peritonitis in a peritoneal dialysis patient. This rare infection was the result of contamination of the dialysis tubing by a pet hamster. We stress the importance of household pets as a source of infection in the peritoneal dialysis population. PMID:11095007

  3. Killing of Staphylococcus aureus in murine macrophages by chloroquine used alone and in combination with ciprofloxacin or azithromycin

    PubMed Central

    Dey, Somrita; Bishayi, Biswadev

    2015-01-01

    This study aimed to determine any alteration in the killing of Staphylococcus aureus in murine peritoneal macrophages when chloroquine (CQ) is used alone compared with when it is used in combination with ciprofloxacin (CIP) or azithromycin (AZM). The study also aimed to find out the implication of reactive oxygen species (ROS) production and cytokine release in the intracellular killing of S. aureus in macrophages. We present here data obtained with a model of S. aureus-infected mouse peritoneal macrophages in which the intracellular growth of the bacteria and the influence of antibiotics was monitored for 30, 60, and 90 minutes in the presence or absence of CQ along with the production of ROS and alteration in levels of antioxidant enzymes and cytokines. It was observed that S. aureus-triggered cytokine response was regulated when macrophages were co-cultured with CQ and AZM as compared with CQ stimulation only. It can be suggested that action of AZM in mediating bacterial killing is enhanced by the presence of CQ, indicating enhanced uptake of AZM during early infection that may be essential for bacteria killing by AZM. Reduction of oxidative stress burden on the S. aureus-infected macrophages may pave the way for better killing of internalized S. aureus by CQ plus ciprofloxacin (CIP) or CQ plus AZM. Based on these observations, one may speculate that in an inflammatory milieu, CQ loaded with AZM elicits a stronger proinflammatory response by increasing the intracellular uptake of AZM or CIP, thus enabling the immune system to mount a more robust and prolonged response against intracellular pathogens. PMID:25653549

  4. Peritoneal manifestations of parasitic infection.

    PubMed

    Kim, So Yeon; Ha, Hyun Kwon

    2008-01-01

    The purpose of this study was to describe of peritoneal manifestations of parasitic infection at CT. A broad spectrum of CT findings can be seen in the peritoneal cavity, including a varying degree of omental or mesenteric infiltration, single or multiple peritoneal mass or nodule, and peritoneal thickening or stranding. Recognition of these findings are crucial for establish an early diagnosis and helps avoid unnecessary surgery. PMID:17924162

  5. Interactions between Streptomyces californicus and Stachybotrys chartarum can induce apoptosis and cell cycle arrest in mouse RAW264.7 macrophages

    SciTech Connect

    Penttinen, Piia . E-mail: Piia.Penttinen@ktl.fi; Pelkonen, Jukka; Huttunen, Kati; Toivola, Mika; Hirvonen, Maija-Riitta

    2005-02-01

    Exposure to complex mixtures of bacteria and fungi in moisture-damaged buildings is a potential cause of inflammatory related symptoms among occupants. The present study assessed interactions between two characteristic moldy house microbes Streptomyces californicus and Stachybotrys chartarum. Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone. The RAW264.7 cells were exposed to six doses (1 x 10{sup 4} to 3 x 10{sup 6} spores/ml) for 24 h, and the time course of the induced responses was evaluated after 4, 8, 16, and 24 h of exposure (1 x 10{sup 6} spores/ml). The cytotoxic potential of the spores was characterized by the MTT test, DNA content analysis, and enzyme assay for caspase-3 activity. The production of cytokines (IL-1{beta}, IL-6, IL-10, TNF{alpha}, and MIP2) was measured immunochemically and nitric oxide by the Griess method. Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G{sub 2}/M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells. In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells.

  6. Anthocyanin-rich fractions from red raspberries attenuate inflammation in both RAW264.7 macrophages and a mouse model of colitis

    PubMed Central

    Li, Li; Wu, Zhiqin; Yao, Lijun; Wu, Yonghou; Huang, Lian; Liu, Kan; Zhou, Xiang; Gou, Deming

    2014-01-01

    Edible berries have a broad spectrum of biomedical functions, including improving immune responses and reducing risk for chronic diseases. In this study, the anti-inflammatory activities of crude extracts (CEs), anthocyanin-rich fractions (ARFs), and des-anthocyanin fractions (DAFs) from seven berries were evaluated based on their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)/IFN-γ-activated RAW264.7 macrophages. ARFs from red raspberries (RR-ARFs) exhibited the highest efficiency in suppressing NO synthesis. The anti-inflammatory properties were also demonstrated by reducing the expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β) and IL-6 in RAW264.7 cells. The luciferase reporter assay demonstrated that the activities of NF-κB and AP-1 signaling pathways were significantly suppressed by RR-ARFs. Further studies showed that RR-ARFs decreased the phosphorylation of IKK, IκBα, p65 and JNK and the nuclear translocation of p65 in LPS/IFN-γ-stimulated RAW264.7 cells. In a mouse colitis model, dextran sulfate sodium (DSS)-induced weight loss and histological damage were significantly ameliorated by RR-ARFs treatment. Taken together, our results indicate that RR-ARFs attenuate inflammation both in vitro and in vivo primarily by inhibiting the activation of NF-κB and MAPKs. The anti-inflammatory of RR-ARFs could be harnessed and applied in animal agriculture, drug and food industries. PMID:25167935

  7. Ghrelin inhibits oxLDL-induced inflammation in RAW264.7 mouse macrophages through down-regulation of LOX-1 expression via NF-κB signaling pathway.

    PubMed

    Sun, N; Wang, H; Wang, L

    2016-01-01

    Oxidized low-density lipoprotein (oxLDL) is one of the many causes of the initiation and progression of atherosclerosis, which can subsequently promote the uptake of oxLDL by macrophages and lead to inflammation in the blood vessels. In the present study, we evaluated the protective effects of ghrelin on oxLDL-induced RAW264.7 mouse macrophages. Ghrelin was able to inhibit the release of several pro-inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, ghrelin also inhibited the expression of Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in oxLDL treated macrophages. Furthermore, we demonstrated that ghrelin could inhibit the expression of p-IκBα, and the inhibitory effects could be blocked by BAY 117082. Taken together, ghrelin possesses anti-inflammatory effects on oxLDL-induced inflammation in macrophages, suggesting that it can prevent or treat atherosclerosis, and deserves to be further studied and developed to be potent drug for treating atherosclerosis. PMID:26950452

  8. Functionalized synchrotron in-line phase-contrast computed tomography: a novel approach for simultaneous quantification of structural alterations and localization of barium-labelled alveolar macrophages within mouse lung samples.

    PubMed

    Dullin, Christian; dal Monego, Simeone; Larsson, Emanuel; Mohammadi, Sara; Krenkel, Martin; Garrovo, Chiara; Biffi, Stefania; Lorenzon, Andrea; Markus, Andrea; Napp, Joanna; Salditt, Tim; Accardo, Agostino; Alves, Frauke; Tromba, Giuliana

    2015-01-01

    Functionalized computed tomography (CT) in combination with labelled cells is virtually non-existent due to the limited sensitivity of X-ray-absorption-based imaging, but would be highly desirable to realise cell tracking studies in entire organisms. In this study we applied in-line free propagation X-ray phase-contrast CT (XPCT) in an allergic asthma mouse model to assess structural changes as well as the biodistribution of barium-labelled macrophages in lung tissue. Alveolar macrophages that were barium-sulfate-loaded and fluorescent-labelled were instilled intratracheally into asthmatic and control mice. Mice were sacrificed after 24 h, lungs were kept in situ, inflated with air and scanned utilizing XPCT at the SYRMEP beamline (Elettra Synchrotron Light Source, Italy). Single-distance phase retrieval was used to generate data sets with ten times greater contrast-to-noise ratio than absorption-based CT (in our setup), thus allowing to depict and quantify structural hallmarks of asthmatic lungs such as reduced air volume, obstruction of airways and increased soft-tissue content. Furthermore, we found a higher concentration as well as a specific accumulation of the barium-labelled macrophages in asthmatic lung tissue. It is believe that XPCT will be beneficial in preclinical asthma research for both the assessment of therapeutic response as well as the analysis of the role of the recruitment of macrophages to inflammatory sites. PMID:25537601

  9. Developmental derivation of embryonic and adult macrophages.

    PubMed

    Sh