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Sample records for co-cultured macrophages activated

  1. Macrophage-mediated osteogenesis activation in co-culture with osteoblast on calcium silicate cement.

    PubMed

    Tu, Ming-Gene; Chen, Yi-Wen; Shie, Ming-You

    2015-12-01

    The use of calcium silicate (CS) cement holds great promise for bone substitute biomaterials. However, the effects of CS on osteoblast and macrophage cells are not fully understood. This study examines cell proliferation and differentiation of mono- or co-cultured MC3T3-E1 and Raw 264.7 cells on CS cement. Very few studies to date have looked at the effects of osteoblast and macrophages on biomaterial-regulated osteogenesis. In this study the proliferation and differentiation of MC3T3-E1, Raw 264.7 and co-cultured MC3T3-E1/Raw 264.7 on CS cements have been analyzed using a PrestoBlue kit and ELISA. In addition, the effect of macrophages on CS-coordinated osteogenesis of MC3T3-E1 has been investigated. Results show that MC3T3-E1, Raw 264.7 and co-cultured MC3T3-E1/Raw 264.7 adhere to and proliferate well on the CS cement. In a co-culture, the CS cements inhibit receptor activator of nuclear factor kappa B ligand expression of both genes and proteins in Raw 264.7 cells when compared to those grown in mono-cultured system. Ca deposition of MC3T3-E1 in the co-culture is higher than that of cells in a mono-culture. Bone morphogenetic protein 2 (BMP2) is also significantly up-regulated by the CS cement stimulation, indicating that macrophages may participate in the CS stimulated osteogenesis. Interestingly, when macrophage are cultured with BMP2 receptor-blocking MC3T3-E1 on the CS cements, the osteogenesis differentiation of the cells is significantly inhibited, indicating the important role of macrophages in biomaterial-induced osteogenesis via BMP2 receptors. It is assumed that it is an increase in the secretion of the BMP2 from the Raw 264.7 cell that is primarily involved in the promotion of the osteogenesis of the MC3T3-E1. These results provide valuable insights into both the mechanism of CS-stimulated osteogenesis, and strategies to optimize the evaluation system for the in vitro osteogenesis capacity of bone substitute biomaterials.

  2. Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication

    PubMed Central

    2009-01-01

    Background Melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM), used as an immune modulator, has been recommended for patients with depressed immune systems. Previous studies in mice have demonstrated that the CHM activates macrophages, induces an increase in the number of leukocytes and improves the murine response against Sarcoma-180. Methods Here we studied the interaction of mouse lymph node lymphocytes, co-cultured in vitro with macrophages in the presence or absence of the CHM, with B16F10 melanoma cells. Results Lymphocytes co-cultured with macrophages in the presence of the CHM had greater anti-melanoma activity, reducing melanoma cell density and increasing the number of lysed tumor cells. There was also a higher proportion of activated (CD25+) lymphocytes with increased viability. Overall, lymphocytes activated by treatment destroyed growing cancer cells more effectively than control lymphocytes. Conclusion Co-culture of macrophages with lymphocytes in the presence of the CHM enhanced the anti-cancer performance of lymphocytes against a very aggressive lineage of melanoma cells. These results suggest that non-toxic therapies using CHMs are a promising alternative approach to the treatment of melanomas. In addition, they are attractive combination-therapy candidates, which may enhance the efficacy of conventional medicines by improving the immune response against tumor cells. PMID:19698142

  3. The effect of activated alveolar macrophages on experimental lung emphysema development. II. The study of fibroblast and alveolar macrophage co-culture.

    PubMed

    Sulkowska, M; Wołczyński, S; Sulkowski, S; Sobaniec-Lotowska, M; Chyczewski, L; Sulik, M; Kulikowski, M; Dziecioł, J; Berger, W

    1995-01-01

    The cell-cell interaction between fibroblasts and alveolar macrophages was examined using a co-culture system. Alveolar macrophages (AM) were harvested from the bronchoalveolar lavages (BAL) of rats with papain induced lung emphysema. The BCG-vaccine was applied as a macrophage mobilizing and activating agent. The morphological examinations carried out in scanning electron microscope (SEM) as well as the evaluation of the uptake of 3H-thymidine did not show any significant differences between respective co-cultures of fibroblasts and AM isolated both from the lungs of control and experimental animals (treated with BCG or papain, and BCG+papain). However, significant growth were noted in 3H-thymidine uptake between fibroblast cultures done with or without cells isolated from the lungs. The results obtained suggest that AM can promote fibroblast proliferation during the progression of experimental lung emphysema.

  4. Tumor-Cell Co-Culture Induced Alternative Activation of Macrophages Is Modulated by Interferons In Vitro

    PubMed Central

    Müller-Quernheim, Ulrike Carolin; Potthast, Lars; Müller-Quernheim, Joachim

    2012-01-01

    Tumor-associated macrophages infiltrate tumors and facilitate tumor growth. Here, we analyzed M1 and M2 marker expression in the course of co-culture-driven macrophage differentiation and investigated the influence of interferons (IFNs) on this differentiation. To generate monocyte-derived macrophages (MDMs) 1×106 monocytes of healthy volunteers were cultivated either with 25×103 adherent A549/mL or in medium containing 50% A549 conditioned medium (CM) for 72 h in the presence or absence of IFN-α, β or γ, respectively. Supernatants were tested for CCL18 (M2 marker) and CXCL10 (M1 marker) by enzyme-linked immunosorbent assay. CCL18 and CXCL10 release by MDM is increased by the presence of A549 cells, but also when cultured in A549 CM. On stimulation with IFN-γ, we observe an increased release of the M1 marker CXCL10 and a decreased release of CCL18. Type I IFNs also increases CXCL10 release. Thus, A549 releases a soluble factor which enhances CCL18 production and M2 polarization, indicating that a localized specific cytokine milieu, as found in the environment of a tumor or in fibrotic lung tissue, favors alternative activation of macrophages. In the presence of IFN-γ, M2 differentiation is attenuated as shown by the decrease of the M2 chemokine CCL18 and by the increase of the M1 chemokine CXCL10. However, CXCL10 levels were also increased by the co-culture, which indicates a simultaneous classical activation (M1) or the formation of a M1/M2 hybrid. PMID:22280057

  5. The effect of squalane-dissolved fullerene-C60 on adipogenesis-accompanied oxidative stress and macrophage activation in a preadipocyte-monocyte co-culture system.

    PubMed

    Xiao, Li; Aoshima, Hisae; Saitoh, Yasukazu; Miwa, Nobuhiko

    2010-08-01

    Effects of squalane-dissolved fullerene-C60 (Sql-fullerene) on macrophage activation and adipose conversion with oxidative stress were studied using an inflammatory adipose-tissue equivalent (ATE) and OP9 mouse stromal preadipocyte-U937 lymphoma cell co-culture systems. Differentiation of OP9 cells was initiated by insulin-rich serum replacement (SR) as an adipogenic stimulant, and then followed by accumulation of intracellular lipid droplets and reactive oxygen species (ROS), both of which were significantly inhibited by Sql-fullerene. In the OP9-U937 cell co-culture system, U937 cells rapidly differentiated to macrophage-like cells during SR-induced adipogenesis in OP9 cells. The ROS accumulation was in the co-culture more marked than in OP9 cells alone, suggesting that the interaction between adipocytes and monocytes/macrophages promotes inflammatory responses. Sql-fullerene significantly inhibited macrophage activation and low-grade adipogenesis in the OP9-U937 co-culture system. We developed a three-dimensional inflammatory adipose-tissue model "ATE" consisting of, characteristically, U937 cells in the culture-wells, and, in addition, mounted a culture insert containing OP9 cells-populated collagen gel. ATE is enabled with suitable stimulation to represent the pathology of inflammatory disorders, such as macrophage infiltration in adipose tissue. Five-day culturing of ATE in SR medium occurred U937 macrophage migration and intracellular oil-droplet accumulation that were significantly inhibited by Sql-fullerene. Our results suggest that Sql-fullerene might be explored as a potential medicine for the treatment of metabolic syndrome or other obesity-related disorders. Copyright 2010 Elsevier Ltd. All rights reserved.

  6. Riboflavin Reduces Pro-Inflammatory Activation of Adipocyte-Macrophage Co-culture. Potential Application of Vitamin B2 Enrichment for Attenuation of Insulin Resistance and Metabolic Syndrome Development.

    PubMed

    Mazur-Bialy, Agnieszka Irena; Pocheć, Ewa

    2016-12-15

    Due to the progressive increase in the incidence of obese and overweight individuals, cardiometabolic syndrome has become a worldwide pandemic in recent years. Given the immunomodulatory properties of riboflavin, the current study was performed to investigate the potency of riboflavin in reducing obesity-related inflammation, which is the main cause of insulin resistance, diabetes mellitus 2 or arteriosclerosis. We determined whether pretreatment with a low dose of riboflavin (10.4-1000 nM) affected the pro-inflammatory activity of adipocyte-macrophage co-culture (3T3 L1-RAW 264.7) following lipopolysaccharide stimulation (LPS; 100 ng/mL) which mimics obesity-related inflammation. The apoptosis of adipocytes and macrophages as well as tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), interleukin 1beta (IL-1β), monocyte chemotactic protein 1 (MCP-1), high-mobility group box 1 (HMGB1), transforming growth factor-beta 1 (TGFβ), interleukin 10 (IL-10), inducible nitric oxide synthase (iNOS), nitric oxide (NO), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1) expression and release, macrophage migration and adipokines (adiponectin and leptin) were determined. Our results indicated an efficient reduction in pro-inflammatory factors (TNFα, IL-6, MCP-1, HMGB1) upon culture with riboflavin supplementation (500-1000 nM), accompanied by elevation in anti-inflammatory adiponectin and IL-10. Moreover, macrophage migration was reduced by the attenuation of chemotactic MCP-1 release and degradation of the extracellular matrix by MMP-9. In conclusion, riboflavin effectively inhibits the pro-inflammatory activity of adipocyte and macrophage co-cultures, and therefore we can assume that its supplementation may reduce the likelihood of conditions associated with the mild inflammation linked to obesity.

  7. Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro

    PubMed Central

    2012-01-01

    Background Solid tumours comprise various cells, including cancer cells, resident stromal cells, migratory haemopoietic cells and other. These cells regulate tumour growth and metastasis. Macrophages constitute probably the most important element of all interactions within the tumour microenvironment. However, the molecular mechanism, that guides tumour environment, still remains unknown. Exploring the underlying molecular mechanisms that orchestrate these phenomena has been the aim of our study. A co-culture of canine mammary cancer cells and macrophages was established and maintained for 72 hrs. Having sorted the cells, gene expression in cancer cells and macrophages, using DNA microarrays, was examined. The results were confirmed using real-time qPCR and confocal microscopy. Moreover, their ability for migration and invasion has been assessed. Results Microarray analysis showed that the up-regulated genes in the cancer cell lines are involved in 15 highly over-manifested pathways. The pathways that drew our diligent attention included: the inflammation pathway mediated by chemokine and cytokine, the Toll receptor signalling pathway and the B cell activation. The up-regulated genes in the macrophages were involved in only 18 significantly over-manifested pathways: the angiogenesis, the p53 pathway feedback loops2 and the Wnt signalling pathway. The microarray analysis revealed that co-culturing of cancer cells with macrophages initiated the myeloid-specific antigen expression in cancer cells, as well as cytokine/chemokine genes expression. This finding was confirmed at mRNA and protein level. Moreover, we showed that macrophages increase cancer migration and invasion. Conclusions The presence of macrophages in the cancer environment induces acquisition of the macrophage phenotype (specific antigens and chemokines/cytokines expression) in cancer cells. We presumed that cancer cells also acquire other myeloid features, such as: capabilities of cell rolling

  8. Design and utilization of macrophage and vascular smooth muscle cell co-culture systems in atherosclerotic cardiovascular disease investigation.

    PubMed

    Zuniga, Mary C; White, Sharla L Powell; Zhou, Wei

    2014-10-01

    Atherosclerotic cardiovascular disease has been acknowledged as a chronic inflammatory condition. Monocytes and macrophages lead the inflammatory pathology of atherosclerosis whereas changes in atheromatous plaque thickness and matrix composition are attributed to vascular smooth muscle cells. Because these cell types are key players in atherosclerosis progression, it is crucial to utilize a reliable system to investigate their interaction. In vitro co-culture systems are useful platforms to study specific molecular mechanisms between cells. This review aims to summarize the various co-culture models that have been developed to investigate vascular smooth muscle cell and monocyte/macrophage interactions, focusing on the monocyte/macrophage effects on vascular smooth muscle cell function.

  9. Sialylation of neurites inhibits complement-mediated macrophage removal in a human macrophage-neuron Co-Culture System.

    PubMed

    Linnartz-Gerlach, Bettina; Schuy, Christine; Shahraz, Anahita; Tenner, Andrea J; Neumann, Harald

    2016-01-01

    The complement system has been implicated in the removal of dysfunctional synapses and neurites during development and in disease processes in the mouse, but it is unclear how far the mouse data can be transferred to humans. Here, we co-cultured macrophages derived from human THP1 monocytes and neurons derived from human induced pluripotent stem cells, to study the role of the complement system in a human model. Components of the complement system were expressed by the human macrophages and human neuronal culture, while receptors of the complement cascade were expressed by human macrophages as shown via gene transcript analysis and flow cytometry. We mimicked pathological conditions leading to an altered glycocalyx by treatment of human neurons with sialidases. Desialylated human neurites were opsonized by the complement component C1q. Furthermore, human neurites with an intact sialic acid cap remained untouched, while desialylated human neurites were removed and ingested by human macrophages. While blockage of the complement receptor 1 (CD35) had no effect, blockage of CD11b as part of the complement receptor 3 (CR3) reversed the effect on macrophage phagocytosis of desialylated human neurites. Data demonstrate that in the human system sialylation of the neuronal glycocalyx serves as an inhibitory flag for complement binding and CR3-mediated phagocytosis by macrophages.

  10. The Dietary Isoflavone Daidzein Reduces Expression of Pro-Inflammatory Genes through PPARα/γ and JNK Pathways in Adipocyte and Macrophage Co-Cultures

    PubMed Central

    Sakamoto, Yuri; Kanatsu, Junko; Toh, Mariko; Naka, Ayano; Kondo, Kazuo; Iida, Kaoruko

    2016-01-01

    Obesity-induced inflammation caused by adipocyte-macrophage interactions plays a critical role in developing insulin resistance, and peroxisome proliferator-activated receptors (PPARs) regulate inflammatory gene expression in these cells. Recently, the soy isoflavone daidzein was reported to act as a PPAR activator. We examined whether daidzein affected adipocyte-macrophage crosstalk via the regulation of PPARs. Co-cultures of 3T3-L1 adipocytes and RAW264 macrophages, or palmitate-stimulated RAW264 macrophages were treated with daidzein in the presence or absence of specific inhibitors for PPARs: GW6471 (a PPARα antagonist), and GW9662 (a PPARγ antagonist). Inflammatory gene expression was then determined. Daidzein significantly decreased chemokine (C-C motif) ligand 2 (Ccl2, known in humans as monocyte chemo-attractant protein 1 (MCP1)) and interleukin 6 (Il6) mRNA levels induced by co-culture. In 3T3-L1 adipocytes, daidzein inversed the attenuation of adiponectin gene expression by co-culture, and these effects were inhibited by the PPAR-γ specific inhibitor. Daidzein also decreased Ccl2 and Il6 mRNA levels in RAW264 macrophages stimulated with palmitate or conditioned medium (CM) from hypertrophied 3T3-L1 adipocytes. This inhibitory effect on Il6 expression was abrogated by a PPAR-α inhibitor. Additionally, we examined the activation of nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways and found that daidzein significantly inhibited palmitate-induced phosphorylation of JNK. Our data suggest that daidzein regulates pro-inflammatory gene expression by activating PPAR-α and -γ and inhibiting the JNK pathway in adipocyte and macrophage co-cultures. These effects might be favorable in improving adipose inflammation, thus, treatment of daidzein may be a therapeutic strategy for chronic inflammation in obese adipose tissue. PMID:26901838

  11. A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions

    PubMed Central

    Noel, Gaelle; Baetz, Nicholas W.; Staab, Janet F.; Donowitz, Mark; Kovbasnjuk, Olga; Pasetti, Marcela F.; Zachos, Nicholas C.

    2017-01-01

    Integration of the intestinal epithelium and the mucosal immune system is critical for gut homeostasis. The intestinal epithelium is a functional barrier that secludes luminal content, senses changes in the gut microenvironment, and releases immune regulators that signal underlying immune cells. However, interactions between epithelial and innate immune cells to maintain barrier integrity and prevent infection are complex and poorly understood. We developed and characterized a primary human macrophage-enteroid co-culture model for in-depth studies of epithelial and macrophage interactions. Human intestinal stem cell-derived enteroid monolayers co-cultured with human monocyte-derived macrophages were used to evaluate barrier function, cytokine secretion, and protein expression under basal conditions and following bacterial infection. Macrophages enhanced barrier function and maturity of enteroid monolayers as indicated by increased transepithelial electrical resistance and cell height. Communication between the epithelium and macrophages was demonstrated through morphological changes and cytokine production. Intraepithelial macrophage projections, efficient phagocytosis, and stabilized enteroid barrier function revealed a coordinated response to enterotoxigenic and enteropathogenic E. coli infections. In summary, we have established the first primary human macrophage-enteroid co-culture system, defined conditions that allow for a practical and reproducible culture model, and demonstrated its suitability to study gut physiology and host responses to enteric pathogens. PMID:28345602

  12. Pancreatic cancer cell/fibroblast co-culture induces M2 like macrophages that influence therapeutic response in a 3D model.

    PubMed

    Kuen, Janina; Darowski, Diana; Kluge, Tobias; Majety, Meher

    2017-01-01

    Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs and monocytes. Using this model, we analyzed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analyzing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated. 3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activated spheroid polarized MDMs or block immune suppressive factors such as Arginase-I.

  13. Suppression of inflammation-associated factors by indole-3-carbinol in mice fed high-fat diets and in isolated, co-cultured macrophages and adipocytes

    PubMed Central

    Chang, H-P; Wang, M-L; Hsu, C-Y; Liu, M-E; Chan, M-H; Chen, Y-H

    2011-01-01

    Aims: This study investigated the effects of indole-3-carbinol (I3C), a compound from cruciferous vegetables, on various parameters related to obesity, in particular, the parameters of infiltration by macrophages and of inflammatory cytokines expressed during the co-culture of adipocytes and macrophages. Methods: Male C57BL/6 mice were fed with a control diet (C group), high-fat diet (HF group) and HF+5 mg kg−1 I3C (HFI group). The I3C was intraperitoneally injected (HFI group) for 12 weeks. Epididymal adipose tissue (AT) was collected and stained for F4/80, a marker of macrophages. Results: The immunohistochemical staining for F4/80 indicated a greater presence of macrophages in the HF group than in AT from the control and HFI groups. Furthermore, I3C treatment, in an in vitro cell culture system, decreased expression of inducible nitric oxide synthase (iNOS), decreased nitrite content and enhanced expression of peroxisome proliferator-activated receptor (PPAR-γ). Moreover, in vitro cell culture studies revealed that I3C inhibited intracellular lipid accumulation in hypertrophied adipocytes. In macrophage and primary adipocyte co-culture, I3C inhibited expression of interleukin-6 (IL-6). Conclusions: In vivo treatment with I3C reduced the infiltration of macrophages in AT, and in vitro addition of I3C to co-cultured macrophages and adipocytes reduced nitrite production and IL-6 expression. With cultures of adipocytes only, I3C inhibited accumulation of intracellular lipid, either by disrupting differentiation, or by directly inhibiting triglyceride synthesis. PMID:21343904

  14. Inhibition of human arterial smooth muscle cell growth by human monocyte/macrophages: a co-culture study.

    PubMed

    Proudfoot, D; Fitzsimmons, C; Torzewski, J; Bowyer, D E

    1999-07-01

    Monocyte/macrophages produce a variety of substances which may influence the function of smooth muscle cells (SMC). During atherogenesis, macrophages are thought to modulate SMC migration, proliferation and synthesis of extracellular matrix. Such modulation is the balance between stimulatory and inhibitory influences. Thus, for example, our earlier studies have shown that macrophages not only secrete mitogens, but also produce small molecular weight inhibitors of SMC proliferation. In the present study, we have used a co-culture system in which human monocyte/macrophages were separated from human arterial SMC (hSMC) by a filter with the optional addition of a 12 kDa cut-off dialysis membrane, in order to assess their effect on hSMC growth. We have found that human peripheral blood-derived monocytes produced a substance of < 12 kDa that inhibited hSMC growth in the co-culture system. The monocyte-derived factor causing this effect was completely blocked by indomethacin, indicating that growth-inhibitory factors produced by the monocytes were cyclooxygenase products. We have shown that PGE1 and PGE2 inhibit hSMC growth, making them likely candidates for the effector molecules released from monocytes in our co-culture system.

  15. The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture.

    PubMed

    Chen, Gui; Shen, Yuexin; Li, Xiyue; Jiang, Qin; Cheng, Shanshan; Gu, Yuxiu; Liu, Liangliang; Cao, Yi

    2017-03-01

    It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures.

  16. Regulation of matrix metalloproteinase secretion by green tea catechins in a three-dimensional co-culture model of macrophages and gingival fibroblasts.

    PubMed

    Morin, Marie-Pierre; Grenier, Daniel

    2017-03-01

    Elevated levels of matrix metalloproteinases (MMPs) have been associated with the active phases of tissue and bone destruction in periodontitis, an inflammatory disease characterized by a significant breakdown of tooth support. In the present study, we used a three-dimensional (3D) co-culture model of macrophages and gingival fibroblasts to investigate the ability of a green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) to regulate the secretion of MMP-3, -8, and -9. The 3D co-culture model was composed of gingival fibroblasts embedded in a type I collagen matrix overlaid with macrophages. Two arbitrary ratios were tested. The ratio composed of 1 macrophage to 10 fibroblasts was used to mimic a slightly inflamed periodontal site while the ratio composed of 10 macrophages to 1 fibroblast was used to mimic a severely inflamed periodontal site. The 3D co-culture model was pre-treated for 2h with either the green tea extract or EGCG. It was then stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). The model was also first stimulated with LPS for 2h and then incubated with the green tea extract or EGCG. The concentrations of secreted MMP-3, -8, and -9 were quantified by enzyme-linked immunoassays. When the 3D co-culture model was stimulated with A. actinomycetemcomitans LPS, the 10:1 ratio of macrophages to gingival fibroblasts was associated with a highest secretion of MMP-3 and -9 and, to a lesser extent, MMP-8, than the 1:10 ratio. Non-cytotoxic concentrations of the green tea extract or EGCG reduced the basal secretion levels of all three MMPs. A 2-h treatment with the green tea extract or EGCG prior to the stimulation with LPS resulted in a dose-dependent decrease in MMP secretion, with MMP-9 showing the most significant decrease. A decrease in MMP secretion was also observed when the green tea extract or EGCG was added following a 2-h stimulation with LPS. Our results suggested that green tea catechins, and more

  17. Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.

    PubMed

    Bodet, Charles; Chandad, Fatiha; Grenier, Daniel

    2006-01-01

    Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.

  18. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    NASA Technical Reports Server (NTRS)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  19. Co-culture of bone marrow stem cells and macrophages indicates intermediate mechanism between local inflammation and innate immune system in diabetic periodontitis

    PubMed Central

    Wang, Jia; Li, Hao; Li, Bo; Gong, Qiulin; Chen, Xinmin; Wang, Qi

    2016-01-01

    Diabetic periodontitis (DP), which has been shown to cause alveolar bone loss, is among the most common complications associated with diabetes. The precise mechanisms underlying alveolar bone loss in patients with DP remain unclear. Therefore, the present study established a co-culture system of bone marrow stem cells (BMSCs) and macrophages, in order to investigate the potential mechanisms underlying DP-associated alveolar bone loss in vitro. In addition, Porphyromonas gingivalis (PG) periodontal infection and high glucose levels were used to induce DP in mice. The present study evaluated the protein expression levels of various chemokines and the migration of BMSCs and macrophages. The protein expression levels of extracellular signal-regulated kinase 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) were significantly increased in the BMSCs exposed to high glucose and PG, which may have been due to the activation of MAPK. In addition, DP induction in mice was associated with the release of chemokine (C-C motif) ligand 2 (CCL2) from BMSCs and the secretion of chemokine (C-C Motif) receptor 2 (CCR2) and tumor necrosis factor-α from macrophages, which was associated in turn with enhanced adhesion and chemotaxis of macrophages. The results of the present study suggested that DP led to the upregulation of CCL2 in the periodontal tissues and enhanced macrophage infiltration via the CCL2/CCR2 axis, which in turn promoted alveolar bone loss. PMID:27446245

  20. The Effects of Information Exposure Activities on Attitudinal Changes Among Co-Culturals: Some Preliminary Findings.

    ERIC Educational Resources Information Center

    Civikly, Jean M.; Plax, Timothy G.

    To examine the effect of various activities and interactions on the attitudes of members of co-cultures toward each other was the purpose of this study. The research was conducted in two stages. In the first stage, semantic differential scales were developed for the measurement of attitudes toward co-cultures. Each of five sets of scales was used…

  1. The functional behavior of a macrophage/fibroblast co-culture model derived from normal and diabetic mice with a marine gelatin-oxidized alginate hydrogel.

    PubMed

    Zeng, Qiong; Chen, Weiliam

    2010-08-01

    Tissues/cells-mediated biodegradable material degradation is epitomized by the constantly changing tissues/cell-implant interface, implicating the constant adaptation of the tissues/cells. Macrophages and fibroblasts are multi-functional cells highly involved in the interactions; the two cell types modulates the behaviors of each other, but their combinatorial functional behavior in the presence of interactive bioactive wound dressings has not been adequately examined. The activity is further complicated by the implantation of biodegradable materials, such as hydrogels commonly utilized as wound dressings, in a pathological environment and this is exemplified by the macrophages with a diabetic pathology producing an alternative cytokine profile which is implicated in wound healing delay. In this study, an in situ gelable formable/conformable hydrogel formulated from modified alginate and marine gelatin was used as a model biodegradable interactive wound dressing to elucidate the combinatorial behavior of macrophages/fibroblasts derived from both normal and diabetic hosts. Cell proliferation, migration and distribution were first characterized; this was followed by simultaneous quantitative detection of 40 inflammatory cytokines and chemokines by a protein microarray. The results showed that the macrophages/fibroblasts co-culture promoted fibroblasts proliferation and migration in the presence of the hydrogel; moreover, the expressions of inflammatory cytokines and chemokines were altered when compared with the corresponding fibroblasts or macrophages monocultures. The inflammatory cytokines patterns between the normal and diabetic hosts were considerably different.

  2. Role of the MAPK pathway in the observed bystander effect in lymphocytes co-cultured with macrophages irradiated with γ-rays or carbon ions.

    PubMed

    Dong, Chen; He, Mingyuan; Ren, Ruiping; Xie, Yuexia; Yuan, Dexiao; Dang, Bingrong; Li, Wenjian; Shao, Chunlin

    2015-04-15

    The radiation-induced bystander effect (RIBE) has potential implications in cancer risks from space particle radiation; however, the mechanisms underlying RIBE are unclear. The role of the MAPK pathway in the RIBEs of different linear energy transfer (LET) was investigated. Human macrophage U937 cells were irradiated with γ-rays or carbon ions and then co-cultured with nonirradiated HMy2.CIR (HMy) lymphocytes for different periods. The activation of MAPK proteins and the generation of intracellular nitric oxide (NO) and reactive oxygen species (ROS) in the irradiated U937 cells were measured. Micronuclei (MN) formation in the HMy cells was applied to evaluate the bystander damage. Some U937 cells were pretreated with different MAPK inhibitors before irradiation. Additional MN formation was induced in the HMy cells after co-culturing with irradiated U937 cells, and the yield of this bystander MN formation was dependent on the co-culture period with γ-ray irradiation but remained high after 1h of co-culture with carbon irradiation. Further investigations disclosed that the time response of the RIBEs had a relationship with LET, where ERK played a different role from JNK and p38 in regulating RIBEs by regulating the generation of the bystander signaling factors NO and ROS. The finding that the RIBE of high-LET radiation could persist for a much longer period than that of γ-rays implies that particle radiation during space flight could have a high risk of long-term harmful effects. An appropriate intervention targeting the MAPK pathway may have significant implications in reducing this risk. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Monocyte and monocyte-derived macrophage secretion of MCP-1 in co-culture with autologous malignant and benign control fragment spheroids.

    PubMed

    Heimdal, J H; Olsnes, C; Olofsson, J; Aarstad, H J

    2001-08-01

    This study was performed in order to determine how monocytes and macrophages in co-culture with autologous head and neck squamous cell carcinoma (HNSCC) tumor tissue regulate the secretion of monocyte chemotactic protein-1 (MCP-1). The levels of MCP-1 were measured when autologous monocytes or monocyte-derived macrophages (MDMs) were co-cultured in vitro with autologous fragment (F)-spheroids established from HNSCC tumors or benign mucosa serving as control. MCP-1 secretion from co-culture stimulated monocytes and MDMs was increased compared to spontaneous MCP-1 secretion. With prolonged co-culture, MDMs showed a steady-state MCP-1 secretion above background levels for up to 96 h, even with change of co-culture media every 24 h. Addition of an anti-MCP-1 antibody to the medium decreased co-culture-induced monocyte IL-6 secretion. Addition of lipopolysaccharide (LPS) (1 [microg/ml) reduced MCP-1 secretion compared to spontaneous secretion in monocyte cultures. F-spheroids also secrete MCP-1, but at insignificant levels compared to the MCP-1 secretion from monocytes and MDMs. MCP-1 secretion from monocytes/MDMs is regulated differently when co-culture stimulation is compared to LPS-stimulation. Monocytes and MDMs expressed MCP-1 mRNA at a high level in all tested conditions: stimulated in co-culture, not stimulated or stimulated with LPS, indicating post-transcriptional regulation of MCP-1 secretion. The secretion of MCP-1 from tumor-derived F-spheroids, and the maintenance of co-culture MCP-1 secretion from MDMs in vitro, suggests that tumor-associated macrophages are a source of MCP-1 in HNSCC tumors.

  4. Metabolism Supports Macrophage Activation

    PubMed Central

    Langston, P. Kent; Shibata, Munehiko; Horng, Tiffany

    2017-01-01

    Macrophages are found in most tissues of the body, where they have tissue- and context-dependent roles in maintaining homeostasis as well as coordinating adaptive responses to various stresses. Their capacity for specialized functions is controlled by polarizing signals, which activate macrophages by upregulating transcriptional programs that encode distinct effector functions. An important conceptual advance in the field of macrophage biology, emerging from recent studies, is that macrophage activation is critically supported by metabolic shifts. Metabolic shifts fuel multiple aspects of macrophage activation, and preventing these shifts impairs appropriate activation. These findings raise the exciting possibility that macrophage functions in various contexts could be regulated by manipulating their metabolism. Here, we review the rapidly evolving field of macrophage metabolism, discussing how polarizing signals trigger metabolic shifts and how these shifts enable appropriate activation and sustain effector activities. We also discuss recent studies indicating that the mitochondria are central hubs in inflammatory macrophage activation. PMID:28197151

  5. Substance P induces inflammatory responses involving NF-κB in genetically diabetic mice skin fibroblasts co-cultured with macrophages

    PubMed Central

    Ni, Tao; Liu, Yushu; Peng, Yinbo; Li, Ming; Fang, Yong; Yao, Min

    2016-01-01

    Purpose: Delayed wound healing is an intractable complex of diabetes and substance P (SP) is proved to benefit wound healing, whose functioning mechanism remains elusive. This study aims at revealing whether the influence of SP on diabetic wound healing is dependent on inflammatory responses, particularly NF-κB. Methods: Skin fibroblasts of genetically diabetic mice were co-cultured with bone marrow-derived macrophages, and treated with SP, SP + L703,606 (a neurokinin-1 receptor antagonist), or SP + MG132 (an inhibitor of NF-κB). For macrophages, their migration ability was assessed by Transwell experiments, and their M2 polarization was analyzed by flow cytometry and markers for M2 phenotype. Pro-inflammatory factors in the supernatant were detected by enzyme-linked immunosorbent assay. In fibroblasts, the transcription levels of the four pro-inflammatory factors and the protein levels of NF-κB regulators like inhibitor of NF-κB alpha (IκBα) and IκB kinases (IKKs) were monitored by real-time quantitative PCR and western blot, respectively. Results: SP could significantly induce migration to fibroblasts (P<0.01), M2 polarization (P<0.001) and pro-inflammatory factor concentration (P<0.01) in the co-culture system. It also promotes the transcription process of pro-inflammatory factors in fibroblasts (P<0.01), and induce activation of IKKα/β and phosphorylation of IκBα, which caused NF-κB activation. All these effects were reversed if NF-κB was inhibited. Conclusion: The promoting effects of SP on diabetic wound healing was dependent on enhanced inflammatory responses, especially the activation of NF-κB. This study provided evidence for the potential usage of SP in accelerating diabetic wound healing. PMID:27347325

  6. A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection.

    PubMed

    Schwab, Ute; Caldwell, Shannon; Matychak, Mary-Beth; Felippe, Julia

    2013-07-15

    We developed a 3-D equine bronchial epithelial cell (BEC) culture that fully differentiates into ciliary beating and mucus producing cells. Using this system, we evaluated how mucus affects the phagocytic activity of macrophages. Adult horse monocyte-derived macrophages were incubated with Rhodococcus equi for 4h either in the mucus layer of in vitro generated airway epithelium or on collagen coated membranes. Using light and electron microscopy, we noted that the number of macrophages with intracellular bacteria, and the number of intracellular bacteria per macrophage were lower in the presence of mucus. TNFα measurements revealed that the presence of BECs promoted TNFα production by R. equi-infected macrophages; a decrease in TLR-2 (involved in R. equi recognition) and an increase in EGF-R (involved in mucin production) mRNA expression were also noted. Interestingly, when foal macrophages were added to foal BECs, we made the opposite observation, i.e. many macrophages were loaded with R. equi. Our in vitro bronchial system shows great potential for the identification of mechanisms how BECs and mucus play a role in phagocyte activation and bacterial clearance. Further studies using this system will show whether the airway environment in the foal responds differently to R. equi infection.

  7. A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

    PubMed

    Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

    2017-02-01

    Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca(2+) level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca(2+) sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca(2+) imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca(2+) oscillation and sustained Ca(2+) signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca(2+) signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

  8. Macrophage activation and polarization.

    PubMed

    Martinez, Fernando Oneissi; Sica, Antonio; Mantovani, Alberto; Locati, Massimo

    2008-01-01

    Macrophages are widely distributed immune system cells that play an indispensable role in homeostasis and defense. They can be phenotypically polarized by the microenvironment to mount specific functional programs. Polarized macrophages can be broadly classified in two main groups: classically activated macrophages (or M1), whose prototypical activating stimuli are IFNgamma and LPS, and alternatively activated macrophages (or M2), further subdivided in M2a (after exposure to IL-4 or IL-13), M2b (immune complexes in combination with IL-1beta or LPS) and M2c (IL-10, TGFbeta or glucocorticoids). M1 exhibit potent microbicidal properties and promote strong IL-12-mediated Th1 responses, whilst M2 support Th2-associated effector functions. Beyond infection M2 polarized macrophages play a role in resolution of inflammation through high endocytic clearance capacities and trophic factor synthesis, accompanied by reduced pro-inflammatory cytokine secretion. Similar functions are also exerted by tumor-associated macrophages (TAM), which also display an alternative-like activation phenotype and play a detrimental pro-tumoral role. Here we review the main functions of polarized macrophages and discuss the perspectives of this field.

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

    PubMed

    Jackson, Megan V; Krasnodembskaya, Anna D

    2017-05-05

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

  10. The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions.

    PubMed

    Harris, Sean; Shubin, Sara Pacheco; Wegner, Susanna; Van Ness, Kirk; Green, Foad; Hong, Sung Woo; Faustman, Elaine M

    2016-10-01

    Our 3-dimensional testis co-culture system (3D-TCS) represents a promising model of male reproductive toxicity which captures sensitive processes of male reproductive development and contains the main testes cell types (germ, Leydig and Sertoli cells). Macrophages are another cell type important for testicular function and help to modulate immuno-endocrine processes during testes development. Chemicals such as phthalate esters (PE's) affect macrophage function and testosterone production in the testes in vivo. The aim of this study was to determine whether macrophages were present in the 3D-TCS and investigate responses in our model that may be related to immuno-endocrine functions. We observed consistent expression of the resident macrophage marker ED2 as well as increases in inflammatory cytokines produced by macrophages and testes cells (IL-6, TNF-α and KC/GRO) after exposure to toxic PE's. Pathway analysis of gene expression changes after exposure to PE's showed that IL-6 and TNF-α signaling pathways were enriched after treatment with reproductively toxic, but not non-reproductively toxic phthalates. These results indicate that macrophages and inflammatory processes are captured in the 3D-TCS and that these processes are impacted by exposure to reproductive toxicants. These processes represent a major mode of action for in vivo testis toxicity for a variety of compounds and our novel in vitro model is able to capture toxicant perturbation of immune function.

  11. Setting the proportion of CD4+ and CD8+ T-cells co-cultured with canine macrophages infected with Leishmania chagasi.

    PubMed

    Viana, Kelvinson Fernandes; Aguiar-Soares, Rodrigo Dian Oliveira; Ker, Henrique Gama; Resende, Lucilene Aparecida; Souza-Fagundes, Elaine Maria; Dutra, Walderez Ornelas; Fujiwara, Ricardo Toshio; da Silveira-Lemos, Denise; Sant'Ana, Rita de Cássia Oliveira; Wardini, Amanda Brito; Araújo, Márcio Sobreira Silva; Martins-Filho, Olindo Assis; Reis, Alexandre Barbosa; Giunchetti, Rodolfo Cordeiro

    2015-07-30

    New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. In vitro cytokine release from rat type II pneumocytes and alveolar macrophages following exposure to JP-8 jet fuel in co-culture.

    PubMed

    Wang, Shengjun; Young, R Scotte; Sun, Nina N; Witten, Mark L

    2002-05-01

    Alveolar type II epithelial cells (AIIE) and pulmonary alveolar macrophages (PAM) are involved in pulmonary toxicity of JP-8 jet fuel exposure. To further elucidate their inflammatory mechanisms, the effect(s) of JP-8 jet fuel on cytokine secretion were examined in a transformed rat AIIE cell line (RLE-6TN) culture alone, primary PAM (from Fischer 344 rats) culture alone, and the co-culture of AIIE and primary PAM. A series of JP-8 jet fuel concentrations (0-0.8 microg/ml), which may actually be encountered in alveolar space of lungs exposed in vivo, were placed in cell culture for 24 h. Cultured AIIE alone secreted spontaneously interleukin (IL)-1beta and -6 [below detectable limits for IL-10 and tumor necrosis factor-alpha (TNF-alpha)], whereas cultured PAM alone secreted IL-1beta, -10, and TNF-alpha, in a concentration-dependent manner. These data suggest that the release of cytokines, not only from PAM but also from AIIE cells, may contribute to JP-8 jet fuel-induced inflammatory response in the alveolar space. However, the co-cultures of AIIE and PAM showed no significant changes in IL-1beta, -6, and TNF-alpha at any JP-8 jet fuel concentration compared to control values. These cytokine levels in co-cultures of AIIE and PAM were inversely related to these of cultured AIIE or PAM alone. Interestingly, IL-10 levels in the co-culture system were concentration-dependently increased up to 1058% at JP-8 concentrations of 0.8 microg/ml, although under detectable limits in cultured AIIE alone and no significant concentration change in cultured PAM alone. It appears that PAM may possibly act via paracrine and/or autocrine pathways to signal AIIE cells to regulate cytokine release.

  13. Monocytes and macrophages, implications for breast cancer migration and stem cell-like activity and treatment

    PubMed Central

    Ward, Rebecca; Sims, Andrew H.; Lee, Alexander; Lo, Christina; Wynne, Luke; Yusuf, Humza; Gregson, Hannah; Lisanti, Michael P.; Sotgia, Federica; Landberg, Göran; Lamb, Rebecca

    2015-01-01

    Macrophages are a major cellular constituent of the tumour stroma and contribute to breast cancer prognosis. The precise role and treatment strategies to target macrophages remain elusive. As macrophage infiltration is associated with poor prognosis and high grade tumours we used the THP-1 cell line to model monocyte-macrophage differentiation in co-culture with four breast cancer cell lines (MCF7, T47D, MDA-MB-231, MDA-MB-468) to model in vivo cellular interactions. Polarisation into M1 and M2 subtypes was confirmed by specific cell marker expression of ROS and HLA-DR, respectively. Co-culture with all types of macrophage increased migration of ER-positive breast cancer cell lines, while M2-macrophages increased mammosphere formation, compared to M1-macrophages, in all breast cancer cells lines. Treatment of cells with Zoledronate in co-culture reduced the “pro-tumourigenic” effects (increased mammospheres/migration) exerted by macrophages. Direct treatment of breast cancer cells in homotypic culture was unable to reduce migration or mammosphere formation. Macrophages promote “pro-tumourigenic” cellular characteristics of breast cancer cell migration and stem cell activity. Zoledronate targets macrophages within the microenvironment which in turn, reduces the “pro-tumourigenic” characteristics of breast cancer cells. Zoledronate offers an exciting new treatment strategy for both primary and metastatic breast cancer. PMID:26008983

  14. Profound influence of LDL oxidative status and monocyte co-cultures on baboon endothelial activation

    PubMed Central

    Xiao, Juan; Hondara, Vida; Wang, Xing Li; VandeBerg, John L; Shi, Qiang

    2012-01-01

    Objective To investigate how increased LDL levels interact with endothelial cells by using well-defined LDL preparations to limit experimental biases caused by heterogeneity of LDL preparations. Methods We pooled LDL from multiple subjects and prepared several types of LDL from a single source. Then we observed their effects on cultured endothelial cells with and without monocyte co-culture. Results Native and minimally oxidized LDL did not cause significant cell death under most circumstances, and did not up-regulate cellular adhesion molecule (CAM) expression. Native LDL did result in significant increases of MCP-1 release in five of eight subjects. However, extensively oxidized LDL caused a significant amount of cell death and dramatically decreased MCP-1 secretion. Minimally oxidized LDL elicited a mixed response pattern, with a great deal of variation among subjects. When endothelial cells were co-cultured with monocytes and treated with native LDL, significant up-regulation of CAMs was detected after 24 hours of exposure. Up-regulation was not seen in any treatment group that contained either native LDL or monocytes only, indicating a synergistic effect of LDL and monocytes on endothelial cells. Incubation of cultured monocytes with native LDL also resulted in TNF-α and IL-1β release in a dosage- and time-dependent manner. Neutralization of both TNF-α and IL-1β by 10 μg/mL polyclonal antibodies inhibited the up-regulation of CAMs. Conclusion Our results suggest that varying extents of oxidative modification of LDL lead to fundamentally different cytological effects and that native LDL exhibits greater endothelial activation capacity when it interactively cooperates with monocytes. PMID:22254208

  15. Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

    PubMed

    Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

    2012-01-01

    The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

  16. The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

    NASA Astrophysics Data System (ADS)

    Duan, J.; Lu, X.; He, G.

    2017-01-01

    In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

  17. Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

    PubMed

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Mel'nikov, P A; Cherepanov, S A; Levinsky, A B; Chehonin, V P

    2016-02-01

    The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

  18. Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos.

    PubMed

    Tan, Xiu-Wen; Ma, Suo-Feng; Yu, Jian-Ning; Zhang, Xia; Lan, Guo-Cheng; Liu, Xin-Yong; Han, Zheng-Bin; Tan, Jing-He

    2007-01-01

    An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because

  19. Sustained synchronized neuronal network activity in a human astrocyte co-culture system

    PubMed Central

    Kuijlaars, Jacobine; Oyelami, Tutu; Diels, Annick; Rohrbacher, Jutta; Versweyveld, Sofie; Meneghello, Giulia; Tuefferd, Marianne; Verstraelen, Peter; Detrez, Jan R.; Verschuuren, Marlies; De Vos, Winnok H.; Meert, Theo; Peeters, Pieter J.; Cik, Miroslav; Nuydens, Rony; Brône, Bert; Verheyen, An

    2016-01-01

    Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases. PMID:27819315

  20. Phloretin and phlorizin promote lipolysis and inhibit inflammation in mouse 3T3-L1 cells and in macrophage-adipocyte co-cultures.

    PubMed

    Huang, Wen-Chung; Chang, Wei-Tien; Wu, Shu-Ju; Xu, Pei-Yin; Ting, Nai-Chun; Liou, Chian-Jiun

    2013-10-01

    Previous studies found that phloretin (PT) and phlorizin (PZ) could inhibit glucose transport, with PT being a better inhibitor of lipid peroxidation. This study aimed to evaluate the antiobesity effects of PT and PZ in 3T3-L1 cells and if they can modulate the relationship between adipocytes and macrophages. Differentiated 3T3-L1 cells were treated with PT or PZ. Subsequently, transcription factors of adipogenesis and lipolysis proteins were measured. In addition, RAW 264.7 macrophages treated with PT or PZ were cultured in differentiated media from 3T3-L1 cells to analyze inflammatory mediators and signaling pathways. PT significantly enhanced glycerol release and inhibited the adipogenesis-related transcription factors. PT also promoted phosphorylation of AMP-activated protein kinase and increased activity of adipose triglyceride lipase and hormone-sensitive lipase. PT suppressed the nuclear transcription factor kappa-B and mitogen-activated protein kinase pathways when RAW 264.7 cells were cultured in differentiated media from 3T3-L1 cells. PZ improved lipolysis and inhibited the macrophage inflammatory response less effectively than PT. This study suggests that PT is more effective than PZ at increasing lipolysis in adipocytes. In addition, PT also suppresses inflammatory response in macrophage that is stimulated by differentiated media from 3T3-L1 cells. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Effects of Activated Macrophages on Nocardia asteroides

    PubMed Central

    Filice, Gregory A.; Beaman, Blaine L.; Remington, Jack S.

    1980-01-01

    The mechanism(s) of host resistance against Nocardia asteroides has not been well defined. Since disease due to N. asteroides frequently occurs in patients with impaired cell-mediated immunity, we studied the interaction of N. asteroides with activated and control mouse peritoneal macrophages. Activated macrophages were from mice infected with Toxoplasma gondii or injected with Corynebacterium parvum. N. asteroides in the early stationary phase (>99% in the coccobacillary form) was used for challenge of macrophage monolayers. Growth of two strains of N. asteroides was markedly inhibited in activated macrophages, whereas N. asteroides grew well in control macrophages. Quantitation of macrophage-associated N. asteroides indicated that activated macrophages killed 40 to 50% of N. asteroides within 6 h (P < 0.002). In control macrophage preparations, it appeared as if Nocardia filaments extended from within macrophages to the outside, and many of these filaments appeared to have extended to and then grown through neighboring macrophages. In activated macrophage preparations, Nocardia remained in the coccobacillary form in most macrophages. Control macrophage monolayers were almost completely overgrown with and destroyed by Nocardia 20 h after challenge, whereas activated macrophage monolayers remained intact. Nocardia that grew in control macrophages were not acid-alcohol fast or only weakly so, whereas the few Nocardia that grew in activated macrophages were strongly acid-alcohol fast. Our results indicate that activated macrophages may be important in host defense against N. asteroides. Images Fig. 1 PMID:6991421

  2. Co-culture of spleen stromal cells with bone marrow mononuclear cells leads to the generation of a novel macrophage subset.

    PubMed

    Wang, X; Li, Y; Xiao, H; Zhang, X; Cao, J; Zhang, D; Zhang, J; Li, X; Shen, B; Wang, Q; Shi, Y

    2014-01-01

    Macrophages adopt diverse activation states depending on the microenvironment. Recently, stromal cells have been demonstrated to be organizers of the microenvironment. Here, using splenic stromal cells to mimic the splenic microenvironment in vivo, we show that spleen stromal cells can programme bone marrow-derived mononuclear cells to differentiate and polarize into a novel macrophage subset. These differentiated macrophages (Diff-Mφ) exhibited pronounced production of IL-10, IL-6 and TNF-α, but diminished the production of IL-12 in response to LPS. The generation of Diff-Mφ depended on cell-cell contact as well as on soluble factors. Diff-Mφ directly suppressed the antigen-non-specific (CD3/CD28) CD4(+) T cell proliferative response and induced cell death of activated CD4(+) T cells. As for cytokine production in CD4(+) T cells, Diff-Mφ promoted IL-10 and IL-17 production, whereas inhibited IL-4 production and did not alter IFN-γ production. Besides, Diff-Mφ also expressed iNOS, CD16/CD32, CD54, CD43, CCR7, CD44, PD-L1 and FasL, which might be involved in the function of Diff-Mφ. These results suggest that splenic microenvironment may physiologically induce a novel type of macrophages differentiation. © 2013 John Wiley & Sons Ltd.

  3. A new cyclopeptide with antifungal activity from the co-culture broth of two marine mangrove fungi.

    PubMed

    Li, Chunyuan; Wang, Jinhua; Luo, Cuiping; Ding, Weijia; Cox, Daniel G

    2014-01-01

    A new cyclic tetrapeptide, cyclo-(L-leucyl-trans-4-hydroxy-L-prolyl-D-leucyl-trans-4-hydroxy-L-proline) (1), was isolated from the co-culture broth of two mangrove fungi Phomopsis sp. K38 and Alternaria sp. E33. The structure of 1 was determined by analysis of spectroscopic data and Marfey's analytic method. Primary bioassay demonstrated that compound 1 exhibited moderate to high inhibitory activity against four crop-threatening fungi including Gaeumannomyces graminis, Rhizoctonia cerealis, Helminthosporium sativum and Fusarium graminearum as compared with triadimefon.

  4. Impairing autophagy in retinal pigment epithelium leads to inflammasome activation and enhanced macrophage-mediated angiogenesis

    PubMed Central

    Liu, Jian; Copland, David A.; Theodoropoulou, Sofia; Chiu, Hsi An Amy; Barba, Miriam Durazo; Mak, Ka Wang; Mack, Matthias; Nicholson, Lindsay B.; Dick, Andrew D.

    2016-01-01

    Age-related decreases in autophagy contribute to the progression of age-related macular degeneration (AMD). We have now studied the interaction between autophagy impaired in retinal pigment epithelium (RPE) and the responses of macrophages. We find that dying RPE cells can activate the macrophage inflammasome and promote angiogenesis. In vitro, inhibiting rotenone-induced autophagy in RPE cells elicits caspase-3 mediated cell death. Co-culture of damaged RPE with macrophages leads to the secretion of IL-1β, IL-6 and nitrite oxide. Exogenous IL-6 protects the dysfunctional RPE but IL-1β causes enhanced cell death. Furthermore, IL-1β toxicity is more pronounced in dysfunctional RPE cells showing reduced IRAK3 gene expression. Co-culture of macrophages with damaged RPE also elicits elevated levels of pro-angiogenic proteins that promote ex vivo choroidal vessel sprouting. In vivo, impaired autophagy in the eye promotes photoreceptor and RPE degeneration and recruitment of inflammasome-activated macrophages. The degenerative tissue environment drives an enhanced pro-angiogenic response, demonstrated by increased size of laser-induced choroidal neovascularization (CNV) lesions. The contribution of macrophages was confirmed by depletion of CCR2+ monocytes, which attenuates CNV in the presence of RPE degeneration. Our results suggest that the interplay between perturbed RPE homeostasis and activated macrophages influences key features of AMD development. PMID:26847702

  5. HMGB1 enhances the protumoral activities of M2 macrophages by a RAGE-dependent mechanism.

    PubMed

    Rojas, Armando; Delgado-López, Fernando; Perez-Castro, Ramón; Gonzalez, Ileana; Romero, Jacqueline; Rojas, Israel; Araya, Paulina; Añazco, Carolina; Morales, Erik; Llanos, Jorge

    2016-03-01

    The monocyte-macrophage lineage shows a high degree of diversity and plasticity. Once they infiltrate tissues, they may acquire two main functional phenotypes, being known as the classically activated type 1 macrophages (M1) and the alternative activated type 2 macrophages (M2). The M1 phenotype can be induced by bacterial products and interferon-γ and exerts a cytotoxic effect on cancer cells. Conversely, the alternatively activated M2 phenotype is induced by Il-4/IL13 and promotes tumor cell growth and vascularization. Although receptor for advanced glycation end-products (RAGE) engagement in M1 macrophages has been reported by several groups to promote inflammation, nothing is known about the functionality of RAGE in M2 macrophages. In the current study, we demonstrate that RAGE is equally expressed in both macrophage phenotypes and that RAGE activation by high-mobility group protein box1 (HMGB1) promotes protumoral activities of M2 macrophages. MKN45 cells co-cultured with M2 macrophages treated with HMGB1 at different times displayed higher invasive abilities. Additionally, conditioned medium from HMGB1-treated M2 macrophages promotes angiogenesis in vitro. RAGE-targeting knockdown abrogates these activities. Overall, the present findings suggest that HMGB1 may contribute, by a RAGE-dependent mechanism, to the protumoral activities of the M2 phenotype.

  6. Alternatively activated RAW264.7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma.

    PubMed

    Zhang, Bicheng; Wang, Jun; Gao, Juan; Guo, Yan; Chen, Xi; Wang, Baocheng; Gao, Jianfei; Rao, Zhiguo; Chen, Zhengtang

    2009-05-01

    Tumor-associated macrophages (TAMs) have been implicated in promoting tumor progression and invasion. The onset and maintenance of tumor angiogenesis and lymphangiogenesis also seem to be partly driven by a group of polarized alternatively activated macrophages (aaMphi) in lung adenocarcinoma. Here, the aaMphi and classically activated macrophages (caMphi) were obtained using RAW264.7 cells via IL-4 and IFN-gamma + LPS treatment, respectively. Co-inoculation of aaMphi with Lewis lung carcinoma (LLC) cells promoted tumor growth, increased lymph node metastasis, and reduced the survival in C57BL/6 mice bearing LLC. Furthermore, the effects of the activated macrophages on the lymphangiogenesis-related properties of lymphatic endothelial cells (LECs) were investigated in vitro. When LECs were cultured in macrophages conditioned medium or in a co-culture system of macrophages and LECs, aaMphi significantly promoted proliferation, migration, and tube-like formation of LECs. We identified high VEGF-C expression in aaMphi and low expression in caMphi as well as unactivated macrophages by ELISA and Western blotting. In LECs, co-culture with aaMphi resulted in a significant increase of mRNA levels of specific lymphatic marker VEGF receptor-3 and the homeobox gene Prox-1, as well as lymphangiogenic factor VEGF-C rather than VEGF-D by quantitative RT-PCR. Furthermore, enhanced LECs migration and capillary formation by co-culture with aaMphi were significantly inhibited by rVEGF receptor-3/Fc chimera. In conclusion, these data show that aaMphi play a critical role in tumor-induced lymphangiogenesis through up-regulating VEGF-C and increasing lymphangiogenesis-related behavior of LECs, which may contribute to lymphatic invasion in lung adenocarcinoma.

  7. Assessment of the cytotoxicity of a mineral trioxide aggregate-based sealer with respect to macrophage activity.

    PubMed

    Braga, Julia Mourão; Oliveira, Ricardo Reis; de Castro Martins, Renata; Vieira, Leda Quercia; Sobrinho, Antonio Paulino Ribeiro

    2015-10-01

    To assess the influence of co-culture with mineral trioxide aggregate (MTA) and MTA Fillapex (FLPX) on the viability, adherence, and phagocytosis activity of peritoneal macrophages from two mouse strains. Cellular viability, adherence, and phagocytosis of Saccharomyces boulardii were assayed in the presence of capillaries containing MTA and MTA Fillapex. The data were analyzed using parametric (Student's t) and non-parametric (Mann-Whitney) tests. FLPX was severely cytotoxic and decreased cell viability, adherence, and phagocytic activity of both macrophage subtypes. Cells that were treated with MTA Fillapex remained viable (>80%) for only 4 h after stimulation. Macrophages from C57BL/6 mice presented higher adherence and higher phagocytic activity compared with macrophages from BALB/c mice. Comparison of MTA and FLPX effects upon macrophages indicates that FLPX may impair macrophage activity and viability, while MTA seems to increase phagocytic activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Glucagon-like peptide-1 (GLP-1) induces M2 polarization of human macrophages via STAT3 activation.

    PubMed

    Shiraishi, Daisuke; Fujiwara, Yukio; Komohara, Yoshihiro; Mizuta, Hiroshi; Takeya, Motohiro

    2012-08-24

    It is known that glucagon-like peptide-1 (GLP-1) is a hormone secreted postprandially from the L-cells of the small intestine and regulates glucose homeostasis. GLP-1 is now used for the treatment of diabetes because of its beneficial role against insulin resistance. The GLP-1 receptor (GLP-1R) is expressed on many cell types, including macrophages, and GLP-1 suppresses the development of atherosclerosis by inhibiting macrophage function. However, there have so far been few studies that have investigated the significance of GLP-1/GLP-1R signaling in macrophage activation. In the present study, we examined the effect of GLP-1 and exenatide, a GLP-1R agonist, on human monocyte-derived macrophage (HMDM) activation. We found that GLP-1 induced signal transducer and activator of transcription 3 (STAT3) activation. Silencing of GLP-1R suppressed the GLP-1-induced STAT3 activation. In addition, alternatively activated (M2) macrophage-related molecules, such as IL-10, CD163, and CD204 in HMDM, were significantly upregulated by GLP-1. Furthermore, the co-culture of 3T3-L1 adipocytes with GLP-1-treated RAW 264.7 macrophages increased the secretion of adiponectin compared to co-culture of the 3T3-L1 adipocytes with untreated RAW 264.7 macrophages. Our results demonstrate that GLP-1 induces macrophage polarization toward the M2 phenotype, which may contribute to the protective effects of GLP-1 against diabetes and cardiovascular diseases.

  9. Osteopontin expression in co-cultures of human squamous cell carcinoma-derived cells and osteoblastic cells and its effects on the neoplastic cell phenotype and osteoclastic activation.

    PubMed

    Teixeira, Lucas Novaes; de Castro Raucci, Larissa Moreira Spinola; Alonso, Gabriela Caroline; Coletta, Ricardo Della; Rosa, Adalberto Luiz; de Oliveira, Paulo Tambasco

    2016-09-01

    This study evaluated the temporal expression of osteopontin (OPN) in co-cultures of human osteoblastic cells (SAOS-2) and oral squamous cell carcinoma (OSCC)-derived cells (SCC9) and examined the effects of osteoblast-derived OPN on the neoplastic cell phenotype. Additionally, the effects of these co-cultures on subsequent osteoclastic activity were explored. SCC9 cells were plated on Transwell® membranes that were either coated or not coated with Matrigel and were then co-cultured with SAOS-2 cells during the peak of OPN expression. SCC9 cells exposed to OPN-silenced SAOS-2 cultures and SCC9 cells cultured alone served as controls. SCC9 cells were quantitatively evaluated for cell adhesion, proliferation, migration, and invasion into Matrigel. The impact of co-culturing SAOS-2 and SCC9 cells on the resorptive capacity of U-937-derived osteoclastic cells was also investigated. Furthermore, a reciprocal induction of SAOS-2 and SCC9 cells in terms of OPN expression over the co-culture interval was identified. SAOS-2-secreted OPN altered the SCC9 cell phenotype, leading to enhanced cell adhesion and proliferation and higher Matrigel invasion. This invasion was also enhanced, albeit to a lesser degree, by co-culture with OPN-silenced SAOS-2 cells. Cell migration was not affected. Co-culture with SAOS-2 cells-mainly during the period of peak OPN expression-promoted over-expression of IL-6 and IL-8 by SCC9 cells and enhanced the resorptive capacity of osteoclastic cells. Taken together, these results suggest that osteoblast-derived OPN affects the interactions among OSCC-derived epithelial cells, osteoblasts, and osteoclasts, which could contribute to the process of bone destruction during bone invasion by OSCC.

  10. The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sorting.

    PubMed

    Zhang, Yu; Schedle, Andreas; Matejka, Michael; Rausch-Fan, Xiaohui; Andrukhov, Oleh

    2010-12-01

    The interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts. We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63 cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial cells could have merit for further co-culture research.

  11. Mesenchymal stem cells ameliorate rhabdomyolysis-induced acute kidney injury via the activation of M2 macrophages.

    PubMed

    Geng, Yanqiu; Zhang, Li; Fu, Bo; Zhang, Jianrong; Hong, Quan; Hu, Jie; Li, Diangeng; Luo, Congjuan; Cui, Shaoyuan; Zhu, Fei; Chen, Xiangmei

    2014-06-24

    The mortality of rhabdomyolysis-induced acute kidney injury (AKI) is still high, as there is no effective therapy. It has been shown that bone marrow-derived mesenchymal stem cells (MSCs) can induce M2 macrophages, which mediate MSC protection in other experimental inflammation-related organ injury. This study was designed to investigate the protective effects of macrophage activation in MSC therapy of rhabdomyolysis-induced AKI. MSCs were injected into glycerol-induced rhabdomyolysis mice. Renal injury was evaluated using the serum creatinine, urea nitrogen, renal pathology and acute tubular necrosis score. The distribution of MSCs was detected using two-photon fluorescence confocal imaging. Immunofluorescence of anti-F4/80 and anti-CD206 was performed to determine macrophages and M2 macrophages in the tissues of the kidney, and M2 macrophage infiltration was also evaluated using western blotting analyses. After depletion of macrophages using clodronate liposomes at the phase of kidney repair, renal injury was re-evaluated. RAW 264.7 macrophages were incubated with lipopolysaccharide and co-cultured with MSCs and subsequently visualised using immunofluorescence staining and flow cytometry analysis. Finally, disparate phenotype macrophages, including normal macrophages (M0), lipopolysaccharide-stimulated macrophages (M1), and MSC-co-cultured macrophages (M2), were infused into mice with AKI, which were pre-treated with liposomal clodronate. In vivo infusion of MSCs protected AKI mice from renal function impairment and severe tubular injury, which was accompanied by a time-dependent increase in CD206-positive M2 macrophage infiltration. In addition, depleting macrophages with clodronate delayed restoration of AKI. In vitro, macrophages co-cultured with MSCs acquired an anti-inflammatory M2 phenotype, which was characterised by an increased expression of CD206 and the secretory cytokine interleukin (IL)-10. The concentrations of IL-10, IL-6 and tumor necrosis factor

  12. Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

    SciTech Connect

    Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong

    2013-11-15

    During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC.

  13. Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes

    SciTech Connect

    Ryu, Jae-Sung; Jung, Yeon-Hwa; Cho, Mi-Young; Yeo, Jee Eun; Choi, Yun-Jin; Kim, Yong Il; Koh, Yong-Gon

    2014-05-16

    Highlights: • Co-culture of hSDMSCs with SNP-stimulated chondrocytes improves anti-inflammation. • Co-culture system produces IGF-1. • Co-culture system suppresses inflammatory genes expression. • Co-culture system improves cell proliferation. • Exogenous IGF-1 inhibits inflammatory activity in SNP-stimulated chondrocytes. - Abstract: Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.

  14. Effects of cell type and configuration on anabolic and catabolic activity in 3D co-culture of mesenchymal stem cells and nucleus pulposus cells.

    PubMed

    Ouyang, Ann; Cerchiari, Alec E; Tang, Xinyan; Liebenberg, Ellen; Alliston, Tamara; Gartner, Zev J; Lotz, Jeffrey C

    2017-01-01

    Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61-73, 2017. © 2016 The Authors. Journal of Orthopaedic Research

  15. Morphine Modulates Interleukin-4- or Breast Cancer Cell-induced Pro-metastatic Activation of Macrophages.

    PubMed

    Khabbazi, Samira; Goumon, Yannick; Parat, Marie-Odile

    2015-06-16

    Interactions between cancer cells and stromal cells in the tumour microenvironment play a key role in the control of invasiveness, metastasis and angiogenesis. Macrophages display a range of activation states in specific pathological contexts and alternatively activated (M2) macrophages can promote tumour aggressiveness. Opioids are able to modulate tumour growth and metastasis. We tested whether morphine modulates the activation of macrophages induced by (i) interleukin-4 (IL-4), the prototypical M2 polarization-inducing cytokine, or (ii) coculture with breast cancer cells. We showed that IL-4 causes increased MMP-9 production and expression of the alternative activation markers arginase-1 and MRC-1. Morphine prevented IL-4-induced increase in MMP-9 in a naloxone- and methylnaltrexone-reversible fashion. Morphine also prevented IL-4-elicited alternative activation of RAW264.7 macrophages. Expression of MMP-9 and arginase-1 were increased when RAW264.7 were subjected to paracrine activation by 4T1 cells, and this effect was prevented by morphine via an opioid receptor-mediated mechanism. Morphine further decreased 4T1 breast cancer cell invasion elicited by co-culture with RAW264.7. Reduction of MMP-9 expression and alternative activation of macrophages by morphine was confirmed using mouse bone marrow-derived macrophages. Taken together, our results indicate that morphine may modulate tumour aggressiveness by regulating macrophage protease production and M2 polarization within the tumour microenvironment.

  16. A co-culture system with three different primary human cell populations reveals that biomaterials and MSC modulate macrophage-driven fibroblast recruitment.

    PubMed

    Caires, Hugo R; da Silva, Patrícia Barros; Barbosa, Mário A; Almeida, Catarina R

    2017-09-01

    The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least three cell types that play a role in the host response upon scaffold implantation. With this system it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, Mesenchymal Stromal/Stem Cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not PLA. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of pro-regenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome. This article is protected by copyright. All rights reserved.

  17. Activated adult microglia influence retinal progenitor cell proliferation and differentiation toward recoverin-expressing neuron-like cells in a co-culture model.

    PubMed

    Xu, Yunhe; Balasubramaniam, Balini; Copland, David A; Liu, Jian; Armitage, M John; Dick, Andrew D

    2015-07-01

    Microglia contribute to immune homeostasis of the retina, and thus act as a potential regulator determining successful repair or retinal stem cell transplantation. We investigated the interaction between human microglia and retinal progenitor cells in cell co-culture to further our exploration on developing a new therapeutic strategy for retinal degeneration. Microglia and retinal progenitor cultures were developed using CD11b(+) and CD133(+), respectively, from adult donor retina. Microglia activation was developed using interferon-gamma and lipopolysaccharide. Retinal progenitor differentiation was analysed in co-culture with or without microglial activation. Retinal progenitor proliferation was analysed in presence of conditioned medium from activated microglia. Phenotype and function of adult human retinal cell cultures were examined using cell morphology, immunohistochemistry and real-time PCR. By morphology, neuron-like cells generated in co-culture expressed photoreceptor marker recoverin. Neurospheres derived from retinal progenitor cells showed reduced growth in the presence of conditioned medium from activated microglia. Delayed retinal progenitor cell migration and reduced cellular differentiation was observed in co-cultures with activated microglia. In independent experiments, activated microglia showed enhanced mRNA expression of CXCL10, IL-27, IL-6, and TNF-alpha compared to controls. Adult human retina retains retinal progenitors or potential to reprogram cells to then proliferate and differentiate into neuron-like cells in vitro. Human microglia support retinal progenitor differentiation into neuron-like cells, but such capacity is altered following microglial activation. Modulating microglia activity is a potential approach to promote retinal repair and facilitate success of stem-cell transplantation.

  18. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    SciTech Connect

    Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  19. Pulmonary macrophages activity in CO intoxication.

    PubMed

    Pieri, Maria; Giugliano, Pasquale; Vacchiano, Giuseppe

    2016-02-01

    The presence of macrophages and their activation on the pulmonary tissues of 21 subjects deceased after CO intoxication has been studied. A notable number of activated macrophages, especially in the interstitial level, have been evidenced, and such phenomenon supports the hypothesis of a possible association between CO intoxication and pulmonary macrophages activity. The highlighted association could be mediated by changes of the surfactant, by impairing of mitochondrial respiration and by release of pro-inflammatory cytokines. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  20. Macrophage activation in human diseases.

    PubMed

    Schultze, Joachim L; Schmieder, Astrid; Goerdt, S

    2015-08-01

    It is becoming increasingly accepted that macrophages play a crucial role in many diseases associated with chronic inflammation, including atherosclerosis, obesity, diabetes, cancer, skin diseases, and even neurodegenerative diseases. It is therefore not surprising that macrophages in human diseases have gained significant interest during the last years. Molecular analysis combined with more sophisticated murine disease models and the application of genome-wide technologies has resulted in a much better understanding of the role of macrophages in human disease. We highlight important gain of knowledge during the last years for tumor-associated macrophages, and for macrophages in atherosclerosis, obesity and wound healing. Albeit these exciting findings certainly pave the way to novel diagnostics and therapeutics, several hurdles still need to be overcome. We propose a general outline for future research and development in disease-related macrophage biology based on integrating (1) genome-wide technologies, (2) direct human sampling, and (3) a dedicated use of in vivo model systems. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Fluoxetine and its active metabolite norfluoxetine disrupt estrogen synthesis in a co-culture model of the feto-placental unit.

    PubMed

    Hudon Thibeault, Andrée-Anne; Laurent, Laetitia; Vo Duy, Sung; Sauvé, Sébastien; Caron, Patrick; Guillemette, Chantal; Sanderson, J Thomas; Vaillancourt, Cathy

    2017-02-15

    The effects of fluoxetine, one of the most prescribed selective serotonin-reuptake inhibitors (SSRIs) during pregnancy, and its active metabolite norfluoxetine were studied on placental aromatase (CYP19) and feto-placental steroidogenesis. Fluoxetine did not alter estrogen secretion in co-culture of fetal-like adrenocortical (H295R) and trophoblast-like (BeWo) cells used as a model of the feto-placental unit, although it induced CYP19 activity, apparently mediated by the serotonin (5-HT)2A receptor/PKC signaling pathway. Norfluoxetine decreased estrogen secretion in the feto-placental co-culture and competitively inhibited catalytic CYP19 activity in BeWo cells. Decreased serotonin transporter (SERT) activity in the co-culture was comparable to 17β-estradiol treatment of BeWo cells. This work shows that the complex interaction of fluoxetine and norfluoxetine with placental estrogen production, involves 5-HT-dependent and -independent mechanisms. Considering the crucial role of estrogens during pregnancy, our results raise concern about the impact of SSRI treatment on placental function and fetal health. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. PEDF increases the tumoricidal activity of macrophages towards prostate cancer cells in vitro

    PubMed Central

    Nelius, Thomas; de Riese, Werner; Volpert, Olga V.

    2017-01-01

    Background Although inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment in vitro, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis. Methods RAW 264.7 and THP-1 cells, and BMDMs were grown in vitro as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry. Results We found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects

  3. Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes.

    PubMed

    Motlík, J; Sutovský, P; Kalous, J; Kubelka, M; Moos, J; Schultz, R M

    1996-08-01

    Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8 h of initial culture) or metaphase I (12 h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8 h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6 h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions

  4. Glioma sensitive or chemoresistant to temozolomide differentially modulate macrophage protumor activities.

    PubMed

    Azambuja, Juliana H; da Silveira, Elita F; de Carvalho, Taíse R; Oliveira, Pathise S; Pacheco, Simone; do Couto, Carlus T; Beira, Fátima T; Stefanello, Francieli M; Spanevello, Rosélia M; Braganhol, Elizandra

    2017-11-01

    Glioblastomas are the most devastating brain tumor characterized by chemoresistance development and poor prognosis. Macrophages are a component of tumor microenvironment related to glioma malignancy. The relation among inflammation, innate immunity and cancer is accepted; however, molecular and cellular mechanisms mediating this relation and chemoresistance remain unresolved. Here we evaluated whether glioma sensitive or resistant to temozolomide (TMZ) modulate macrophage polarization and inflammatory pathways associated. The impact of glioma-macrophage crosstalk on glioma proliferation was also investigated. GL261 glioma chemoresistance was developed by exposing cells to increasing TMZ concentrations over a period of 6months. Mouse peritoneal macrophages were exposed to glioma-conditioned medium or co-cultured directly with glioma sensitive (GL) or chemoresistant (GLTMZ). Macrophage polarization, in vitro and in vivo glioma proliferation, redox parameters, ectonucleotidase activity and ATP cytotoxicity were performed. GLTMZ cells were more effective than GL in induce M2-like macrophage polarization and in promote a strong immunosuppressive environment characterized by high IL-10 release and increased antioxidant potential, which may contribute to glioma chemoresistance and proliferation. Interestingly, macrophage-GLTMZ crosstalk enhanced in vitro and in vivo proliferation of chemoresistant cells, decreased ectonucleotidase activities, which was followed by increased macrophage sensitivity to ATP induced death. Results suggest a differential macrophage modulation by GLTMZ cells, which may favor the maintenance of immunosuppressive tumor microenvironment and glioma proliferation. The induction of immunosuppressive environment and macrophage education by chemoresistant gliomas may be important for tumor recovery after chemotherapy and could be considered to overcome chemoresistance development. Copyright © 2017. Published by Elsevier B.V.

  5. The Many Alternative Faces of Macrophage Activation.

    PubMed

    Hume, David A

    2015-01-01

    Monocytes and macrophages provide the first line of defense against pathogens. They also initiate acquired immunity by processing and presenting antigens and provide the downstream effector functions. Analysis of large gene expression datasets from multiple cells and tissues reveals sets of genes that are co-regulated with the transcription factors that regulate them. In macrophages, the gene clusters include lineage-specific genes, interferon-responsive genes, early inflammatory genes, and genes required for endocytosis and lysosome function. Macrophages enter tissues and alter their function to deal with a wide range of challenges related to development and organogenesis, tissue injury, malignancy, sterile, or pathogenic inflammatory stimuli. These stimuli alter the gene expression to produce "activated macrophages" that are better equipped to eliminate the cause of their influx and to restore homeostasis. Activation or polarization states of macrophages have been classified as "classical" and "alternative" or M1 and M2. These proposed states of cells are not supported by large-scale transcriptomic data, including macrophage-associated signatures from large cancer tissue datasets, where the supposed markers do not correlate with other. Individual macrophage cells differ markedly from each other, and change their functions in response to doses and combinations of agonists and time. The most studied macrophage activation response is the transcriptional cascade initiated by the TLR4 agonist lipopolysaccharide. This response is reviewed herein. The network topology is conserved across species, but genes within the transcriptional network evolve rapidly and differ between mouse and human. There is also considerable divergence in the sets of target genes between mouse strains, between individuals, and in other species such as pigs. The deluge of complex information related to macrophage activation can be accessed with new analytical tools and new databases that provide

  6. CP-25 attenuates the inflammatory response of fibroblast-like synoviocytes co-cultured with BAFF-activated CD4(+) T cells.

    PubMed

    Jia, Xiaoyi; Wei, Fang; Sun, Xiaojing; Chang, Yan; Xu, Shu; Yang, Xuezhi; Wang, Chun; Wei, Wei

    2016-08-02

    Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells. The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells. The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability

  7. In vitro antagonistic activity evaluation of Lactic Acid Bacteria (LAB) combined with cellulase enzyme against campylobacter jejuni growth in co-culture.

    PubMed

    Dubois-Dauphin, Robin; Sabrina, Vandeplas; Isabelle, Didderen; Christopher, Marcq; Andre, Thewis; Philippe, Thonart

    2011-01-01

    The antibacterial effects of nine lactic acid bacteria (LAB) against Campylobacter jejuni were investigated by using agar gel diffusion and co-culture assays. Some differences were recorded between the inhibition effects measured with these two methods. Only two LAB, Lb. pentosus CWBI B78 and E. faecium THT, exhibited a clear anti- Campylobacter activity in co-culture assay with dehydrated poultry excreta mixed with ground straw (DPE/GS) as the only growth substrate source. It was observed that the supplementation of such medium with a cellulase A complex (Beldem S.A.) enhanced the antimicrobial effect of both LAB strains. The co-culture medium acidification and the C. jejuni were positively correlated with the cellulase A concentration. The antibacterial effect was characterized by the lactic acid production from the homofermentative E. faecium THT and the lactic and acetic acids production from the heterofermentative Lb. pentosus CWBI B78. The antagonistic properties of LAB strains and enzyme combination could be used in strategies aiming at the reduction of Campylobacter prevalence in the poultry production chain and consequently the risk of human infection.

  8. The Many Alternative Faces of Macrophage Activation

    PubMed Central

    Hume, David A.

    2015-01-01

    Monocytes and macrophages provide the first line of defense against pathogens. They also initiate acquired immunity by processing and presenting antigens and provide the downstream effector functions. Analysis of large gene expression datasets from multiple cells and tissues reveals sets of genes that are co-regulated with the transcription factors that regulate them. In macrophages, the gene clusters include lineage-specific genes, interferon-responsive genes, early inflammatory genes, and genes required for endocytosis and lysosome function. Macrophages enter tissues and alter their function to deal with a wide range of challenges related to development and organogenesis, tissue injury, malignancy, sterile, or pathogenic inflammatory stimuli. These stimuli alter the gene expression to produce “activated macrophages” that are better equipped to eliminate the cause of their influx and to restore homeostasis. Activation or polarization states of macrophages have been classified as “classical” and “alternative” or M1 and M2. These proposed states of cells are not supported by large-scale transcriptomic data, including macrophage-associated signatures from large cancer tissue datasets, where the supposed markers do not correlate with other. Individual macrophage cells differ markedly from each other, and change their functions in response to doses and combinations of agonists and time. The most studied macrophage activation response is the transcriptional cascade initiated by the TLR4 agonist lipopolysaccharide. This response is reviewed herein. The network topology is conserved across species, but genes within the transcriptional network evolve rapidly and differ between mouse and human. There is also considerable divergence in the sets of target genes between mouse strains, between individuals, and in other species such as pigs. The deluge of complex information related to macrophage activation can be accessed with new analytical tools and new databases

  9. Pro-inflammatory Macrophages suppress PPARγ activity in Adipocytes via S-nitrosylation.

    PubMed

    Yin, Ruiying; Fang, Li; Li, Yingjia; Xue, Peng; Li, Yazi; Guan, Youfei; Chang, Yongsheng; Chen, Chang; Wang, Nanping

    2015-12-01

    Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor and plays an essential role in insulin signaling. Macrophage infiltration into adipose tissue is a character of metabolic inflammation and closely related to insulin resistance in type 2 diabetes. The mechanism by which pro-inflammatory macrophages cause insulin resistance remains to be elucidated. Here we showed that co-culture with macrophages significantly suppressed the transcriptional activity of PPARγ on its target genes in 3T3-L1 preadipocytes and diabetic primary adipocytes, depending on inducible nitric oxide synthase (iNOS). We further showed that PPARγ underwent S-nitrosylation in response to nitrosative stress. Mass-spectrometry and site-directed mutagenesis revealed that S-nitrosylation at cysteine 168 was responsible for the impairment of PPARγ function. Extended exposure to NO instigated the proteasome-dependent degradation of PPARγ. Consistently, in vivo evidence revealed an association of the decreased PPARγ protein level with increased macrophage infiltration in visceral adipose tissue (VAT) of obese diabetic db/db mice. Together, our results demonstrated that pro-inflammatory macrophages suppressed PPARγ activity in adipocytes via S-nitrosylation, suggesting a novel mechanism linking metabolic inflammation with insulin resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. [EFFECT OF PEPTIDE SEMAX ON SYNAPTIC ACTIVITY AND SHORT-TERM PLASTICITY OF GLUTAMATERGIC SYNAPSES OF CO-CULTURED DORSAL ROOT GANGLION AND DORSAL HORN NEURONS].

    PubMed

    Shypshyna, M S; Veselovsky, N S; Myasoedov, N F; Shram, S I; Fedulova, S A

    2015-01-01

    The influence of long-term culturing (12 days in vitro) of dorsal root ganglion (DRG) and dorsal horn (DH) neurons with peptide Semax on the level of synaptic activity at co-cultures, as well as short-term plasticity in sensory synapses were studied. It has been shown that neuronal culturing with peptide at concentrations of 10 and 100 µM led to increasing the frequency of spontaneous glutamatergic postsynaptic currents in DH neurons to 71.7 ± 1.8% and 93.9 ± 3.1% (n = 6; P < 0.001); Semax has a not significant effect on the amplitude and frequency of miniature glutamatergic currents, but causes an increase of the amplitudes of spontaneous postsynaptic currents, as well as elevates the quantum content. The data show the increase of multivesicular glutamate release efficiency in neural networks of co-cultures following incubation with the peptide. Also Semax (10 and 100 µM) induces changes of the basic parameters of short-term plasticity in sensory synapses: (1) increasing the paired-pulse ratio from 0.53 ± 0.028 (n = 8) to 0.91 ± 0.072 (n = 6, P < 0.01) and 0.95 ± 0.026 (n = 7; P < 0.001); (2) reducing the ratio of the coefficients of variation (CV2/ CV1) from 1.49 ± 0.11 (n = 8) to 1.02 ± 0.09 (n = 6; P < 0.05) and 1.11 ± 0.13 (n = 7; P < 0.0) respectively. The results indicate a stimulating effect of Semax on the activity of glutamatergic synapses in neural networks of co-cultures, as well as the ability of the peptide to effectively modulate the short-term plasticity in sensory synapses.

  11. HCV dsRNA-Activated Macrophages Inhibit HCV Replication in Hepatocytes

    PubMed Central

    Wang, Yizhong; Li, Jieliang; Wang, Xu; Zhou, Yu; Zhang, Ting; Ho, Wenzhe

    2015-01-01

    Background: Macrophages play critical roles in innate immune response in the liver. Whether macrophages participate in liver innate immunity against HCV replication is poorly understood Objectives: The aim of this study was to investigate the role of macrophages in liver innate immunity against HCV replication. Materials and Methods: Freshly isolated monocytes were purified from peripheral blood of healthy adult donors. Macrophages refer to 7-day-cultured monocytes in vitro. A hepatoma cell line (Huh7) was infected with HCV JFH-1 to generate in vitro HCV infectious system. RT-PCR was used to determine HCV RNA and mRNA levels of genes expression. ELISA was used to measure the protein level of interferon-α (IFN-α) and western blot was used to determine protein expression level of Toll-like receptor 3 (TLR3). Results: HCV dsRNA induced the expression of type I IFN (IFN-α/β) in monocyte-derived macrophages. HCV dsRNA also induced the expression of TLR3 and IFN regulatory factor-7 (IRF-7), the key regulators of the IFN signaling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with macrophages activated with HCV dsRNA or incubated in media conditioned with supernatant (SN) from HCV dsRNA-activated macrophages, HCV replication was significantly suppressed. This macrophage SN action on HCV inhibition was mediated through type I IFN, which was evidenced by the observation that antibody to type I IFN receptor could neutralize the macrophages-mediated anti-HCV effect. The role of type I IFN in macrophages-mediated anti-HCV activity is further supported by the observation that HCV dsRNA-activated macrophages SN treatment induced the expression of several IFN-stimulated genes (ISGs), ISG15, ISG56, OAS-1, OAS-2, MxA and Viperin in HCV-infected Huh7 cells. Conclusions: Macrophages may play an important role in liver innate immunity against HCV replication through a type I IFN-dependent mechanism. PMID:26322111

  12. Dietary n-3 PUFA affect TcR-mediated activation of purified murine T cells and accessory cell function in co-cultures

    PubMed Central

    CHAPKIN, R S; ARRINGTON, J L; APANASOVICH, T V; CARROLL, R J; MCMURRAY, D N

    2002-01-01

    Diets enriched in n-3 polyunsaturated fatty acids (PUFA) suppress several functions of murine splenic T cells by acting directly on the T cells and/or indirectly on accessory cells. In this study, the relative contribution of highly purified populations of the two cell types to the dietary suppression of T cell function was examined. Mice were fed diets containing different levels of n-3 PUFA; safflower oil (SAF; control containing no n-3 PUFA), fish oil (FO) at 2% and 4%, or 1% purified docosahexaenoic acid (DHA) for 2 weeks. Purified (>90%) T cells were obtained from the spleen, and accessory cells (>95% adherent, esterase-positive) were obtained by peritoneal lavage. Purified T cells or accessory cells from each diet group were co-cultured with the alternative cell type from every other diet group, yielding a total of 16 different co-culture combinations. The T cells were stimulated with either concanavalin A (ConA) or antibodies to the T cell receptor (TcR)/CD3 complex and the costimulatory molecule CD28 (αCD3/αCD28), and proliferation was measured after four days. Suppression of T cell proliferation in the co-cultures was dependent upon the dose of dietary n-3 PUFA fed to mice from which the T cells were derived, irrespective of the dietary treatment of accessory cell donors. The greatest dietary effect was seen in mice consuming the DHA diet (P = 0·034 in the anova; P = 0·0053 in the Trend Test), and was observed with direct stimulation of the T cell receptor and CD28 costimulatory ligand, but not with ConA. A significant dietary effect was also contributed accessory cells (P = 0·033 in the Trend Test). We conclude that dietary n-3 PUFA affect TcR-mediated by T cell activation by both direct and indirect (accessory cell) mechanisms. PMID:12296847

  13. Antibacterial activity of polihexanide formulations in a co-culture of HaCaT keratinocytes and Staphylococcus aureus and at different pH levels.

    PubMed

    Wiegand, Cornelia; Eberlein, Thomas; Andriessen, Anneke

    2017-05-01

    Complex stalled wounds feature an alkaline milieu that favors tissue destruction and microbial growth. The presence of bacteria in turn perpetuates the inflammatory response. However, only limited knowledge exists of pH dependency on the antibacterial efficacy of polyhexamethylene biguanide (PHMB) or the influence of surfactants or delivery vehicle used in antiseptic formulations. So far, PHMB alone has been shown to protect the keratinocytes from bacterial damage in such a co-culture system as well as exhibiting increased antimicrobial activity at higher pH values. Here, the interaction of PHMB with the surfactants macrogolum and undecylenamidopropyl betaine that are most commonly used as additives in antiseptics and rinsing solutions such as Lavasept and Prontosan has been explored in addition to the PHMB-containing biocellulose dressing Suprasorb X + PHMB. Undecylenamidopropyl betaine was found to lower the antimicrobial activity of polihexanide in the co-culture system, while macrogolum and the biocellulose increased polihexanide efficiency to reduce Staphylococcus aureus especially in the presence of serum. The increasing antibacterial efficacy of PHMB with rising pH was not altered by undecylenamidopropyl betaine, macrogolum, or the biocellulose. The results suggest that application of PHMB with macrogolum or by delivery through a biocellulose dressing might be advantageous for management of wound infections. © 2017 by the Wound Healing Society.

  14. Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma.

    PubMed

    Higashi-Kuwata, Nobuyo; Makino, Takamitsu; Inoue, Yuji; Takeya, Motohiro; Ihn, Hironobu

    2009-08-01

    Localized scleroderma is a connective tissue disorder that is limited to the skin and subcutaneous tissue. Macrophages have been reported to be particularly activated in patients with skin disease including systemic sclerosis and are potentially important sources for fibrosis-inducing cytokines, such as transforming growth factor beta. To clarify the features of immunohistochemical characterization of the immune cell infiltrates in localized scleroderma focusing on macrophages, skin biopsy specimens were analysed by immunohistochemistry. The number of cells stained with monoclonal antibodies, CD68, CD163 and CD204, was calculated. An evident macrophage infiltrate and increased number of alternatively activated macrophages (M2 macrophages) in their fibrotic areas were observed along with their severity of inflammation. This study revealed that alternatively activated macrophages (M2 macrophages) may be a potential source of fibrosis-inducing cytokines in localized scleroderma, and may play a crucial role in the pathogenesis of localized scleroderma.

  15. Measurement of Macrophage Activation by Chemiluminescence.

    DTIC Science & Technology

    1986-05-01

    cytotoxicity and oxidative metabolism including those reactions responsible for chemiluminescence (11-13). This activation has been correlated with an...order to assay this event, enhancers of chemiluminescence such as lucigenin and luminol are required to amplify the reaction . Enhancement of CL by...Maleic vinyl ether activation of murine *. macrophages against lung metastasizing tumors. Cancer Res 41:3901-3906 1981 4. Fidler I.J., Sone, S., Fogler

  16. Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

    PubMed

    Choi, Young-Ho; Shin, Taeyoung; Love, Charley C; Johnson, Cindy; Varner, Dickson D; Westhusin, Mark E; Hinrichs, Katrin

    2002-02-01

    We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase

  17. Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages.

    PubMed Central

    Brummer, E; Hanson, L H; Restrepo, A; Stevens, D A

    1989-01-01

    The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis. Images PMID:2744848

  18. NMAAP1 Expressed in BCG-Activated Macrophage Promotes M1 Macrophage Polarization.

    PubMed

    Liu, Qihui; Tian, Yuan; Zhao, Xiangfeng; Jing, Haifeng; Xie, Qi; Li, Peng; Li, Dong; Yan, Dongmei; Zhu, Xun

    2015-10-01

    Macrophages are divided into two subpopulations: classically activated macrophages (M1) and alternatively activated macrophages (M2). BCG (Bacilli Calmette-GuC)rin) activates disabled naC/ve macrophages to M1 macrophages, which act as inflammatory, microbicidal and tumoricidal cells through cell-cell contact and/or the release of soluble factors. Various transcription factors and signaling pathways are involved in the regulation of macrophage activation and polarization. We discovered that BCG-activated macrophages (BAM) expressed a new molecule, and we named it Novel Macrophage Activated Associated Protein 1 (NMAAP1). The current study found that the overexpression of NMAAP1 in macrophages results in M1 polarization with increased expression levels of M1 genes, such as inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-N1), Interleukin 6 (IL-6), Interleukin 12 (IL-12), Monocyte chemoattractant protein-1 (MCP-1) and Interleukin-1 beta (IL-1N2), and decreased expression of some M2 genes, such as Kruppel-like factor 4 (KLF4) and suppressor of cytokine signaling 1 (SOCS1), but not other M2 genes, including arginase-1 (Arg-1), Interleukin (IL-10), transforming growth factor beta (TGF-N2) and found in inflammatory zone 1 (Fizz1). Moreover, NMAAP1 overexpression in the RAW264.7 cell line increased cytotoxicity against MCA207 tumor cells, which depends on increased inflammatory cytokines rather than cell-cell contact. NMAAP1 also substantially enhanced the phagocytic ability of macrophages, which implies that NMAAP1 promoted macrophage adhesive and clearance activities. Our results indicate that NMAAP1 is an essential molecule that modulates macrophages phenotype and plays an important role in macrophage tumoricidal functions.

  19. Structure revision and cytotoxic activity of marinamide and its methyl ester, novel alkaloids produced by co-cultures of two marine-derived mangrove endophytic fungi.

    PubMed

    Zhu, Feng; Chen, Guangying; Wu, Jingshu; Pan, Jiahui

    2013-01-01

    Marinamide (1) and its methyl ester (2) have been previously reported as pyrrolyl 1-isoquinolone alkaloids, which were produced by co-cultures of two marine-derived mangrove endophytic fungi from the South China Sea coast. Recrystallisation of methyl marinamide (2) from pyridine forms the known pesticide, quinolactacide (3). Treatment of 3 with methyl iodide to afford N-methyl quinolactacide (4) was identified by X-ray crystallography. Thus, the structures of 1 and 2 were revised from the previously reported pyrrolyl 1-isoquinolone structures to pyrrolyl 4-quinolone analogues. In the MTT assays, both 1 and 2 exhibited potent cytotoxic activity against HepG2, 95-D, MGC832 and HeLa tumour cell lines.

  20. Macrophage activation and polarization: nomenclature and experimental guidelines.

    PubMed

    Murray, Peter J; Allen, Judith E; Biswas, Subhra K; Fisher, Edward A; Gilroy, Derek W; Goerdt, Sergij; Gordon, Siamon; Hamilton, John A; Ivashkiv, Lionel B; Lawrence, Toby; Locati, Massimo; Mantovani, Alberto; Martinez, Fernando O; Mege, Jean-Louis; Mosser, David M; Natoli, Gioacchino; Saeij, Jeroen P; Schultze, Joachim L; Shirey, Kari Ann; Sica, Antonio; Suttles, Jill; Udalova, Irina; van Ginderachter, Jo A; Vogel, Stefanie N; Wynn, Thomas A

    2014-07-17

    Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Modulation of macrophage activation by prostaglandins

    PubMed Central

    Carnuccio, R.; D'Acquisto, F.; Rosa, M. Di

    1996-01-01

    The effect of prostaglandtn E2, iloprost and cAMP on both nitric oxide and tumour necrosis factor-α release in J774 macrophages has been studied. Both prostaglandin E2 and iloprost inhibited, in a concentration-dependent fashion, the lipopolysaccharide-induced generation of nitric oxide and tumour necrosis factor-α. The inhibitory effect of these prostanoids seems to be mediated by an increase of the second messenger cAMP since it was mimicked by dibutyryl cAMP and potentiated by the selective type IV phosphodiesterase inhibitor RO-20-1724. Our results suggest that the inhibition of nitric oxide release by prostaglandin E2 and iloprost in lipopolysaccharide-activated J774 macrophages may be secondary to the inhibition of tumour necrosis factor-α generation, which in turn is likely to be mediated by cAMP. PMID:18475691

  2. The effect of mineral trioxide aggregate on phagocytic activity and production of reactive oxygen, nitrogen species and arginase activity by M1 and M2 macrophages.

    PubMed

    Rezende, T M B; Vieira, L Q; Cardoso, F P; Oliveira, R R; de Oliveira Mendes, S T; Jorge, M L R; Ribeiro Sobrinho, A P

    2007-08-01

    To assess the influence of co-culture with mineral trioxide aggregate (MTA) on phagocytosis and the production of reactive oxygen intermediates (ROI) and nitrogen (NO) species and the arginase activity by M1 and M2 peritoneal macrophages. Cellular viability, adherence and phagocytosis of Saccharomyces boulardii were assayed in the presence of MTA. Macrophages were stimulated with zymosan for ROI assays and with Fusobacterium nucleatum and Peptostreptococcus anaerobius and IFN-gamma for NO production and arginase activity, when in contact with capillaries containing MTA. Data were analysed by T, anova, Kruskall-Wallis and Mann-Whitney tests. M2 macrophages displayed greater cellular viability in polypropylene tubes, greater ability to ingest yeast and smaller production of ROI and higher arginase activity when compared with M1 macrophages. Both macrophages, M1 and M2, presented similar cell adherence and NO production. The addition of bacterial preparations to macrophages interfered with NO and arginase productions. MTA did not interfere with any of the parameters measured. Phagocytosis and the ability of the two macrophage subtypes to eliminate microbes were not affected by MTA.

  3. Immune Activity of BCG Infected Mouse Macrophages Treated with a Novel Recombinant Mouse Lactoferrin.

    PubMed

    O'Shea, Kelly M; Hwang, Shen-An; Actor, Jeffrey K

    2015-01-01

    Lactoferrin has been investigated for its adjuvant action to boost the BCG vaccine. Previous studies demonstrated that lactoferrin (LF) enhanced efficacy of the Bacillus Calmette-Guérin (BCG) vaccine to protect mice against the virulent Erdman Mycobacterium tuberculosis challenge. The studies here investigate the hypothesis that a novel CHO-derived recombinant mouse LF can modify cytokine production and antigen presentation molecules on macrophages. The mouse LF (rmLF) was examined for effects on bone marrow derived macrophage (BMM) activities when cultured with BCG. Comparisons were made to CHO-derived recombinant human LF (rhLF). Inflammatory cytokine responses were investigated, as were antigen presentation and associated co-stimulatory molecules. Cytokine responses were subsequently measured when these cells were co-cultured with naïve or BCG sensitized CD4+ lymphocytes. While overall responses were similar between mouse, human, and bovine forms, the homologous rmLF treated infected BMMs showed unique activation patterns of cytokine production. These results indicate that species-specific LF can have different effects on mouse macrophages exposed to BCG, thus potentially affecting adjuvant activity when used in models of vaccination in mice.

  4. Direct imaging of macrophage activation during PDT treatment

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Zhou, Feifan; Chen, Wei R.; Xing, Da

    2012-03-01

    Mounting evidence describes a more complex progress of macrophage activation during photodynamic therapy (PDT), which performing distinct immunological functions and different physiologies on surrounding cells and tissues. Macrophage-targeted PDT has been applied in the selective killing of cells involved in inflammation and tumor. We have previously shown that PDT-mediated tumor cells apoptosis can induce a higher level immune response than necrosis, and enhance the macrophage activation. However, the molecular mechanism of macrophage activation during PDT-induced apoptotic cells (AC) still unclear. Here, we use confocal microscopy to image the phagocytosis of tumor cells by macrophages. We also observed that PDT-treated AC can activate Toll-like receptors (TLRs) which are present on macrophages surface. Besides, the increase in nitric oxide (NO) formation in macrophages was detected in real time by a laser scanning microscopy. This study provided more details for understanding the molecular mechanism of the immune response induced by PDT-treated AC.

  5. Direct imaging of macrophage activation during PDT treatment

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Zhou, Feifan; Chen, Wei R.; Xing, Da

    2011-11-01

    Mounting evidence describes a more complex progress of macrophage activation during photodynamic therapy (PDT), which performing distinct immunological functions and different physiologies on surrounding cells and tissues. Macrophage-targeted PDT has been applied in the selective killing of cells involved in inflammation and tumor. We have previously shown that PDT-mediated tumor cells apoptosis can induce a higher level immune response than necrosis, and enhance the macrophage activation. However, the molecular mechanism of macrophage activation during PDT-induced apoptotic cells (AC) still unclear. Here, we use confocal microscopy to image the phagocytosis of tumor cells by macrophages. We also observed that PDT-treated AC can activate Toll-like receptors (TLRs) which are present on macrophages surface. Besides, the increase in nitric oxide (NO) formation in macrophages was detected in real time by a laser scanning microscopy. This study provided more details for understanding the molecular mechanism of the immune response induced by PDT-treated AC.

  6. The macrophage in HIV-1 infection: from activation to deactivation?

    PubMed

    Herbein, Georges; Varin, Audrey

    2010-04-09

    Macrophages play a crucial role in innate and adaptative immunity in response to microorganisms and are an important cellular target during HIV-1 infection. Recently, the heterogeneity of the macrophage population has been highlighted. Classically activated or type 1 macrophages (M1) induced in particular by IFN-gamma display a pro-inflammatory profile. The alternatively activated or type 2 macrophages (M2) induced by Th-2 cytokines, such as IL-4 and IL-13 express anti-inflammatory and tissue repair properties. Finally IL-10 has been described as the prototypic cytokine involved in the deactivation of macrophages (dM). Since the capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines, this review shows how modulation of macrophage activation by cytokines impacts the capacity to support productive HIV-1 infection. Based on the activation status of macrophages we propose a model starting with M1 classically activated macrophages with accelerated formation of viral reservoirs in a context of Th1 and proinflammatory cytokines. Then IL-4/IL-13 alternatively activated M2 macrophages will enter into the game that will stop the expansion of the HIV-1 reservoir. Finally IL-10 deactivation of macrophages will lead to immune failure observed at the very late stages of the HIV-1 disease.

  7. THE ENHANCEMENT OF MACROPHAGE BACTERIOSTASIS BY PRODUCTS OF ACTIVATED LYMPHOCYTES

    PubMed Central

    Fowles, Robert E.; Fajardo, Ileana M.; Leibowitch, Jacques L.; David, John R.

    1973-01-01

    It was reported previously that the incubation of normal guinea pig macrophages with partially purified products of activated lymphocytes resulted in altered macrophage function including increased cell adherence to culture vessels, spreading, phagocytosis, and glucose carbon-1 oxidation. Studies reported here demonstrate that such macrophages also exhibit enhanced bacteriostasis. Lymphocytes were stimulated with concanavalin A, the culture supernatant was chromatographed over Sephadex G-100 and the fraction of mol wt 25,000–55,000, rich in lymphocyte mediators, was cultured with normal guinea pig macrophages for 1–3 days. Macrophages incubated with fractions from unstimulated lymphocyte cultures served as controls. The resulting macrophage monolayers were infected with Listeria monocytogenes. Macrophages incubated with mediator-rich fractions exhibited 2- to 10-fold enhanced bacteriostasis compared to controls. Further studies indicate that this enhancement was attributable to intrinsic changes in the macrophages and not simply a consequence of the number of macrophages on the monolayers. The studies support the concept that macrophage bacteriostasis can be enhanced by lymphocyte mediators. However, macrophages, which have been preincubated directly with sensitive lymphocytes and antigen exhibit even greater bacteriostasis and sometimes bactericidal capacity, suggesting that either a labile lymphocyte factor or direct lymphocyte macrophage interaction may also be involved in bactericidal activity. PMID:4200649

  8. Human Mesenchymal stem cells program macrophage plasticity by altering their metabolic status via a PGE2-dependent mechanism

    PubMed Central

    Vasandan, Anoop Babu; Jahnavi, Sowmya; Shashank, Chandanala; Prasad, Priya; Kumar, Anujith; Prasanna, S. Jyothi

    2016-01-01

    Mesenchymal stem cells (MSCs) are speculated to act at macrophage-injury interfaces to mediate efficient repair. To explore this facet in-depth this study evaluates the influence of MSCs on human macrophages existing in distinct functional states. MSCs promoted macrophage differentiation, enhanced respiratory burst and potentiated microbicidal responses in naïve macrophages (Mφ). Functional attenuation of inflammatory M1 macrophages was associated with a concomitant shift towards alternatively activated M2 state in MSC-M1 co-cultures. In contrast, alternate macrophage (M2) activation was enhanced in MSC-M2 co-cultures. Elucidation of key macrophage metabolic programs in Mo/MSC, M1/MSC and M2/MSC co-cultures indicated changes in Glucose transporter1 (GLUT1 expression/glucose uptake, IDO1 protein/activity, SIRTUIN1 and alterations in AMPK and mTOR activity, reflecting MSC-instructed metabolic shifts. Inability of Cox2 knockdown MSCs to attenuate M1 macrophages and their inefficiency in instructing metabolic shifts in polarized macrophages establishes a key role for MSC-secreted PGE2 in manipulating macrophage metabolic status and plasticity. Functional significance of MSC-mediated macrophage activation shifts was further validated on human endothelial cells prone to M1 mediated injury. In conclusion, we propose a novel role for MSC secreted factors induced at the MSC-macrophage interface in re-educating macrophages by manipulating metabolic programs in differentially polarized macrophages. PMID:27910911

  9. Activated macrophages down-regulate expression of E-cadherin in hepatocellular carcinoma cells via NF-κB/Slug pathway.

    PubMed

    Wang, Xianteng; Wang, Hao; Li, Guosheng; Song, Yonghong; Wang, Shurong; Zhu, Faliang; Guo, Chun; Zhang, Lining; Shi, Yongyu

    2014-09-01

    Hepatocellular carcinomas are an aggressive malignancy mainly due to metastasis or postsurgical recurrence. Expression of E-cadherin is strongly reduced in Hepatocellular carcinoma (HCC) tissues, and its downregulation is connected to invasiveness and metastasis in hepatocellular carcinomas. The previous study showed that the supernatant from activated macrophages can downregulate the expression of E-cadherin in HCC cells. The partial known molecular mechanism is that tyrosine kinases c-Src- and EGFR phosphorylate β-catenin and E-cadherin leading to destabilization of E-cadherin/β-catenin complex. The aim of this study is to clarify other mechanism by which activated macrophages downregulate the expression of E-cadherin. We detect the expression of E-cadherin and macrophage infiltration in hepatocellular carcinoma tissues by double-staining immunohistochemistry and evaluate the relationship between macrophages and E-cadherin expression in hepatocellular carcinoma cells in vitro experiments. We found that reduced expression of E-cadherin was associated with macrophage infiltration along the border between the tumor nest and stroma in hepatocellular carcinoma tissues. Besides, protein expression of E-cadherin was significantly decreased in hepatocellular carcinoma cells co-cultured with macrophages derived from THP-1 cells. Consistently, mRNA expression of E-cadherin was also decreased in cancer cells co-cultured with THP-1-differentiated macrophages. Moreover, the downregulation of E-cadherin expression was companied by upregulation of Slug expression in cancer cells with conditional medium from THP-1-differentiated macrophage culture. The change in expression of E-cadherin and Slug was abrogated when NF-κB signaling pathway was blocked. All the findings suggested that macrophages contributed to the decreased expression of E-cadherin by NF-κB/Slug pathway in hepatocellular carcinomas.

  10. D-penicillamine-induced autoimmunity: relationship to macrophage activation.

    PubMed

    Li, Jinze; Uetrecht, Jack P

    2009-09-01

    Idiosyncratic drug reactions represent a serious health problem, and they remain unpredictable largely due to our limited understanding of the mechanisms involved. Penicillamine-induced autoimmunity in Brown Norway (BN) rats represents one model of an idiosyncratic reaction, and this drug can also cause autoimmune reactions in humans. We previously demonstrated that penicillamine binds to aldehydes on the surface of macrophages. There is evidence that an imine bond formed by aldehyde groups on macrophages and amine groups on T cells is one type of interaction between these two cells that is involved in the induction of an immune response. We proposed that the binding of penicillamine with aldehyde groups on macrophages could lead to their activation and in some patients could lead to autoimmunity. In this study, the transcriptome profile of spleen macrophages 6 h after penicillamine treatment was used to detect effects of penicillamine on macrophages with a focus on 20 genes known to be macrophage activation biomarkers. One biological consequence of macrophage activation was investigated by determining mRNA levels for IL-15 and IL-1 beta which are crucial for NK cell activation, as well as levels of mRNA for selected cytokines in spleen NK cells. Up-regulation of the macrophage activating cytokines, IFN-gamma and GM-CSF, and down-regulation of IL-13 indicated activation of NK cells, which suggests a positive feedback loop between macrophages and NK cells. Furthermore, treatment of a murine macrophage cell line, RAW264.7, with penicillamine increased the production of TNF-alpha, IL-6, and IL-23, providing additional evidence that penicillamine activates macrophages. Hydralazine and isoniazid cause a lupus-like syndrome in humans and also bind to aldehyde groups. These drugs were also found to activate RAW264.7 macrophages. Together, these data support the hypothesis that drugs that bind irreversibly with aldehydes lead to macrophage activation, which in some

  11. Polarization dictates iron handling by inflammatory and alternatively activated macrophages

    PubMed Central

    Corna, Gianfranca; Campana, Lara; Pignatti, Emanuele; Castiglioni, Alessandra; Tagliafico, Enrico; Bosurgi, Lidia; Campanella, Alessandro; Brunelli, Silvia; Manfredi, Angelo A.; Apostoli, Pietro; Silvestri, Laura; Camaschella, Clara; Rovere-Querini, Patrizia

    2010-01-01

    Background Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. Design and Methods Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. Results M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize – albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. Conclusions Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with

  12. Alternatively activated macrophages in infection and autoimmunity

    PubMed Central

    Fairweather, DeLisa; Cihakova, Daniela

    2009-01-01

    Macrophages are innate immune cells that play an important role in activation of the immune response and wound healing. Pathogens that require T helper-type 2 (Th2) responses for effective clearance, such as parasitic worms, are strong inducers of alternatively activated or M2 macrophages. However, infections such as bacteria and viruses that require Th1-type responses may induce M2 as a strategy to evade the immune system. M2 are particularly efficient at scavenging self tissues following injury through receptors like the mannose receptor and scavenger receptor-A. Thus, M2 may increase autoimmune disease by presenting self tissue to T cells. M2 may also exacerbate immune complex (IC)-mediated pathology and fibrosis, a hallmark of autoimmune disease in women, due to the release of profibrotic factors such as interleukin (IL)-1β, transforming growth factor-β, fibronectin and matrix metalloproteinases. We have found that M2 comprise anywhere from 30% to 70% of the infiltrate during acute viral or experimental autoimmune myocarditis, and shifts in M2 populations correlate with increased IC-deposition, fibrosis and chronic autoimmune pathology. Thus, women may be at an increased risk of M2-mediated autoimmunity due to estrogen’s ability to increase Th2 responses. PMID:19819674

  13. Functional modifications of macrophage activity after sublethal irradiation. [Toxoplasma gondii

    SciTech Connect

    Swartz, R.P.

    1982-01-01

    The modifications of macrophage activity following sublethal irradiation, both in vivo and in vitro, were studied using spreading and C3b-receptor-mediated ingestion assays. Nonelicited peritoneal washout cells were examined for changes in activity and selected population characteristics. The cells from irradiated mice were from a resident peritoneal population and not immigrating cells. The macrophage population showed enhanced activity early with a refractory period (24-48) when the macrophages were unresponsive to stimulation by irradiated lymphocytes. The enhanced activity was inversely dose dependent on macrophage. The lymphocytes showed a regulatory function(s) on the time post irradiation at which they were examined. Early lymphocytes exhibited the ability to enhance the activity of normal macrophages while lymphocytes removed 24 hours post irradiation could suppress the activity of already activated macrophages. The effect(s) of the various lymphocyte populations were reproduced with cell-free supernatants which was indicative of the production of lymphokines. Separation on nylon wool columns indicated that the activity resided primarily in the T-cell population of lymphocytes. In vitro irradiation indicated that stimulation of the lymphocytes is macrophage dependent. Additional work indicated that sublethally irradiated macrophages did not inhibit replication of the coccidian protozoon Toxoplasma gondii although they did show increased phagocytosis. Examination of the serum from whole body irradiated mice showed the presence of a postirradiation substance which enhanced the phagocytosis of normal macrophages. It was not present in the serum of normal mice and was not endotoxin.

  14. Modulation of macrophage activation and programming in immunity.

    PubMed

    Liu, Guangwei; Yang, Hui

    2013-03-01

    Macrophages are central mediators of the immune, contributing both to the initiation and the resolution of inflammation. The concept of macrophage activation and program has stimulated interest in its definition, and functional significance in homeostasis and diseases. It has been known that macrophages could be differently activated and programmed into different functional subtypes in response to different types of antigen stumuli or different kinds of cytokines present in the microenvironment and could thus profoundly influence immune responses, but little is known about the state and exact regulatory mechanism of macrophage activation and program from cell or molecular signaling level in immunity. In this review, we summarize the recent finding regarding the regulatory mechanism of macrophage activation and program toward M1 and M2, especially on M2 macrophages.

  15. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response.

    PubMed

    Haenseler, Walther; Sansom, Stephen N; Buchrieser, Julian; Newey, Sarah E; Moore, Craig S; Nicholls, Francesca J; Chintawar, Satyan; Schnell, Christian; Antel, Jack P; Allen, Nicholas D; Cader, M Zameel; Wade-Martins, Richard; James, William S; Cowley, Sally A

    2017-06-06

    Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Tributyltin chloride induced testicular toxicity by JNK and p38 activation, redox imbalance and cell death in sertoli-germ cell co-culture.

    PubMed

    Mitra, Sumonto; Srivastava, Ankit; Khandelwal, Shashi

    2013-12-06

    The widespread use of tributyltin (TBT) as biocides in antifouling paints and agricultural chemicals has led to environmental and marine pollution. Human exposure occurs mainly through TBT contaminated seafood and drinking water. It is a well known endocrine disruptor in mammals, but its molecular mechanism in testicular damage is largely unexplored. This study was therefore, designed to ascertain effects of tributyltin chloride (TBTC) on sertoli-germ cell co-culture in ex-vivo and in the testicular tissue in-vivo conditions. An initial Ca(2+) rise followed by ROS generation and glutathione depletion resulted in oxidative damage and cell death. We observed p38 and JNK phosphorylation, stress proteins (Nrf2, MT and GST) induction and mitochondrial depolarization leading to caspase-3 activation. Prevention of TBTC reduced cell survival and cell death by Ca(2+) inhibitors and free radical scavengers specify definitive role of Ca(2+) and ROS. Sertoli cells were found to be more severely affected which in turn can hamper germ cells functionality. TBTC exposure in-vivo resulted in increased tin content in the testis with enhanced Evans blue leakage into the testicular tissue indicating blood-testis barrier disruption. Tesmin levels were significantly diminished and histopathological studies revealed marked tissue damage. Our data collectively indicates the toxic manifestations of TBTC on the male reproductive system and the mechanisms involved.

  17. Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

    PubMed

    Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

    2017-08-01

    Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (<24 hours; OXVASC [Oxford Vascular Study]). Isolated EV fractions were subjected to untargeted proteomic analysis by liquid chromatography mass-spectrometry/mass-spectrometry and then applied to macrophages in culture to investigate inflammatory gene expression. EV number, but not size, is significantly increased in stroke patients when compared to age-matched controls. Proteomic analysis reveals an overall increase in acute phase proteins, including C-reactive protein. EV fractions applied to monocyte-differentiated macrophage cultures induced inflammatory gene expression. Together these data show that EVs from stroke patients are proinflammatory in nature and are capable of inducing inflammation in immune cells. © 2017 American Heart Association, Inc.

  18. Immunomodulation by Blastomyces dermatitidis: functional activity of murine peritoneal macrophages.

    PubMed Central

    McDaniel, L S; Cozad, G C

    1983-01-01

    Cell-mediated immunity plays the dominant role in the immune response of mice to Blastomyces dermatitidis infections. Since macrophages play an important role in cell-mediated immunity, the interactions between sensitized murine peritoneal macrophages and the yeast phase of B. dermatitidis were investigated. Scanning electron microscopy showed that the sensitized macrophages readily phagocytized B. dermatitidis yeast cells. In addition, there appeared to be activation of metabolic pathways within the sensitized macrophages, as indicated by increased chemiluminescence activity during phagocytosis. Sensitized macrophages were significantly better at controlling intracellular proliferation of the yeast cells when compared to nonsensitized cells. This was determined by disruption of macrophages and plating for viable yeasts. Scanning electron microscope observations offered further substantiation. Experiments with Candida albicans indicated that B. dermatitidis non-specifically activated macrophages. At 2 h postphagocytosis, 30% fewer C. albicans in B. dermatitidis-activated macrophages were able to form germ tubes. These studies demonstrated the multiple potential of activated macrophages with regard to their functional activity. Images PMID:6840859

  19. Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system.

    PubMed

    Wewering, Franziska; Jouy, Florent; Wissenbach, Dirk K; Gebauer, Scarlett; Blüher, Matthias; Gebhardt, Rolf; Pirow, Ralph; von Bergen, Martin; Kalkhof, Stefan; Luch, Andreas; Zellmer, Sebastian

    2017-02-01

    Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

  20. Human umbilical cord blood-stem cells direct macrophage polarization and block inflammasome activation to alleviate rheumatoid arthritis.

    PubMed

    Shin, Tae-Hoon; Kim, Hyung-Sik; Kang, Tae-Wook; Lee, Byung-Chul; Lee, Hwa-Yong; Kim, Yoon-Jin; Shin, Ji-Hee; Seo, Yoojin; Won Choi, Soon; Lee, Seunghee; Shin, Kichul; Seo, Kwang-Won; Kang, Kyung-Sun

    2016-12-22

    Rheumatoid arthritis (RA) is a long-lasting intractable autoimmune disorder, which has become a substantial public health problem. Despite widespread use of biologic drugs, there have been uncertainties in efficacy and long-term safety. Mesenchymal stem cells (MSCs) have been suggested as a promising alternative for the treatment of RA because of their immunomodulatory properties. However, the precise mechanisms of MSCs on RA-related immune cells are not fully elucidated. The aim of this study was to investigate the therapeutic potential of human umbilical cord blood-derived MSCs (hUCB-MSCs) as a new therapeutic strategy for patients with RA and to explore the mechanisms underlying hUCB-MSC-mediated immunomodulation. Mice with collagen-induced arthritis (CIA) were administered with hUCB-MSCs after the onset of disease, and therapeutic efficacy was assessed. Systemic delivery of hUCB-MSCs significantly ameliorated the severity of CIA to a similar extent observed in the etanercept-treated group. hUCB-MSCs exerted this therapeutic effect by regulating macrophage function. To verify the regulatory effects of hUCB-MSCs on macrophages, macrophages were co-cultured with hUCB-MSCs. The tumor necrosis factor (TNF)-α-mediated activation of cyclooxygenase-2 and TNF-stimulated gene/protein 6 in hUCB-MSCs polarized naive macrophages toward an M2 phenotype. In addition, hUCB-MSCs down-regulated the activation of nucleotide-binding domain and leucine-rich repeat pyrin 3 inflammasome via a paracrine loop of interleukin-1β signaling. These immune-balancing effects of hUCB-MSCs were reproducible in co-culture experiments using peripheral blood mononuclear cells from patients with active RA. hUCB-MSCs can simultaneously regulate multiple cytokine pathways in response to pro-inflammatory cytokines elevated in RA microenvironment, suggesting that treatment with hUCB-MSCs could be an attractive candidate for patients with treatment-refractory RA.

  1. Titanium particles that have undergone phagocytosis by macrophages lose the ability to activate other macrophages.

    PubMed

    Xing, Zhiqing; Schwab, Luciana P; Alley, Carie F; Hasty, Karen A; Smith, Richard A

    2008-04-01

    Titanium particles derived from the wear of the orthopaedic implant surfaces can activate macrophages to secrete cytokines and stimulate osteoclastic bone resorption, causing osteolysis around orthopaedic implants. However, what happens to the titanium particles after being phagocytosed by macrophages is not known. We prepared titanium particles (as received, clean, and LPS-coated), and exposed them to macrophages in culture. Free particles were washed away after 24 h and the intracellular particles were kept in culture for additional 48 h until being harvested by lysing the cells. Particles that had been cell treated or noncell treated were examined by scanning electronic microscopy to analyze the shape, size, and concentration of the particles. The cell treated and noncell treated particles were exposed to macrophages in culture with a particle to cell ratio of 300:1. After 18 h, the levels of TNF-alpha in culture medium and the viability of the cells were examined. Clean particles did not stimulate TNF-alpha secretion by macrophages, while LPS-coated particles dramatically increased that response. Phagocytosis by macrophages did not change the shape and size of the particles, but depleted the ability of the particles to stimulate TNF-alpha secretion by macrophages. This indicates that macrophages are capable of rendering titanium particles inactive without degrading the particles, possibly by altering the surface chemistry of the particles.

  2. Collagenase Production by Endotoxin-Activated Macrophages

    PubMed Central

    Wahl, Larry M.; Wahl, Sharon M.; Mergenhagen, Stephan E.; Martin, George R.

    1974-01-01

    Peritoneal exudate macrophages, when exposed to bacterial lipopolysaccharide in culture, were found to produce collagenase (EC 3.4.24.3). This enzyme was not detected in extracts of the macrophages or in media from nonstimulated macrophage cultures. Lipidcontaining fractions of the lipopolysaccharide, including a glycolipid from the rough mutant of Salmonella minnesota (R595) and lipid A, were potent stimulators of collagenase production. The lipid-free polysaccharide fraction had no effect. Cycloheximide prevented the production of collagenase by endotoxin-treated macrophages, suggesting that it was newly synthesized. Images PMID:4372628

  3. Exopolysaccharide from Trichoderma pseudokoningii induces macrophage activation.

    PubMed

    Wang, Guodong; Zhu, Lei; Yu, Bo; Chen, Ke; Liu, Bo; Liu, Jun; Qin, Guozheng; Liu, Chunyan; Liu, Huixia; Chen, Kaoshan

    2016-09-20

    In this study, we evaluated the immunomodulatory activity of an exopolysaccharide (EPS) derived from Trichoderma pseudokoningii and investigated the molecular mechanism of EPS-mediated activation of macrophages. Results revealed that EPS could significantly induce the production of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-1β and enhance phagocytic activity in RAW 264.7 cells. Immunofluorescence staining indicated that EPS promoted the nuclear translocation of nuclear factor (NF)-κB p65 subunit. Western blot analysis showed that EPS increased the expression of inducible nitric oxide synthase (iNOS) protein, the degradation of IκB-α and the phosphorylation of mitogen-activated protein kinases (MAPKs). Furthermore, pretreatment of RAW 264.7 cells with specific inhibitors of NF-κB and MAPKs significantly attenuated EPS-induced TNF-α and IL-1β production. EPS also induced the inhibition of cytokine secretion by special antibodies against Toll-like receptor-4 (TLR4) and Dectin-1. These data suggest that EPS from Trichoderma pseudokoningii activates RAW 264.7 cells through NF-κB and MAPKs signaling pathways via TLR4 and Dectin-1.

  4. Cellular and Molecular Mechanisms Underpinning Macrophage Activation during Remyelination

    PubMed Central

    Lloyd, Amy F.; Miron, Veronique E.

    2016-01-01

    Remyelination is an example of central nervous system (CNS) regeneration, whereby myelin is restored around demyelinated axons, re-establishing saltatory conduction and trophic/metabolic support. In progressive multiple sclerosis, remyelination is limited or fails altogether which is considered to contribute to axonal damage/loss and consequent disability. Macrophages have critical roles in both CNS damage and regeneration, such as remyelination. This diverse range in functions reflects the ability of macrophages to acquire tissue microenvironment-specific activation states. This activation is dynamically regulated during efficient regeneration, with a switch from pro-inflammatory to inflammation-resolution/pro-regenerative phenotypes. Although, some molecules and pathways have been implicated in the dynamic activation of macrophages, such as NFκB, the cellular and molecular mechanisms underpinning plasticity of macrophage activation are unclear. Identifying mechanisms regulating macrophage activation to pro-regenerative phenotypes may lead to novel therapeutic strategies to promote remyelination in multiple sclerosis. PMID:27446913

  5. CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.

    PubMed

    Kwon, Min Jung; Shin, Hae Young; Cui, Yuexian; Kim, Hyosil; Thi, Anh Hong Le; Choi, Jun Young; Kim, Eun Young; Hwang, Dong Hoon; Kim, Byung Gon

    2015-12-02

    CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regeneration-associated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

  6. Amphiregulin may be a new biomarker of classically activated macrophages.

    PubMed

    Meng, Chen; Liu, Guilin; Mu, Honglan; Zhou, Miaomiao; Zhang, Shihai; Xu, Younian

    2015-10-23

    Amphiregulin (Areg) participates in tissue repair and inflammation regulation. As important effector cells in inflammation, macrophages can be polarized to classically (M1) or alternatively (M2) activated phenotype with diverse functions in immunity. However, the relationship between Areg expression and macrophage activation is poorly understood. Here we report that Areg was significantly expressed in M1 but not in M2 macrophages. This was confirmed by analyses of RT-PCR and ELISA in peritoneal macrophages, and by evaluating protein expression in alveolar macrophages and RAW264.7 cells. Selective inhibitors of TLR4 (CLI-095) and MAP kinase, including Erk1/2 (PD98059), JNK (SP600125) and p38 (SB203580), significantly reduced Areg expression in M1 macrophages, suggesting that M1 macrophages produce Areg mainly through the TLR4-MAPK pathway, which is involved in the mechanism of M1 activation. When compared with productions of classical biomarkers of M1 macrophages, Areg expression was highly consistent in time series. Taken together, Areg may be an effective new biomarker of M1 macrophages.

  7. Osmoprotectants and carriers for formulating co-cultures of Gram-negative biocontrol agents active against potato dry rot in storage

    USDA-ARS?s Scientific Manuscript database

    Pseudomonas fluorescens strains S11:P:12, P22:Y:05, and S22:T:04 suppress four important storage potato maladies; dry rot, late blight, pink rot, and sprouting. When grown as a three-strain co-culture, the efficacy and consistency of the strains are enhanced over blends of individually cultured str...

  8. Control of tumor-associated macrophage alternative activation by MIF

    PubMed Central

    Yaddanapudi, Kavitha; Putty, Kalyani; Rendon, Beatriz E.; Lamont, Gwyneth J.; Faughn, Jonathan D.; Satoskar, Abhay; Lasnik, Amanda; Eaton, John W.; Mitchell, Robert A.

    2013-01-01

    Tumor stromal alternatively activated macrophages are important determinants of anti-tumor T lymphocyte responses, intratumoral neovascularization and metastatic dissemination. Our recent efforts to investigate the mechanism of macrophage migration inhibitory factor (MIF) in antagonizing anti-melanoma immune responses reveal that macrophage-derived MIF participates in macrophage alternative activation in melanoma-bearing mice. Both peripheral and tumor-associated macrophages (TAMs) isolated from melanoma bearing MIF-deficient mice display elevated pro-inflammatory cytokine expression and reduced anti-inflammatory, immunosuppressive and pro-angiogenic gene products compared to macrophages from tumor bearing MIF wildtype mice. Moreover, TAMs and myeloid-derived suppressor cells (MDSCs) from MIF-deficient mice exhibit reduced T lymphocyte immunosuppressive activities than do those from their wildtype littermates. Corresponding with reduced tumor immunosuppression and neoangiogenic potential by TAMs, MIF-deficiency confers protection against transplantable subcutaneous melanoma outgrowth and melanoma lung metastatic colonization. Finally, we report for the first time that our previously discovered MIF small molecule antagonist, 4-iodo-6-phenylpyrimidine (4-IPP), recapitulates MIF-deficiency in vitro and in vivo and attenuates tumor polarized macrophage alternative activation, immunosuppression, neoangiogenesis and melanoma tumor outgrowth. These studies describe an important functional contribution by MIF to tumor-associated macrophage alternative activation and provide justification for immunotherapeutic targeting of MIF in melanoma patients. PMID:23390297

  9. Targeting macrophage anti-tumor activity to suppress melanoma progression

    PubMed Central

    Yang, Luhong; Liu, Chengfang; Zhang, Qi; Zhang, Linjing

    2017-01-01

    By phagocytosing cancer cells and their cellular debris, macrophages play a critical role in nonspecific defense (innate immunity) and, as antigen presenters, they help initiate specific defense mechanisms (adaptive immunity). Malignant melanoma is a lethal disease due to its aggressive capacity for metastasis and resistance to therapy. For decades, considerable effort has gone into development of an effective immunotherapy for treatment of metastatic melanoma. In this review, we focus on the anti-tumor activities of macrophages in melanoma and their potential as therapeutic targets in melanoma. Although macrophages can be re-educated through intercellular signaling to promote tumor survival owing to their plasticity, we expect that targeting the anti-tumor activity of macrophages remains a promising strategy for melanoma inhibition. The combination of tumoricidal macrophage activation and other treatments such as surgery, chemotherapy, and radiotherapy, may provide an effective and comprehensive anti-melanoma strategy. PMID:28060744

  10. Sodium-activated macrophages: the salt mine expands.

    PubMed

    Lucca, Liliana E; Hafler, David A

    2015-08-01

    High sodium consumption has been raising interest as a putative environmental factor linking Western lifestyle to the growing epidemic of autoimmune and inflammatory diseases. Now Zhang and colleagues show that high sodium drives macrophage to acquire a new proinflammatory effector phenotype with a distinct signature, paving the path to assess the role of salt-activated macrophages in human disease.

  11. EGFR regulates macrophage activation and function in bacterial infection.

    PubMed

    Hardbower, Dana M; Singh, Kshipra; Asim, Mohammad; Verriere, Thomas G; Olivares-Villagómez, Danyvid; Barry, Daniel P; Allaman, Margaret M; Washington, M Kay; Peek, Richard M; Piazuelo, M Blanca; Wilson, Keith T

    2016-09-01

    EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

  12. Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

    PubMed

    Cuda, Carla M; Misharin, Alexander V; Khare, Sonal; Saber, Rana; Tsai, FuNien; Archer, Amy M; Homan, Philip J; Haines, G Kenneth; Hutcheson, Jack; Dorfleutner, Andrea; Budinger, G R Scott; Stehlik, Christian; Perlman, Harris

    2015-10-16

    Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/fl) mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. Cre (LysM) Casp8 (fl/fl) mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann-Whitney U test. Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8-deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates

  13. Macrophage Activation Syndrome in Paediatric Rheumatic Diseases.

    PubMed

    Islam, M I; Talukder, M K; Islam, M M; Laila, K; Rahman, S A

    2017-04-01

    Macrophage activation syndrome (MAS) is a potentially fatal complication of rheumatic disorders, which commonly occurs in systemic juvenile idiopathic arthritis (sJIA).This study was carried out with the aims of describing the clinical features, laboratory findings and outcomes of MAS associated with paediatric rheumatic diseases in the Department of Paediatrics, Bangabandhu Sheikh Mujib Medical University (BSMMU) and compare these results with previous studies on MAS. This retrospective study was conducted in the paediatric rheumatology wing of the Department of Paediatrics, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh. Clinical and laboratory profile of all the diagnosed cases of MAS were analyzed from the medical records from January 2010 to July 2015. Among 10 MAS patients, 6 were female and 4 were male. Seven patients of systemic JIA, two patients of SLE and one patient with Kawasaki Disease developed MAS in their course of primary disease. Mean duration of primary disease prior to development of MAS was 2.9 years and mean age of onset was 9.1 years. High continued fever and new onset hepatosplenomegaly were the hallmark of the clinical presentation. White blood cell count and platelet count came down from the mean of 16.2 to 10.2×10⁹/L and 254 to 90×10⁹/L. Mean erythrocyte sedimentation rate was dropped from 56 to 29 mm/hr. Six patients had abnormal liver enzyme level (ALT) and 5 had evidence of coagulopathy (prolonged prothrombin time and APTT) at the onset of disease. Hyperferritinnemia were found in all the patients. Bone marrow study was done in 5 patients but features of hamophagocytosis were found only in 2 patients. All patients received intravenous steroid and 3 patients who did not respond to steroid received additional cyclosporine. Mortality rate was 30% in this series. Macrophage activation syndrome is a fatal complication of paediatric rheumatic diseases among which s-JIA was predominant. Early diagnosis and

  14. Alternative activation modifies macrophage resistance to Mycobacterium bovis.

    PubMed

    Castillo-Velázquez, Uziel; Aranday-Cortés, Elihú; Gutiérrez-Pabello, José A

    2011-07-05

    The aim of this study was to evaluate the influence of macrophage alternative activation in the intracellular pathogen natural disease resistance phenotype of the host. Macrophage monolayers from resistant (R) (3) or susceptible (S) (3) cattle donors were treated with 10 ng/ml of bovine recombinant IL-4 (rbIL-4), and infected with virulent and avirulent Mycobacterium bovis (MOI 10:1). Bactericidal assays were performed to assess the bacterial phagocytic index and intracellular survival. Total RNA was reverse transcribed and used to analyze the relative changes in gene expression of IL-10, IL-12, IL-18 IL-1β, TNF-α, MCP-1, MCP-2, IL-6, MIP-1, MIP-3, iNOS, ARGII and SLAM by real time PCR. Cell supernatants were collected and nitric oxide and arginase production was assessed. Apoptosis induction was measured by TUNEL. IL-4 treatment increased the phagocytic index in both R and S macrophages; however intracellular survival was augmented mainly in S macrophages. Alternative activation decreased gene expression of pro-inflammatory cytokines, nitric oxide production and DNA fragmentation mainly in R macrophages. On the other hand, arginase production was not different between R and S macrophages. Alternative activation modifies the macrophage response against M. bovis. IL-4 treatment minimized the functional differences that exist between R and S macrophages. Copyright © 2011. Published by Elsevier B.V.

  15. Retinal Pigment Epithelial Cells Suppress Phagolysosome Activation in Macrophages

    PubMed Central

    Wang, Eric; Choe, Yoona; Ng, Tat Fong; Taylor, Andrew W.

    2017-01-01

    Purpose The eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is retinal pigment epithelial cells (RPE) altering phagocytosis in macrophages. Methods The conditioned media of RPE eyecups from eyes of healthy mice and mice with experimental autoimmune uveitis (EAU) were used to treat primary macrophage phagocytizing pHrodo bacterial bioparticles. In addition, the neuropeptides were depleted from the conditioned media of healthy RPE eyecups and used to treat phagocytizing macrophages. The conditioned media from healthy and EAU RPE eyecups were assayed for IL-6, and IL-6 was added to the healthy conditioned media, and neutralized in the EAU conditioned media. The macrophages were treated with the conditioned media and assayed for fluorescence. The macrophages were imaged, and the fluorescence intensity, relative to active phagolysosomes, was measured. Also, the macrophages were assayed using fluorescent viability dye staining. Results The conditioned media from healthy, but not from EAU RPE eyecups suppressed phagolysosome activation. Depletion of the neuropeptides alpha-melanocyte–stimulating hormone and neuropeptide Y from the healthy RPE eyecup conditioned media resulted in macrophage death. In the EAU RPE eyecup conditioned media was 0.96 ± 0.18 ng/mL of IL-6, and when neutralized the conditioned media suppressed phagolysosome activation. Conclusions The healthy RPE through soluble molecules, including alpha-melanocyte–stimulating hormone and neuropeptide Y, suppresses the activation of the phagolysosome in macrophages. In EAU, the IL-6 produced by the RPE promotes the activation of phagolysosomes in macrophages. These results demonstrate that under healthy conditions, RPE promotes an altered pathway of phagocytized material in macrophages with implications on antigen processing and clearance. PMID:28241314

  16. The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production.

    PubMed

    Caron-Beaudoin, Elyse; Viau, Rachel; Hudon-Thibeault, Andrée-Anne; Vaillancourt, Cathy; Sanderson, J Thomas

    2017-10-01

    Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages

    PubMed Central

    Soldano, Stefano; Pizzorni, Carmen; Paolino, Sabrina; Trombetta, Amelia Chiara; Montagna, Paola; Brizzolara, Renata; Ruaro, Barbara; Sulli, Alberto; Cutolo, Maurizio

    2016-01-01

    Background Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Methods Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. Results ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1

  18. Molecular and epigenetic basis of macrophage polarized activation.

    PubMed

    Porta, Chiara; Riboldi, Elena; Ippolito, Alessandro; Sica, Antonio

    2015-08-01

    Macrophages are unique cells for origin, heterogeneity and plasticity. At steady state most of macrophages are derived from fetal sources and maintained in adulthood through self-renewing. Despite sharing common progenitors, a remarkable heterogeneity characterized tissue-resident macrophages indicating that local signals educate them to express organ-specific functions. Macrophages are extremely plastic: chromatin landscape and transcriptional programs can be dynamically re-shaped in response to microenvironmental changes. Owing to their ductility, macrophages are crucial orchestrators of both initiation and resolution of immune responses and key supporters of tissue development and functions in homeostatic and pathological conditions. Herein, we describe current understanding of heterogeneity and plasticity of macrophages using the M1-M2 dichotomy as operationally useful simplification of polarized activation. We focused on the complex network of signaling cascades, metabolic pathways, transcription factors, and epigenetic changes that control macrophage activation. In particular, this network was addressed in sepsis, as a paradigm of a pathological condition determining dynamic macrophage reprogramming.

  19. Toxoplasma gondii Chitinase Induces Macrophage Activation

    PubMed Central

    Almeida, Fausto; Sardinha-Silva, Aline; da Silva, Thiago Aparecido; Pessoni, André Moreira; Pinzan, Camila Figueiredo; Alegre-Maller, Ana Claudia Paiva; Cecílio, Nerry Tatiana; Moretti, Nilmar Silvio; Damásio, André Ricardo Lima; Pedersoli, Wellington Ramos; Mineo, José Roberto; Silva, Roberto Nascimento; Roque-Barreira, Maria Cristina

    2015-01-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50°C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection. PMID:26659253

  20. Effects of lipopolysaccharide on the catabolic activity of macrophages

    SciTech Connect

    Cluff, C.; Ziegler, H.K.

    1986-03-05

    The ability of macrophages to degrade and catabolize antigens is of relevance both as a means to process complex antigens prior to presentation to T cells, as well as a way to down regulate immune responses by destroying the antigenicity of polypeptides. With these considerations, the authors have investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of /sup 125/-I labelled surface components of heat-killed Listeria monocytogenes (HKLM) subsequent to their uptake by macrophages. They have compared the catabolic activity of macrophages from peritoneal exudates of mice injected i.p. with saline or LPS and have found that LPS-elicited macrophages display a greatly enhanced (3 fold) rate of catabolism. This increase in catabolic activity peaks 3 days after LPS injection and steadily declines thereafter, approaching a baseline level after 3 weeks. The enhancement of catabolic activity is under LPS gene control. LPS-elicited macrophages rapidly destroy the antigenicity of bacterial antigens and function poorly as antigen presenting cells in vitro. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions with negative regulatory effects on the induction of specific immune responses.

  1. Antiorthostatic suspension stimulates profiles of macrophage activation in mice

    NASA Technical Reports Server (NTRS)

    Miller, E. S.; Bates, R. A.; Koebel, D. A.; Sonnenfeld, G.

    1999-01-01

    The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

  2. Antiorthostatic suspension stimulates profiles of macrophage activation in mice

    NASA Technical Reports Server (NTRS)

    Miller, E. S.; Bates, R. A.; Koebel, D. A.; Sonnenfeld, G.

    1999-01-01

    The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

  3. Activation of NK cell cytotoxicity by aerosolized CpG-ODN/poly(I:C) against lung melanoma metastases is mediated by alveolar macrophages.

    PubMed

    Sommariva, Michele; Le Noci, Valentino; Storti, Chiara; Bianchi, Francesca; Tagliabue, Elda; Balsari, Andrea; Sfondrini, Lucia

    2017-03-01

    Controversies remain about NK cells direct responsiveness to Toll-like receptor (TLR) agonists or dependence on macrophages. In a melanoma lung metastasis model, aerosolized TLR9 and TLR3 agonists have been reported to induce antitumor immunity through NK cells activation. In the current study, we demonstrated that in vitro TLR9/TLR3 stimulation induced IFN-γ secretion by NK cells, but an increase in their cytotoxicity was detected only after NK cells co-culture with in vitro TLR9/TLR3 agonists pretreated alveolar macrophages. Alveolar macrophages from melanoma lung metastases-bearing mice, treated with aerosolized TLR agonists, also promoted NK cell cytotoxicity. Activated NK cells from lungs of melanoma metastases-bearing mice that were given aerosolized TLR9/TLR3 agonists were able to polarize naive alveolar macrophages toward a M1-like phenotype. Our results demonstrate that activation of NK cells in the lung after TLR engagement is mediated by alveolar macrophages and that activated NK cells shape macrophage behavior.

  4. M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells.

    PubMed

    Cui, Jiajun; Xu, Wenhua; Chen, Jian; Li, Hui; Dai, Lu; Frank, Jacqueline A; Peng, Shaojun; Wang, Siying; Chen, Gang

    2017-03-28

    The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages.

  5. M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells

    PubMed Central

    Li, Hui; Dai, Lu; Frank, Jacqueline A.; Peng, Shaojun; Wang, Siying; Chen, Gang

    2017-01-01

    The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages. PMID:28423485

  6. Inhibitory effect of deferoxamine or macrophage activation on transformation of Paracoccidioides brasiliensis conidia ingested by macrophages: reversal by holotransferrin.

    PubMed

    Cano, L E; Gomez, B; Brummer, E; Restrepo, A; Stevens, D A

    1994-04-01

    Conidia of P. brasiliensis ingested by murine macrophages at 37 degrees C showed enhanced transformation to yeast cells and further intracellular growth compared with conidia in culture medium alone. Treatment of macrophages with the iron chelator deferoxamine inhibited the intracellular conidium-to-yeast transformation. Cytokine-activated macrophages could also exert this inhibitory effect. Holotransferrin reversed the inhibitory effect of either deferoxamine or activated macrophages on intracellular conidium-to-yeast transformation. These results indicate that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested conidia and growth of yeast cells.

  7. Inhibitory effect of deferoxamine or macrophage activation on transformation of Paracoccidioides brasiliensis conidia ingested by macrophages: reversal by holotransferrin.

    PubMed Central

    Cano, L E; Gomez, B; Brummer, E; Restrepo, A; Stevens, D A

    1994-01-01

    Conidia of P. brasiliensis ingested by murine macrophages at 37 degrees C showed enhanced transformation to yeast cells and further intracellular growth compared with conidia in culture medium alone. Treatment of macrophages with the iron chelator deferoxamine inhibited the intracellular conidium-to-yeast transformation. Cytokine-activated macrophages could also exert this inhibitory effect. Holotransferrin reversed the inhibitory effect of either deferoxamine or activated macrophages on intracellular conidium-to-yeast transformation. These results indicate that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested conidia and growth of yeast cells. PMID:8132359

  8. Extract from Calotropis procera latex activates murine macrophages.

    PubMed

    Seddek, Abdel latif Shaker; Mahmoud, Motamed Elsayed; Shiina, Takahiko; Hirayama, Haruko; Iwami, Momoe; Miyazawa, Seiji; Nikami, Hideki; Takewaki, Tadashi; Shimizu, Yasutake

    2009-07-01

    Calotropis procera latex has long been used in traditional medicines. Extracts from C. procera latex have been reported to have various pharmacological actions, including protection from myocardial infarction, hepatoprotective action, antitumor activity, antinociceptive, and pro- and anti-inflammatory actions. To evaluate the immunomodulatory functions of the water-soluble C. procera extract (CPE), we investigated its ability to activate macrophages-effector cells in inflammatory and immune responses. Intraperitoneal injection of CPE in mice (2 mg/mouse) induced migration of macrophages to the intraperitoneal cavity, confirming the proinflammatory effects of water-soluble CPE. The direct effects of CPE on macrophages were then assessed by measuring the production of nitric oxide (NO) as an indicator for macrophage activation. Addition of CPE (1-10 microg/ml) to the culture medium of the murine monocyte/macrophage cell line RAW264.7 caused an increase in NO production in a time- and dose-dependent manner. CPE-elicited NO production was blocked by application of an inhibitor of inducible nitric oxide synthase (iNOS). Expression of iNOS mRNA was induced by treatment of cultured macrophages with CPE. Injection of CPE in mice also resulted in an increase in plasma NO level. The results suggest that CPE activates macrophages and facilitates NO production via up-regulation of iNOS gene expression.

  9. Therapeutic potential of carbohydrates as regulators of macrophage activation.

    PubMed

    Lundahl, Mimmi L E; Scanlan, Eoin M; Lavelle, Ed C

    2017-09-08

    It is well established for a broad range of disease states, including cancer and Mycobacterium tuberculosis infection, that pathogenesis is bolstered by polarisation of macrophages towards an anti-inflammatory phenotype, known as M2. As these innate immune cells are relatively long-lived, their re-polarisation to pro-inflammatory, phagocytic and bactericidal "classically activated" M1 macrophages is an attractive therapeutic approach. On the other hand, there are scenarios where the resolving inflammation, wound healing and tissue remodelling properties of M2 macrophages are beneficial - for example the successful introduction of biomedical implants. Although there are numerous endogenous and exogenous factors that have an impact on the macrophage polarisation spectrum, this review will focus specifically on prominent macrophage-modulating carbohydrate motifs with a view towards highlighting structure-function relationships and therapeutic potential. Copyright © 2017. Published by Elsevier Inc.

  10. AKT mediated glycolytic shift regulates autophagy in classically activated macrophages.

    PubMed

    Matta, Sumit Kumar; Kumar, Dhiraj

    2015-09-01

    Autophagy is considered as an innate defense mechanism primarily due to its role in the targeting of intracellular pathogens for lysosomal degradation. Here we report inhibition of autophagy as an adaptive response in classically activated macrophages that helps achieve high cellular ROS production and cell death-another hallmark of innate mechanisms. We show prolonged classical activation of Raw 264.7 macrophages by treating them with IFN-γ and LPS inhibited autophagy. The inhibition of autophagy was dependent on nitric oxide (NO) production which activated the AKT-mTOR signaling, the known negative regulators of autophagy. Autophagy inhibition in these cells was accompanied with a shift to aerobic glycolysis along with a decline in the mitochondrial membrane potential (MOMP). The decline in MOMP coupled with autophagy inhibition led to increased mitochondrial content and considerably elevated cellular ROS, eventually causing cell death. Next, using specific siRNA mediated knockdowns we show AKT was responsible for the glycolytic shift and autophagy inhibition in activated macrophages. Surprisingly, AKT knockdown in activated macrophages also rescued them from cell death. Finally we show that AKT mediated autophagy inhibition in the activated macrophages correlated with the depletion of glucose from the extracellular medium, and glucose supplementation not only rescued autophagy levels and reversed other phenotypes of activated macrophages, but also inhibited cell death. Thus we report here a novel link between AKT mediated glycolytic metabolism and autophagy in the activated macrophages, and provide a possible mechanism for sustained macrophage activation in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Peroxidase activity in monocytes and tissue macrophages of mice.

    PubMed

    Ogawa, T; Koerten, H K; Daems, W T

    1978-04-28

    A description is given of the distribution of peroxidatic (PO) activity in murine monocytes of blood and peritoneal cavity, and in murine macrophages residing in the unstimulated peritoneal cavity as well as in liver, spleen, bone marrow, and small intestine. In the monocytes, PO activity is restricted to some of the cytoplasmic granules; in the tissue (or resident) macrophages present in peritoneal cavity, liver, spleen, and small intestine, the PO activity is located in the nuclear envelope and the rough endoplasmic reticulum. Macrophages in the bone marrow are PO-negative. In the spleen and bone marrow, reticulum cells show PO activity in the nuclear envelope and the RER. Transitional forms between monocytes and tissue macrophages were not observed.

  12. Adipogenic miR-27a in adipose tissue upregulates macrophage activation via inhibiting PPARγ of insulin resistance induced by high-fat diet-associated obesity.

    PubMed

    Yao, Fan; Yu, Yang; Feng, Linjing; Li, Junnan; Zhang, Meishuang; Lan, Xiaoxin; Yan, Xin; Liu, Yilun; Guan, Fengying; Zhang, Ming; Chen, Li

    2017-03-30

    Chronic low degree inflammation caused by macrophage activation is a crucial factor underlying insulin resistance induced by obesity. To illustrate the mechanism of regulating of macrophage activation in adipose tissue, the role of adipogenic miR-27a activating M1 macrophage polarization via blocking PPARγ was evaluated. Obese mice model and miR-27a overexpression or knockdown mice model were established and related biochemical index were examined. Raw264.7 and 3T3-L1 were cultured and co-cultured for mimicking the microenvironment of local inflammation. Macrophage infiltration was observed. MiR-27a and cytokines levels in serum and adipose tissue were measured. Macrophage polarization markers and protein expression in insulin or inflammatory signaling pathways were observed. Impaired glucose tolerance and insulin tolerance was observed in 4w, 8w and 12w of high fat diet and miR-27a overexpression mice. Concurrently, miR-27a was increased in serum in a time-dependent manner, along with M1 cytokines and M1 macrophages increasing in adipose tissue clearly. Insulin signaling pathway was blocked, and PPARγ was suppressed. However, NF-κB was activated. On the other hand, activated macrophages and hypertrophic adipocytes induced by miR-27a could increase the ratio of Raw264.7 migration, including improving cytokines generation, and blocking PPARγ expression markedly. The present studies are conducted to clarify that miR-27a has increased along with up-regulation in the process of proinflammatory cytokines generation, macrophage influx and M1 macrophage polarization in obesity. These indicate that miR-27a gives the novel target of intervention for inflammation and insulin resistance in obesity.

  13. Jacalin-Activated Macrophages Exhibit an Antitumor Phenotype

    PubMed Central

    Danella Polli, Cláudia; Pereira Ruas, Luciana; Chain Veronez, Luciana; Herrero Geraldino, Thais; Rossetto de Morais, Fabiana; Roque-Barreira, Maria Cristina; Pereira-da-Silva, Gabriela

    2016-01-01

    Tumor-associated macrophages (TAMs) have an ambiguous and complex role in the carcinogenic process, since these cells can be polarized into different phenotypes (proinflammatory, antitumor cells or anti-inflammatory, protumor cells) by the tumor microenvironment. Given that the interactions between tumor cells and TAMs involve several players, a better understanding of the function and regulation of TAMs is crucial to interfere with their differentiation in attempts to skew TAM polarization into cells with a proinflammatory antitumor phenotype. In this study, we investigated the modulation of macrophage tumoricidal activities by the lectin jacalin. Jacalin bound to macrophage surface and induced the expression and/or release of mainly proinflammatory cytokines via NF-κB signaling, as well as increased iNOS mRNA expression, suggesting that the lectin polarizes macrophages toward the antitumor phenotype. Therefore, tumoricidal activities of jacalin-stimulated macrophages were evaluated. High rates of tumor cell (human colon, HT-29, and breast, MCF-7, cells) apoptosis were observed upon incubation with supernatants from jacalin-stimulated macrophages. Taken together, these results indicate that jacalin, by exerting a proinflammatory activity, can direct macrophages to an antitumor phenotype. Deep knowledge of the regulation of TAM functions is essential for the development of innovative anticancer strategies. PMID:27119077

  14. Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation

    PubMed Central

    Melton, Andrew C.; Melrose, Jennifer; Alajoki, Liisa; Privat, Sylvie; Cho, Hannah; Brown, Naomi; Plavec, Ana Marija; Nguyen, Dat; Johnston, Elijah D.; Yang, Jian; Polokoff, Mark A.; Plavec, Ivan; Berg, Ellen L.; O'Mahony, Alison

    2013-01-01

    Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F. PMID:23505568

  15. Modeling long-term host cell-Giardia lamblia interactions in an in vitro co-culture system.

    PubMed

    Fisher, Bridget S; Estraño, Carlos E; Cole, Judith A

    2013-01-01

    Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.

  16. Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

    PubMed Central

    Fisher, Bridget S.; Estraño, Carlos E.; Cole, Judith A.

    2013-01-01

    Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions. PMID:24312526

  17. Alternatively activated macrophages promote pancreatic fibrosis in chronic pancreatitis

    PubMed Central

    Xue, Jing; Sharma, Vishal; Hsieh, Michael H.; Chawla, Ajay; Murali, Ramachandran; Pandol, Stephen J.; Habtezion, Aida

    2015-01-01

    Chronic pancreatitis (CP) is a progressive and irreversible inflammatory and fibrotic disease with no cure. Unlike acute pancreatitis, we find that alternatively activated macrophages (AAMs) are dominant in mouse and human CP. AAMs are dependent on IL-4 and IL-13 signaling and we show that mice lacking IL-4Rα, myeloid specific IL-4Rα, and IL-4/IL-13 were less susceptible to pancreatic fibrosis. Furthermore, we demonstrate that mouse and human pancreatic stellate cells (PSCs) are a source of IL-4/IL-13. Notably, we show that pharmacologic inhibition of IL-4/IL-13 in human ex-vivo studies as well as in established mouse CP decreases pancreatic AAMs and fibrosis. We identify a critical role for macrophages in pancreatic fibrosis and in turn PSCs as important inducers of macrophage alternative activation. Our study challenges and identifies pathways involved in cross talk between macrophages and PSCs that can be targeted to reverse or halt pancreatic fibrosis progression. PMID:25981357

  18. Cytotoxic macrophage-released tumour necrosis factor-alpha (TNF-α) as a killing mechanism for cancer cell death after cold plasma activation

    NASA Astrophysics Data System (ADS)

    Kaushik, Nagendra Kumar; Kaushik, Neha; Min, Booki; Choi, Ki Hong; Hong, Young June; Miller, Vandana; Fridman, Alexander; Choi, Eun Ha

    2016-03-01

    The present study aims at studying the anticancer role of cold plasma-activated immune cells. The direct anti-cancer activity of plasma-activated immune cells against human solid cancers has not been described so far. Hence, we assessed the effect of plasma-treated RAW264.7 macrophages on cancer cell growth after co-culture. In particular, flow cytometer analysis revealed that plasma did not induce any cell death in RAW264.7 macrophages. Interestingly, immunofluorescence and western blot analysis confirmed that TNF-α released from plasma-activated macrophages acts as a tumour cell death inducer. In support of these findings, activated macrophages down-regulated the cell growth in solid cancer cell lines and induced cell death in vitro. Together our findings suggest plasma-induced reactive species recruit cytotoxic macrophages to release TNF-α, which blocks cancer cell growth and can have the potential to contribute to reducing tumour growth in vivo in the near future.

  19. Peptides conjugated to gold nanoparticles induce macrophage activation.

    PubMed

    Bastús, Neus G; Sánchez-Tilló, Ester; Pujals, Silvia; Farrera, Consol; Kogan, Marcelo J; Giralt, Ernest; Celada, Antonio; Lloberas, Jorge; Puntes, Victor

    2009-02-01

    Macrophages that react against pathogenic organisms can also be activated with artificial nanometric units consisting of gold nanoparticles (Au NPs) with a peptide coating. Using bone marrow-derived macrophages, here we show that these cells have the capacity to recognize Au NPs once conjugated to two biomedically relevant peptides, the amyloid growth inhibitory peptide (AGIP) and the sweet arrow peptide (SAP), while they do not recognize peptides or NPs alone. The recognition of these conjugates by macrophages is mediated by a pattern recognition receptor, the TLR-4. Consequently, pro-inflammatory cytokines such as TNF-alpha, IL-1 beta and IL-6, as well as nitric oxide synthase were induced and macrophage proliferation was stopped when exposed to the peptide-conjugated Au NPs. Contamination by lipopolysaccharide in our experimental system was excluded. Furthermore, macrophage activation appeared to be independent of peptide length and polarity. As a result of macrophage activation, conjugated Au NPs were internalized and processed. These results open up a new avenue in the world of adjuvants and illustrate the basic requirements for the design of NP conjugates that efficiently reach their target.

  20. CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages

    PubMed Central

    Ball, Michael S.; Shipman, Emilie P.; Kim, Hyunjung; Liby, Karen T.; Pioli, Patricia A.

    2016-01-01

    Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer. PMID:26918785

  1. Update on the role of alternatively activated macrophages in asthma.

    PubMed

    Jiang, Zhilong; Zhu, Lei

    2016-01-01

    Lung macrophages link innate and adaptive immune responses during allergic airway inflammatory responses. Alveolar macrophages (AMs) and interstitial macrophages are two different phenotypes that differentially exert immunological function under physiological and pathological conditions. Exposure to pathogen induces polarization of AM cells into classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells). M1 cells dominantly express proinflammatory cytokines such as TNF-α and IL-1 β and induce lung inflammation and tissue damage. M2 cells are further divided into M2a and M2c subsets. M2a cells dominantly produce allergic cytokines IL-4 and IL-13, but M2c cells dominantly produce anti-inflammatory cytokine IL-10. M2a and M2c cells are differently involved in initiation, inflammation resolution, and tissue remodeling in the different stages of asthma. Microenvironment dynamically influences polarization of AM cells. Cytokines, chemokines, and immune-regulatory cells interplay and affect the balance between the polarization of M1 and M2 cells, subsequently influencing disease progression. Thus, modulation of AM phenotypes through molecular intervention has therapeutic potential in the treatment of asthma and other allergic inflammatory diseases. This review updated recent advances in polarization and functional specialization of these macrophage subtypes with emphasis on modulation of polarization of M2 cells in asthma of human subjects and animal models.

  2. Update on the role of alternatively activated macrophages in asthma

    PubMed Central

    Jiang, Zhilong; Zhu, Lei

    2016-01-01

    Lung macrophages link innate and adaptive immune responses during allergic airway inflammatory responses. Alveolar macrophages (AMs) and interstitial macrophages are two different phenotypes that differentially exert immunological function under physiological and pathological conditions. Exposure to pathogen induces polarization of AM cells into classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells). M1 cells dominantly express proinflammatory cytokines such as TNF-α and IL-1 β and induce lung inflammation and tissue damage. M2 cells are further divided into M2a and M2c subsets. M2a cells dominantly produce allergic cytokines IL-4 and IL-13, but M2c cells dominantly produce anti-inflammatory cytokine IL-10. M2a and M2c cells are differently involved in initiation, inflammation resolution, and tissue remodeling in the different stages of asthma. Microenvironment dynamically influences polarization of AM cells. Cytokines, chemokines, and immune-regulatory cells interplay and affect the balance between the polarization of M1 and M2 cells, subsequently influencing disease progression. Thus, modulation of AM phenotypes through molecular intervention has therapeutic potential in the treatment of asthma and other allergic inflammatory diseases. This review updated recent advances in polarization and functional specialization of these macrophage subtypes with emphasis on modulation of polarization of M2 cells in asthma of human subjects and animal models. PMID:27350756

  3. Coordinated Regulation of Signaling Pathways during Macrophage Activation.

    PubMed

    Lawrence, Toby

    2016-10-01

    The functional and phenotypic diversity of macrophages has long been appreciated, and it is now clear that it reflects a complex interplay between hard-wired differentiation pathways and instructive signals in specific tissues (Lawrence T, Natoli G. 2011, Nat Rev Immunol11:750-761). Recent studies have begun to unravel the molecular basis for the integration of these intrinsic developmental pathways with extracellular signals from the tissue microenvironment that confer the distinct phenotypes of tissue-resident macrophages (Lavin Y et al. 2014. Cell159:1312-1326; Gosselin D et al. 2014. Cell159:1327-1340). Macrophage phenotype and function is particularly dynamic during inflammation or infection, as blood monocytes are recruited into tissues and differentiate into macrophages, and depending on the nature of the inflammatory stimulus, they may acquire distinct functional phenotypes (Xue J et al. 2014. Immunity40:274-288; Murray PJ et al. 2014. Immunity41:14-20). Furthermore, these functional activation states can be rapidly modified in response to a changing microenvironment. Here we will discuss several key signaling pathways that drive macrophage activation during the inflammatory response and discuss how these pathways are integrated to "fine-tune" macrophage phenotype and function.

  4. Monocyte/macrophage cytokine activity regulates vascular smooth muscle cell function within a degradable polyurethane scaffold.

    PubMed

    Battiston, K G; Ouyang, B; Labow, R S; Simmons, C A; Santerre, J P

    2014-03-01

    Tissue engineering strategies rely on the ability to promote cell proliferation and migration into porous biomaterial constructs, as well as to support specific phenotypic states of the cells in vitro. The present study investigated the use of released factors from monocytes and their derived macrophages (MDM) and the mechanism by which they regulate vascular smooth muscle cell (VSMC) response in a VSMC-monocyte co-culture system within a porous degradable polyurethane (D-PHI) scaffold. VSMCs cultured in monocyte/MDM-conditioned medium (MCM), generated from the culture of monocytes/MDM on D-PHI scaffolds for up to 28 days, similarly affected VSMC contractile marker expression, growth and three-dimensional migration when compared to direct VSMC-monocyte co-culture. Monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) were identified as two cytokines present in MCM, at concentrations that have previously been shown to influence VSMC phenotype. VSMCs cultured alone on D-PHI scaffolds and exposed to MCP-1 (5 ng ml(-1)) or IL-6 (1 ng ml(-1)) for 7 days experienced a suppression in contractile marker expression (with MCP-1 or IL-6) and increased growth (with MCP-1) compared to no cytokine medium supplementation. These effects were also observed in VSMC-monocyte co-culture on D-PHI. Neutralization of IL-6, but not MCP-1, was subsequently shown to decrease VSMC growth and enhance calponin expression for VSMC-monocyte co-cultures on D-PHI scaffolds for 7 days, implying that IL-6 mediates VSMC response in monocyte-VSMC co-cultures. This study highlights the use of monocytes and their derived macrophages in conjunction with immunomodulatory biomaterials, such as D-PHI, as agents for regulating VSMC response, and demonstrates the importance of monocyte/MDM-released factors, such as IL-6 in particular, in this process.

  5. Active autophagy but not lipophagy in macrophages with defective lipolysis

    PubMed Central

    Schlager, Stefanie; Chandak, Prakash G.; Korbelius, Melanie; Gottschalk, Benjamin; Leopold, Christina; Obrowsky, Sascha; Rainer, Silvia; Doddapattar, Prakash; Aflaki, Elma; Wegscheider, Martin; Sachdev, Vinay; Graier, Wolfgang F.; Kolb, Dagmar; Radovic, Branislav; Kratky, Dagmar

    2015-01-01

    During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages. PMID:26143381

  6. Endogenous Epoxygenases Are Modulators of Monocyte/Macrophage Activity

    PubMed Central

    Sugden, Mary C.; Holness, Mark J.; Swales, Karen E.; Warner, Timothy D.; Edin, Matthew L.; Zeldin, Darryl C.; Gilroy, Derek W.; Bishop-Bailey, David

    2011-01-01

    Background Arachidonic acid is metabolized through three major metabolic pathways, the cyclooxygenase, lipoxygenase and CYP450 enzyme systems. Unlike cyclooxygenase and lipoxygenases, the role of CYP450 epoxygenases in monocyte/macrophage-mediated responses is not known. Methodology/Principal Findings When transfected in vitro, CYP2J2 is an efficient activator of anti-inflammatory pathways through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) α. Human monocytes and macrophages contain PPARα and here we show they express the epoxygenases CYP2J2 and CYP2C8. Inhibition of constitutive monocyte epoxygenases using the epoxygenase inhibitor SKF525A induces cyclooxygenase (COX)-2 expression and activity, and the release of TNFα, and can be reversed by either add back of the endogenous epoxygenase products and PPARα ligand 11,12- epoxyeicosatrienoic acid (EET) or the addition of the selective synthetic PPARα ligand GW7647. In alternatively activated (IL-4-treated) monocytes, in contrast to classically activated cells, epoxygenase inhibition decreased TNFα release. Epoxygenases can be pro-inflammatory via superoxide anion production. The suppression of TNFα by SKF525A in the presence of IL-4 was associated with a reduction in superoxide anion generation and reproduced by the superoxide dismutase MnCl2. Similar to these acute activation studies, in monocyte derived macrophages, epoxygenase inhibition elevates M1 macrophage TNFα mRNA and further decreases M2 macrophage TNFα. Conclusions/Significance In conclusion, epoxygenase activity represents an important endogenous pathway which limits monocyte activation. Moreover endogenous epoxygenases are immuno-modulators regulating monocyte/macrophage activation depending on the underlying activation state. PMID:22028915

  7. Galectins distinctively regulate central monocyte and macrophage function.

    PubMed

    Paclik, Daniela; Werner, Lael; Guckelberger, Olaf; Wiedenmann, Bertram; Sturm, Andreas

    2011-01-01

    Monocytes and macrophages link the innate and adaptive immune systems and protect the host from the outside world. In inflammatory disorders their activation leads to tissue damage. Galectins have emerged as central regulators of the immune system. However, if they regulate monocyte/macrophage physiology is still unknown. Binding of Gal-1, Gal-2, Gal-3 and Gal-4 to monocytes/macrophages, activation, cytokine secretion and apoptosis were determined by FACS, migration by Transwell system and phagocytosis by phagotest. Supernatants from macrophages co-cultured with galectins revealed their influence on T-cell function. In our study Gal-1, Gal-2, Gal-4, and partly Gal-3 bound to monocytes/macrophages. Galectins prevented Salmonella-induced MHCII upregulation. Cytokine release was distinctly induced by different galectins. T-cell activation was significantly restricted by supernatants of macrophages co-cultured in the presence of Gal-2 or Gal-4. Furthermore, all galectins tested significantly inhibited monocyte migration. Finally, we showed for the first time that galectins induce potently monocyte, but not macrophage apoptosis. Our study provides evidence that galectins distinctively modulate central monocyte/macrophage function. By inhibiting T-cell function via macrophage priming, we show that galectins link the innate and adaptive immune systems and provide new insights into the action of sugar-binding proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent.

    PubMed

    Hohensinner, Philipp J; Baumgartner, Johanna; Kral-Pointner, Julia B; Uhrin, Pavel; Ebenbauer, Benjamin; Thaler, Barbara; Doberer, Konstantin; Stojkovic, Stefan; Demyanets, Svitlana; Fischer, Michael B; Huber, Kurt; Schabbauer, Gernot; Speidl, Walter S; Wojta, Johann

    2017-10-01

    Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1(-/-) bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence

  9. ERK5 Activation in Macrophages Promotes Efferocytosis and Inhibits Atherosclerosis

    PubMed Central

    Heo, Kyung-Sun; Cushman, Hannah J.; Akaike, Masashi; Woo, Chang-Hoon; Wang, Xin; Qiu, Xing; Fujiwara, Keigi; Abe, Jun-ichi

    2015-01-01

    Background Efferocytosis is a process by which dead and dying cells are removed by phagocytic cells. Efferocytosis by macrophages is thought to curb the progression of atherosclerosis, but the mechanistic insight of this process is lacking. Methods and Results When macrophages were fed apoptotic cells or treated with pitavastatin in vitro, efferocytosis-related signaling and phagocytic capacity were upregulated in an ERK5 activity–dependent manner. Macrophages isolated from macrophage-specific ERK5-null mice exhibited reduced efferocytosis and levels of gene and protein expression of efferocytosis-related molecules. When these mice were crossed with low-density lipoprotein receptor−/− mice and fed a high-cholesterol diet, atherosclerotic plaque formation was accelerated, and the plaques had more advanced and vulnerable morphology. Conclusions Our results demonstrate that ERK5, which is robustly activated by statins, is a hub molecule that upregulates macrophage efferocytosis, thereby suppressing atherosclerotic plaque formation. Molecules that upregulate ERK5 and its signaling in macrophages may be good drug targets for suppressing cardiovascular diseases. PMID:25001623

  10. Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation.

    PubMed

    Duarte, Tonya Azevedo; Noronha-Dutra, Alberto Augusto; Nery, Joilda Silva; Ribeiro, Samantha Brum; Pitanga, Thassila Nogueira; Lapa E Silva, José R; Arruda, Sérgio; Boéchat, Neio

    2012-05-01

    The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis

    PubMed Central

    Nguyen, Khoa D.; Qiu, Yifu; Cui, Xiaojin; Goh, Y.P. Sharon; Mwangi, Julia; David, Tovo; Mukundan, Lata; Brombacher, Frank; Locksley, Richard M.; Chawla, Ajay

    2011-01-01

    All homeotherms utilize thermogenesis to maintain core body temperature, ensuring that cellular functions and physiologic processes can ensue in cold environments1-3. In the prevailing model, when the hypothalamus senses cold temperatures, it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue (BAT) and white adipose tissue (WAT)4,5. Acting via the β3-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes6, whereas it stimulates the expression of thermogenic genes, such as PPARγ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1), and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes7-9. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report an unexpected requirement for the interleukin 4 (IL4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Cold exposure rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in BAT and lipolysis in WAT. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL4 increased thermogenic gene expression, fatty acid mobilization, and energy expenditure, all in a macrophage-dependent manner. We have thus discovered a surprising role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold. PMID:22101429

  12. Autophagy-induced RelB/p52 activation mediates tumour-associated macrophage repolarisation and suppression of hepatocellular carcinoma by natural compound baicalin

    PubMed Central

    Tan, H-Y; Wang, N; Man, K; Tsao, S-W; Che, C-M; Feng, Y

    2015-01-01

    The plasticity of tumour-associated macrophages (TAMs) has implicated an influential role in hepatocellular carcinoma (HCC). Repolarisation of TAM towards M1 phenotype characterises an immune-competent microenvironment that favours tumour regression. To investigate the role and mechanism of TAM repolarisation in suppression of HCC by a natural compound baicalin, Orthotopic HCC implantation model was used to investigate the effect of baicalin on HCC; liposome-clodronate was introduced to suppress macrophage populations in mice; bone marrow-derived monocytes (BMDMs) were induced to unpolarised, M1-like, M2-like macrophages and TAM using different conditioned medium. We observed that oral administration of baicalin (50 mg/kg) completely blocked orthotopic growth of implanted HCC. Suppression of HCC by baicalin was diminished when mice macrophage was removed by clodronate treatment. Baicalin induced repolarisation of TAM to M1-like phenotype without specific toxicity to either phenotype of macrophages. Baicalin initiated TAM reprogramming to M1-like macrophage, and promoted pro-inflammatory cytokines production. Co-culturing of HCC cells with baicalin-treated TAMs resulted in reduced proliferation and motility in HCC. Baicalin had minimal effect on derivation of macrophage polarisation factors by HCC cells, while directly induced repolarisation of TAM and M2-like macrophage. This effect was associated with elevated autophagy, and transcriptional activation of RelB/p52 pathway. Suppression of autophagy or RelB abolished skewing of baicalin-treated TAM. Autophagic degradation of TRAF2 in baicalin-treated TAM might be responsible for RelB/p52 activation. Our findings unveil the essential role of TAM repolarisation in suppressive effect of baicalin on HCC, which requires autophagy-associated activation of RelB/p52. PMID:26492375

  13. An inducible transgene reports activation of macrophages in live zebrafish larvae.

    PubMed

    Sanderson, Leslie E; Chien, An-Tzu; Astin, Jonathan W; Crosier, Kathryn E; Crosier, Philip S; Hall, Christopher J

    2015-11-01

    Macrophages are the most functionally heterogenous cells of the hematopoietic system. Given many diseases are underpinned by inappropriate macrophage activation, macrophages have emerged as a therapeutic target to treat disease. A thorough understanding of what controls macrophage activation will likely reveal new pathways that can be manipulated for therapeutic benefit. Live imaging fluorescent macrophages within transgenic zebrafish larvae has provided a valuable window to investigate macrophage behavior in vivo. Here we describe the first transgenic zebrafish line that reports macrophage activation, as evidenced by induced expression of an immunoresponsive gene 1(irg1):EGFP transgene. When combined with existing reporter lines that constitutively mark macrophages, we reveal this unique transgenic line can be used to live image macrophage activation in response to the bacterial endotoxin lipopolysaccharide and xenografted human cancer cells. We anticipate the Tg(irg1:EGFP) line will provide a valuable tool to explore macrophage activation and plasticity in the context of different disease models.

  14. 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

  15. Periodontitis-activated monocytes/macrophages cause aortic inflammation

    PubMed Central

    Miyajima, Shin-ichi; Naruse, Keiko; Kobayashi, Yasuko; Nakamura, Nobuhisa; Nishikawa, Toru; Adachi, Kei; Suzuki, Yuki; Kikuchi, Takeshi; Mitani, Akio; Mizutani, Makoto; Ohno, Norikazu; Noguchi, Toshihide; Matsubara, Tatsuaki

    2014-01-01

    A relationship between periodontal disease and atherosclerosis has been suggested by epidemiological studies. Ligature-induced experimental periodontitis is an adequate model for clinical periodontitis, which starts from plaque accumulation, followed by inflammation in the periodontal tissue. Here we have demonstrated using a ligature-induced periodontitis model that periodontitis activates monocytes/macrophages, which subsequently circulate in the blood and adhere to vascular endothelial cells without altering the serum TNF-α concentration. Adherent monocytes/macrophages induced NF-κB activation and VCAM-1 expression in the endothelium and increased the expression of the TNF-α signaling cascade in the aorta. Peripheral blood-derived mononuclear cells from rats with experimental periodontitis showed enhanced adhesion and increased NF-κB/VCAM-1 in cultured vascular endothelial cells. Our results suggest that periodontitis triggers the initial pathogenesis of atherosclerosis, inflammation of the vasculature, through activating monocytes/macrophages. PMID:24893991

  16. In vitro evidence for metallopeptidase participation in hepatocyte damage induced by Leishmania chagasi-infected macrophages.

    PubMed

    Costa, Juliana Dias; Nogueira de Melo, Ana Cristina; Vermelho, Alane Beatriz; Meirelles, Maria de Nazareth; Porrozzi, Renato

    2008-06-01

    Leishmania (Leishmania) chagasi infection activates macrophages, which release several microbicidal agents, including peptidases, to eliminate the parasite. Leishmanicidal mediators released in large amounts may cause morphological and/or functional injuries to the liver. In order to investigate the involvement of peptidases in this phenomenon, an in vitro co-culture model of peritoneal macrophages infected with L. chagasi and hepatocytes was used. High levels of released hepatic transaminases were found in supernatants from infected co-cultures at the same time point in which alterations in hepatocyte morphology and maximum proteolytic activity were observed. The largest proteolytic activity being at pH 10 as well as the greatest efficiency of treatment with 1,10-phenantroline observed in supernatants from the infected co-cultures suggests the presence of metallopeptidases during the leishmanicidal activity by infected macrophages. Furthermore, TNF-alpha levels and high levels of TGF-beta were increased at this time point, and this can be related to the synthesis of metallopeptidases and the conversion of the latent form to the active form. Metallopeptidase activities were detected by gelatin SDS-PAGE in higher amounts in infected macrophages and co-culture supernatant; moreover, one metallopeptidase migrating at 85 kDa produced in excess (41% more) by infected macrophages was identified as MMP-9. This metallopeptidase may be participating in this phenomenon together with other leishmanicidal factors released by these host cells.

  17. 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.

  18. Macrophage Activation by Ursolic and Oleanolic Acids during Mycobacterial Infection.

    PubMed

    López-García, Sonia; Castañeda-Sanchez, Jorge Ismael; Jiménez-Arellanes, Adelina; Domínguez-López, Lilia; Castro-Mussot, Maria Eugenia; Hernández-Sanchéz, Javier; Luna-Herrera, Julieta

    2015-08-06

    Oleanolic (OA) and ursolic acids (UA) are triterpenes that are abundant in vegetables, fruits and medicinal plants. They have been described as active moieties in medicinal plants used for the treatment of tuberculosis. In this study, we analyzed the effects of these triterpenes on macrophages infected in vitro with Mycobacterium tuberculosis (MTB). We evaluated production of nitric oxide (NO), reactive oxygen species (ROS), and cytokines (TNF-α and TGF-β) as well as expression of cell membrane receptors (TGR5 and CD36) in MTB-infected macrophages following treatment with OA and UA. Triterpenes caused reduced MTB growth in macrophages, stimulated production of NO and ROS in the early phase, stimulated TNF-α, suppressed TGF-β and caused over-expression of CD36 and TGR5 receptors. Thus, our data suggest immunomodulatory properties of OA and UA on MTB infected macrophages. In conclusion, antimycobacterial effects induced by these triterpenes may be attributable to the conversion of macrophages from stage M2 (alternatively activated) to M1 (classically activated).

  19. [Application of cell co-culture techniques in medical studies].

    PubMed

    Luo, Yun; Sun, Gui-Bo; Qin, Meng; Yao, Fan; Sun, Xiao-Bo

    2012-11-01

    As the cell co-culture techniques can better imitate an in vivo environment, it is helpful in observing the interactions among cells and between cells and the culture environment, exploring the effect mechanisms of drugs and their possible targets and filling the gaps between the mono-layer cell culture and the whole animal experiments. In recently years, they has attracted much more attention from the medical sector, and thus becoming one of research hotspots in drug research and development and bio-pharmaceutical fields. The cell co-culture techniques, including direct and indirect methods, are mainly used for studying pathological basis, new-type treatment methods and drug activity screening. Existing cell co-culture techniques are used for more pharmacological studies on single drug and less studies on interaction of combined drugs, such as collaborative compatibility and attenuation and synergistic effect among traditional Chinese medicines (TCMs). In line with the action characteristics of multi-component and multi-target, the cell co-culture techniques provide certain reference value for future studies on the effect and mechanism of combined TCMs on organisms as well as new methods for studies on TCMs and their compounds. This essay summarizes cell co-culture methods and their application and look into the future of their application in studies on TCMs and compounds.

  20. Proatherogenic macrophage activities are targeted by the flavonoid quercetin.

    PubMed

    Lara-Guzman, Oscar J; Tabares-Guevara, Jorge H; Leon-Varela, Yudy M; Álvarez, Rafael M; Roldan, Miguel; Sierra, Jelver A; Londoño-Londoño, Julian A; Ramirez-Pineda, Jose R

    2012-11-01

    Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1β secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid.

  1. Human umbilical cord blood-stem cells direct macrophage polarization and block inflammasome activation to alleviate rheumatoid arthritis

    PubMed Central

    Shin, Tae-Hoon; Kim, Hyung-Sik; Kang, Tae-Wook; Lee, Byung-Chul; Lee, Hwa-Yong; Kim, Yoon-Jin; Shin, Ji-Hee; Seo, Yoojin; Won Choi, Soon; Lee, Seunghee; Shin, Kichul; Seo, Kwang-Won; Kang, Kyung-Sun

    2016-01-01

    Rheumatoid arthritis (RA) is a long-lasting intractable autoimmune disorder, which has become a substantial public health problem. Despite widespread use of biologic drugs, there have been uncertainties in efficacy and long-term safety. Mesenchymal stem cells (MSCs) have been suggested as a promising alternative for the treatment of RA because of their immunomodulatory properties. However, the precise mechanisms of MSCs on RA-related immune cells are not fully elucidated. The aim of this study was to investigate the therapeutic potential of human umbilical cord blood-derived MSCs (hUCB-MSCs) as a new therapeutic strategy for patients with RA and to explore the mechanisms underlying hUCB-MSC-mediated immunomodulation. Mice with collagen-induced arthritis (CIA) were administered with hUCB-MSCs after the onset of disease, and therapeutic efficacy was assessed. Systemic delivery of hUCB-MSCs significantly ameliorated the severity of CIA to a similar extent observed in the etanercept-treated group. hUCB-MSCs exerted this therapeutic effect by regulating macrophage function. To verify the regulatory effects of hUCB-MSCs on macrophages, macrophages were co-cultured with hUCB-MSCs. The tumor necrosis factor (TNF)-α-mediated activation of cyclooxygenase-2 and TNF-stimulated gene/protein 6 in hUCB-MSCs polarized naive macrophages toward an M2 phenotype. In addition, hUCB-MSCs down-regulated the activation of nucleotide-binding domain and leucine-rich repeat pyrin 3 inflammasome via a paracrine loop of interleukin-1β signaling. These immune-balancing effects of hUCB-MSCs were reproducible in co-culture experiments using peripheral blood mononuclear cells from patients with active RA. hUCB-MSCs can simultaneously regulate multiple cytokine pathways in response to pro-inflammatory cytokines elevated in RA microenvironment, suggesting that treatment with hUCB-MSCs could be an attractive candidate for patients with treatment-refractory RA. PMID:28005072

  2. Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

    PubMed Central

    Kodali, Vamsi; Littke, Matthew H.; Tilton, Susan C.; Teeguarden, Justin G.; Shi, Liang; Frevert, Charles W.; Wang, Wei; Pounds, Joel G.; Thrall, Brian D.

    2013-01-01

    Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen Streptococcus pneumoniae is altered by ENP pre-treatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pre-treatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pre-treatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from a M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNFα production, and diminished phagocytic activity toward S. pneumoniae. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and S. pneumonia, and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Nanotoxicology screening strategies

  3. Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

    SciTech Connect

    Kodali, Vamsi; Littke, Matthew H.; Tilton, Susan C.; Teeguarden, Justin G.; Shi, Liang; Frevert, Charles W.; Wang, Wei; Pounds, Joel G.; Thrall, Brian D.

    2013-08-27

    Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen Streptococcus pneumoniae is altered by ENP pretreatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pretreatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pretreatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from an M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNFα production, and diminished phagocytic activity toward S. pneumoniae. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and S. pneumonia and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Finally, nanotoxicology screening

  4. Effects of Histoplasma capsulatum on murine macrophage functions: inhibition of macrophage priming, oxidative burst, and antifungal activities.

    PubMed

    Wolf, J E; Abegg, A L; Travis, S J; Kobayashi, G S; Little, J R

    1989-02-01

    Histoplasma capsulatum yeast cells fail to trigger an oxidative burst response in normal murine macrophages. The results of this study, in which an in vitro assay of macrophage antifungal effects was used, extend these findings. During 18 h of incubation, unprimed elicited murine macrophages inhibited H. capsulatum growth only when macrophages were present in great excess. Gamma interferon (IFN-gamma)-primed macrophages showed enhanced fungal growth inhibition but a similar requirement for an excess of phagocytes. Macrophages containing heat-killed H. capsulatum exhibited diminished antifungal effects toward viable H. capsulatum and Saccharomyces cerevisiae cells. Parallel experiments showed no comparable effect of ingested latex particles on macrophage antifungal activity. Using chemiluminescence as a measure of the oxidative burst, we found that macrophages primed in vitro with IFN-gamma alone failed to exhibit a significant response to triggering by H. capsulatum yeast cells unless a second priming agent (tumor necrosis factor alpha or bacterial lipopolysaccharide) was added to IFN-gamma. Furthermore, macrophage priming with single agents was blocked by the prior ingestion of heat-killed H. capsulatum. These studies provide evidence that ingestion of H. capsulatum yeast cells can induce a prompt and enduring deactivation of murine macrophages.

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

    PubMed Central

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

    2016-01-01

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

  6. Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

    NASA Technical Reports Server (NTRS)

    Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

    2016-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by

  7. Macrophage heterogeneity and cholesterol homeostasis: classically-activated macrophages are associated with reduced cholesterol accumulation following treatment with oxidized LDL.

    PubMed

    Chu, Eugene M; Tai, Daven C; Beer, Jennifer L; Hill, John S

    2013-02-01

    Macrophages are centrally involved during atherosclerosis development and are the predominant cell type that accumulates cholesterol in the plaque. Macrophages however, are heterogeneous in nature reflecting a variety of microenvironments and different phenotypes may be more prone to contribute towards atherosclerosis progression. Using primary human monocyte-derived macrophages, we sought to evaluate one aspect of atherogenic potential of different macrophage phenotypes by determining their propensity to associate with and accumulate oxidized low density lipoprotein (oxLDL). Classically-activated macrophages treated simultaneously with interferon γ (IFNγ) and tumor necrosis factor α (TNFα) associated with less oxLDL and accumulated less cholesterol compared to untreated controls. The combined treatment of IFNγ and TNFα reduced the mRNA expression of CD36 and the expression of both cell surface CD36 and macrophage scavenger receptor 1 (MSR1) protein. Under oxLDL loaded conditions, IFNγ and TNFα did not reduce macrophage protein expression of the transcription factor peroxisome proliferator-actived receptor γ (PPARγ) which is known to positively regulate CD36 expression. However, macrophages treated with IFNγ attenuated the ability of the PPARγ-specific agonist rosiglitazone from upregulating cell surface CD36 protein expression. Our results demonstrate that the observed reduction of cholesterol accumulation in macrophages treated with IFNγ and TNFα following oxLDL treatment was due at least in part to reduced cell surface CD36 and MSR1 protein expression. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Neurotrophic effects of the pineal gland: role of non-neuronal cells in co-cultures of the pineal gland and superior cervical ganglia.

    PubMed

    McNulty, J A; Tsai, S Y; Fox, L M; Madsen, T M; Silberman, S; Tonder, N

    1995-08-01

    The pineal gland (PG) is a source of several trophic factors. In this study, PG and superior cervical ganglia (SCG) from Sprague-Dawley neonates (1-day-old) were co-cultured to test the hypothesis that endogenous release of PG NGF (or an NGF-like cytokine) is sufficient to promote survival of SCG neurons. Neuronal density of SCG neurons was significantly enhanced when co-cultured with PG for 7 days compared to SCG cultured alone. SCG survival and neurite formation in PG co-cultures was less than in SCG treated with exogenous NGF (100 ng/ml). The neurotrophic effect of PG co-cultures was abolished when 1% anti-NGF was added to the medium. Co-cultures of SCG neurons with established 7-day PG cultures induced extensive SCG neurite formation within 24 hr compared to SCG co-cultured with 1-day PG cultures. This suggests that PG neurotrophic effects are due to PG non-neuronal cells (nnc) that proliferate to confluency by 7 days in culture. S-antigen-positive pinealocytes did not proliferate in culture. There was decreased SCG survival when neurons were seeded onto PG cultures that had been previously killed by drying, which suggests that the neurotrophic effects of nnc are not substrate-dependent. Immunocytochemical characterization of PG nnc revealed a heterogenous mixture of astrocytes, macrophage/microglia, and fibroblasts. These findings support the hypothesis that NGF is actively secreted by PG and that nnc are the principal source of this neurotophin.

  9. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    PubMed Central

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  10. Carbon nanohorns allow acceleration of osteoblast differentiation via macrophage activation

    NASA Astrophysics Data System (ADS)

    Hirata, Eri; Miyako, Eijiro; Hanagata, Nobutaka; Ushijima, Natsumi; Sakaguchi, Norihito; Russier, Julie; Yudasaka, Masako; Iijima, Sumio; Bianco, Alberto; Yokoyama, Atsuro

    2016-07-01

    Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the proof-of-concept on the osteoblast differentiation capacity by CNHs will allow future studies focused on CNHs as ideal therapeutic materials for bone regeneration.Carbon nanohorns (CNHs), formed by a rolled graphene structure and terminating in a cone, are promising nanomaterials for the development of a variety of biological applications. Here we demonstrate that alkaline phosphatase activity is dramatically increased by coculture of human monocyte derived macrophages (hMDMs) and human mesenchymal stem cells (hMSCs) in the presence of CNHs. CNHs were mainly localized in the lysosome of macrophages more than in hMSCs during coculturing. At the same time, the amount of Oncostatin M (OSM) in the supernatant was also increased during incubation with CNHs. Oncostatin M (OSM) from activated macrophage has been reported to induce osteoblast differentiation and matrix mineralization through STAT3. These results suggest that the macrophages engulfed CNHs and accelerated the differentiation of mesenchymal stem cells into the osteoblast via OSM release. We expect that the

  11. Dynamics of lung macrophage activation in response to helminth infection

    USDA-ARS?s Scientific Manuscript database

    Most of our understanding of the development and phenotype of alternatively activated macrophages (AAM) has been obtained from studies investigating the response of bone marrow- and peritoneal-derived cells to IL-4 or IL-13 stimulation. Comparatively little is known about the development of the AAM...

  12. Diet Modifies the Neuroimmune System by Influencing Macrophage Activation

    ERIC Educational Resources Information Center

    Sherry, Christina Lynn

    2009-01-01

    It has long been appreciated that adequate nutrition is required for proper immune function and it is now recognized that dietary components contribute to modulation of immune cells, subsequently impacting the whole body's response during an immune challenge. Macrophage activation plays a critical role in the immune system and directs the…

  13. Proteomic analysis of macrophage activated with salmonella lipopolysaccharide

    USDA-ARS?s Scientific Manuscript database

    Macrophages play pivotal role in immunity. They are activated by many pathogen derived molecules such as lipopolysaccharides (LPS) which trigger the production of various proteins and peptides that drive and resolve inflammation. There are numerous studies on the effect of LPS at the genome level bu...

  14. Diet Modifies the Neuroimmune System by Influencing Macrophage Activation

    ERIC Educational Resources Information Center

    Sherry, Christina Lynn

    2009-01-01

    It has long been appreciated that adequate nutrition is required for proper immune function and it is now recognized that dietary components contribute to modulation of immune cells, subsequently impacting the whole body's response during an immune challenge. Macrophage activation plays a critical role in the immune system and directs the…

  15. Immunity to Schistosoma mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae. T-cell activation of macrophages for larval killing.

    PubMed Central

    Gordon, J R; McLaren, D J

    1988-01-01

    This study addresses macrophage activation in guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosoma mansoni. Peritoneal exudate macrophages elicited in vaccinated animals by mineral oil injection were activated to kill larval schistosomes in vitro. Killing efficiency is dependent upon the cell: target ratio employed and is enhanced by, but is not strictly dependent on, the presence of specific antibodies. Macrophages co-cultured with parasites release superoxide radicals and hydrogen peroxide, but the use of inhibitors has shown that neither of these reactive oxygen intermediates are the causal agents of cellular cytotoxicity in this system. Oil-elicited macrophages from naive guinea-pigs do not show comparable activation; they can, however, be activated in vitro by incubation with culture supernatant fluids from schistosome antigen-stimulated spleen, or lymph node cells harvested from vaccinated guinea-pigs. Naive macrophages activated in this way kill schistosomula in vitro and release the activation markers IL-1 and superoxide anion. The macrophage-activating factor (MAF) present in spleen cell culture supernatant fluids has a MW of 35,000-55,000, but does not have the chemical characteristics of gamma-interferon. In this study MAF is shown to be released by a population of lymph node cells that does not adhere to nylon-wool columns, that responds well in proliferation assays to schistosome antigens and to the T-cell mitogen concanavalin A, but does not respond to the B-cell mitogen lipopolysaccharide. These cells have been identified as small lymphocytes. PMID:2832308

  16. Hypocholesterolaemic Activity of Lupin Peptides: Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells

    PubMed Central

    Lammi, Carmen; Zanoni, Chiara; Ferruzza, Simonetta; Ranaldi, Giulia; Sambuy, Yula; Arnoldi, Anna

    2016-01-01

    Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a) to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b) to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion. PMID:27455315

  17. An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

    PubMed

    Hasegawa, Ai; Oyanagi, Tomoya; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2014-11-01

    A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

  18. IKKβ Activity Drives Fetal Lung Macrophage Maturation Along a Non-M1/M2 Paradigm

    PubMed Central

    Stouch, Ashley N.; Zaynagetdinov, Rinat; Barham, Whitney J.; Stinnett, Amanda M.; Slaughter, James C.; Yull, Fiona E.; Hoffman, Hal M.; Blackwell, Timothy S.; Prince, Lawrence S.

    2014-01-01

    In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. While macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2 (FGL2), and indolamine-2, 3-dioxygenase (Ido1) were developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IKKβ transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11bloF4/80hi cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms. PMID:24981452

  19. Indoleamine 2,3-dioxygenase-1 (IDO1) in human endometrial stromal cells induces macrophage tolerance through interleukin-33 in the progression of endometriosis

    PubMed Central

    Mei, Jie; Xie, Xue-Xin; Li, Ming-Qing; Wei, Chun-Yan; Jin, Li-Ping; Li, Da-Jin; Zhu, Xiao-Yong

    2014-01-01

    In the peritoneal fluid, macrophages and their secretory cytokines are essential for endometriosis, but the factors that favor their involvement in the endometriosis-associated inflammatory response are still elusive. Given the anomalous expression of indoleamine 2,3-dioxygenase-1 (IDO1) in endometrial stromal cells (ESCs) and its potentially important roles in immune modulation, we aimed to determine the effects of IDO1 in ESCs on macrophages and the mechanism of those effects. Normal ESCs and ectopic ESCs transfected with the SD11-IDO1 shRNA (short hairpin RNA) or vector-only plasmid SD11 were subsequently co-cultured with peripheral blood (PB)-derived monocytes (PBMC)-driven macrophages directly and indirectly. Cytokine production was determined by analyzing the supernatant of the co-culture unit by enzyme-linked immunosorbent assay (ELISA). The phenotypes and the phagocytic ability of the macrophages were determined by flow cytometry. Compared to normal ESCs, the PBMC-driven macrophages co-cultured with ectopic ESCs displayed lower phagocytic ability. Additionally, macrophages co-cultured with ectopic ESCs exhibited higher levels of CD163 and CD209 and lower levels of HLA-DR and CD11c. Moreover, both the intracellular expression and extracellular secretion of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) were significantly increased, while that of IL-12p70 was decreased in macrophages after being co-cultured with ectopic ESCs. However, there was no significant difference in macrophage phagocytic ability, immunophenotype or cytokine secretion between the direct and indirect co-culture units. Reversely, SD11-IDO1 shRNA transfection of ectopic ESCs could abrogate the decreased phagocytic ability and alternative activation of macrophages in ectopic ESC-macrophage co-culture unit, suggesting that higher IDO1 in ectopic ESCs was indispensable for the induction of macrophage tolerance. Furthermore, the decrease in phagocytic macrophages and

  20. Modulation of nitric oxide synthase activity in macrophages

    PubMed Central

    Jorens, P. G.; Matthys, K. E.

    1995-01-01

    L-Arginine is converted to the highly reactive and unstable nitric oxide (NO) and L-citrulline by an enzyme named nitric oxide synthase (NOS). NO decomposes into other nitrogen oxides such as nitrite (NO2-) and nitrate (NO2-), and in the presence of superoxide anion to the potent oxidizing agent peroxynitrite (ONOO−). Activated rodent macrophages are capable of expressing an inducible form of this enzyme (iNOS) in response to appropriate stimuli, i.e., lipopolysaccharide (LPS) and interferon-γ (IFNγ). Other cytokines can modulate the induction of NO biosynthesis in macrophages. NO is a major effector molecule of the anti-microbial and cytotoxic activity of rodent macrophages against certain micro-organisms and tumour cells, respectively. The NO synthesizing pathway has been demonstrated in human monocytes and other cells, but its role in host defence seems to be accessory. A delicate functional balance between microbial stimuli, host-derived cytokines and hormones in the microenvironment regulates iNOS expression. This review will focus mainly on the known and proposed mechanisms of the regulation of iNOS induction, and on agents that can modulate NO release once the active enzyme has been expressed in the macrophage. PMID:18475620

  1. Macrophage-released ADAMTS1 promotes muscle stem cell activation.

    PubMed

    Du, Hongqing; Shih, Chung-Hsuan; Wosczyna, Michael N; Mueller, Alisa A; Cho, Joonseok; Aggarwal, Abhishek; Rando, Thomas A; Feldman, Brian J

    2017-09-22

    Coordinated activation of muscle stem cells (known as satellite cells) is critical for postnatal muscle growth and regeneration. The muscle stem cell niche is central for regulating the activation state of satellite cells, but the specific extracellular signals that coordinate this regulation are poorly understood. Here we show that macrophages at sites of muscle injury induce activation of satellite cells via expression of Adamts1. Overexpression of Adamts1 in macrophages in vivo is sufficient to increase satellite cell activation and improve muscle regeneration in young mice. We demonstrate that NOTCH1 is a target of ADAMTS1 metalloproteinase activity, which reduces Notch signaling, leading to increased satellite cell activation. These results identify Adamts1 as a potent extracellular regulator of satellite cell activation and have significant implications for understanding the regulation of satellite cell activity and regeneration after muscle injury.Satellite cells are crucial for growth and regeneration of skeletal muscle. Here the authors show that in response to muscle injury, macrophages secrete Adamts1, which induces satellite cell activation by modulating Notch1 signaling.

  2. Macrophages make me sick: how macrophage activation states influence sickness behavior.

    PubMed

    Moon, Morgan L; McNeil, Leslie K; Freund, Gregory G

    2011-11-01

    The macrophage (MΦ) is an essential cellular first responder in the innate immune system, sensing, alerting, removing and destroying intrinsic and extrinsic pathogens. While congenital aplasia of granulocytes, T or B lymphocytes leads to serious disease, lack of MΦs is incompatible with life. The MΦ, however, is not a monomorphic entity. These constructers, repairers and defenders of the body are diverse in form and function. What controls MΦ phenotype is beginning to be understood and involves a complex interplay of origination, location and microenvironment. Common to all MΦ developmental pathways are pro-inflammatory and anti-inflammatory cytokines. MΦs respond to these bioactives in distinct ways developing recently recognized activation phenotypes that canonically support bacterial clearance (classical activation), parasite defense/tissue repair (alternative activation) and anti-inflammation (deactivation). Critically, the same cytokines which orchestrate immune defense and homeostasis dramatically impact sense of well being and cognition by eliciting sickness symptoms. Such behaviors are the manifestation of pro/anti-inflammatory cytokine action in the brain and are a direct consequence of MΦ function. This review describes the "new" archetypal MΦ activation states, delineates microglia phenotypic plasticity and explores the importance of these macrophage activation states to sickness behavior.

  3. Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis

    PubMed Central

    Wu, Weicheng; Mosteller, Raymond D.; Broek, Daniel

    2004-01-01

    Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-κB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria. PMID:15314148

  4. Role of activated macrophages in experimental Fusarium solani keratitis.

    PubMed

    Hu, Jianzhang; Hu, Yingfeng; Chen, Shikun; Dong, Chenhuan; Zhang, Jingjin; Li, Yanling; Yang, Juan; Han, Xiaoli; Zhu, Xuejun; Xu, Guoxing

    2014-12-01

    Macrophages under the conjunctival tissue are the first line defender cells of the corneas. Elimination of these cells would lead to aggravation of fungal keratitis. To determine how the course of fungal keratitis would be altered after the activation of these macrophages, a murine model was achieved by intrastromal instillation of latex beads before the corneas were infected with Fusarium solani. The keratitis was observed and clinically scored daily. Infected corneas were homogenized for colony counts. The levels of the IL-12, IL-4, MPO, MIF and iNOS cytokines were measured in the corneas using real-time polymerase chain reactions and enzyme-linked immunosorbent assays. CD3+, CD4+ and CD8+ lymphocytes in the corneas, submaxillary lymph nodes and peripheral blood were detected using immunohistochemistry and flow cytometry, respectively. The latex bead-treated mice exhibited aggravated keratitis. Substantially increased macrophage and polymorphonuclear leukocyte infiltration was detected in the corneas, although few colonies were observed. There was a marked increase in the IL-12, IL-4, MPO, MIF and iNOS expression in the corneas. The numbers of CD3+, CD4+ and CD8+ lymphocytes and the CD4+/CD8+ ratio were significantly enhanced in the corneas and submaxillary lymph nodes. However, the number of CD4+ lymphocytes was decreased in the peripheral blood, while the number of CD8+ lymphocytes increased. Collectively, our data demonstrate that the activation of macrophages in the cornea may cause an excessive immune response. Macrophages appear to play a critical role in regulating the immune response to corneal infections with F. solani.

  5. Rickettsia australis Activates Inflammasome in Human and Murine Macrophages

    PubMed Central

    Smalley, Claire; Bechelli, Jeremy; Rockx-Brouwer, Dedeke; Saito, Tais; Azar, Sasha R.; Ismail, Nahed; Walker, David H.; Fang, Rong

    2016-01-01

    Rickettsiae actively escape from vacuoles and replicate free in the cytoplasm of host cells, where inflammasomes survey the invading pathogens. In the present study, we investigated the interactions of Rickettsia australis with the inflammasome in both mouse and human macrophages. R. australis induced a significant level of IL-1β secretion by human macrophages, which was significantly reduced upon treatment with an inhibitor of caspase-1 compared to untreated controls, suggesting caspase-1-dependent inflammasome activation. Rickettsia induced significant secretion of IL-1β and IL-18 in vitro by infected mouse bone marrow-derived macrophages (BMMs) as early as 8–12 h post infection (p.i.) in a dose-dependent manner. Secretion of these cytokines was accompanied by cleavage of caspase-1 and was completely abrogated in BMMs deficient in caspase-1/caspase-11 or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), suggesting that R. australis activate the ASC-dependent inflammasome. Interestingly, in response to the same quantity of rickettsiae, NLRP3-/- BMMs significantly reduced the secretion level of IL-1β compared to wild type (WT) controls, suggesting that NLRP3 inflammasome contributes to cytosolic recognition of R. australis in vitro. Rickettsial load in spleen, but not liver and lung, of R. australis-infected NLRP3-/- mice was significantly greater compared to WT mice. These data suggest that NLRP3 inflammasome plays a role in host control of bacteria in vivo in a tissue-specific manner. Taken together, our data, for the first time, illustrate the activation of ASC-dependent inflammasome by R. australis in macrophages in which NLRP3 is involved. PMID:27362650

  6. Rickettsia australis Activates Inflammasome in Human and Murine Macrophages.

    PubMed

    Smalley, Claire; Bechelli, Jeremy; Rockx-Brouwer, Dedeke; Saito, Tais; Azar, Sasha R; Ismail, Nahed; Walker, David H; Fang, Rong

    2016-01-01

    Rickettsiae actively escape from vacuoles and replicate free in the cytoplasm of host cells, where inflammasomes survey the invading pathogens. In the present study, we investigated the interactions of Rickettsia australis with the inflammasome in both mouse and human macrophages. R. australis induced a significant level of IL-1β secretion by human macrophages, which was significantly reduced upon treatment with an inhibitor of caspase-1 compared to untreated controls, suggesting caspase-1-dependent inflammasome activation. Rickettsia induced significant secretion of IL-1β and IL-18 in vitro by infected mouse bone marrow-derived macrophages (BMMs) as early as 8-12 h post infection (p.i.) in a dose-dependent manner. Secretion of these cytokines was accompanied by cleavage of caspase-1 and was completely abrogated in BMMs deficient in caspase-1/caspase-11 or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), suggesting that R. australis activate the ASC-dependent inflammasome. Interestingly, in response to the same quantity of rickettsiae, NLRP3-/- BMMs significantly reduced the secretion level of IL-1β compared to wild type (WT) controls, suggesting that NLRP3 inflammasome contributes to cytosolic recognition of R. australis in vitro. Rickettsial load in spleen, but not liver and lung, of R. australis-infected NLRP3-/- mice was significantly greater compared to WT mice. These data suggest that NLRP3 inflammasome plays a role in host control of bacteria in vivo in a tissue-specific manner. Taken together, our data, for the first time, illustrate the activation of ASC-dependent inflammasome by R. australis in macrophages in which NLRP3 is involved.

  7. ROS is Required for Alternatively Activated Macrophage Differentiation | Center for Cancer Research

    Cancer.gov

    Macrophages are key regulators in host inflammatory responses. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are responsible for inducing macrophage differentiation from monocytes. GM-CSF or M-CSF-differentiated macrophages can be further differentiated, or polarized, to more specialized cells. Classically activated, or M1, macrophages have immune-stimulatory properties and cytotoxic function against tumor cells. Alternatively activated, or M2, macrophages have low cytotoxic function but high tissue-remodeling activity. There are also M2-like cells called tumor-associated macrophages (TAMs) that are responsible for many tumor-promoting activities. Blocking the function of TAMs inhibits tumorigenesis.

  8. Role of Chemokines in Shaping Macrophage Activity in AMD.

    PubMed

    Rutar, Matt; Provis, Jan M

    2016-01-01

    Age-related macular degeneration (AMD) is a multifactorial disorder that affects millions of individuals worldwide. While the advent of anti-VEGF therapy has allowed for effective treatment of neovascular 'wet' AMD, no treatments are available to mitigate the more prevalent 'dry' forms of the disease. A role for inflammatory processes in the progression of AMD has emerged over a period of many years, particularly the characterisation of leukocyte infiltrates in AMD-affected eyes, as well as in animal models. This review focuses on the burgeoning understanding of chemokines in the retina, and their potential role in shaping the recruitment and activation of macrophages in AMD. Understanding the mechanisms which promote macrophage activity in the degenerating retina may be key to controlling the potentially devastating consequences of inflammation in diseases such as AMD.

  9. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

    PubMed

    Murthy, Shubha; Larson-Casey, Jennifer L; Ryan, Alan J; He, Chao; Kobzik, Lester; Carter, A Brent

    2015-08-01

    Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-β1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention.

  10. Phospholipase A2-modified low-density lipoprotein activates macrophage peroxisome proliferator-activated receptors.

    PubMed

    Namgaladze, Dmitry; Morbitzer, Daniel; von Knethen, Andreas; Brüne, Bernhard

    2010-02-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors modulating metabolic and inflammatory responses of phagocytes to stimuli such as fatty acids and their metabolites. We studied the role of PPARs in macrophages exposed to low-density lipoprotein (LDL) modified by secretory phospholipase A(2) (PLA). By analyzing PPAR ligand-binding domain luciferase reporter activation, we observed that PLA-LDL transactivates PPARalpha and PPARdelta, but not PPARgamma. We confirmed that PLA-LDL induced PPAR response element reporter activation by endogenous PPARalpha and PPARdelta in human THP-1 macrophages. By using THP-1 cells with a stable knockdown of PPARalpha and PPARdelta, we showed that PLA-LDL-activated PPARdelta altered macrophage gene expression related to lipid metabolism and lipid droplet formation. Although PPARalpha/delta silencing did not affect cholesterol and triglyceride accumulation in PLA-LDL-treated macrophages, PPARdelta activation by PLA-LDL attenuated macrophage inflammatory gene expression induced by interferon gamma and lipopolysaccharide. PPARdelta activation by PLA-LDL does not influence lipid accumulation in PLA-LDL-treated macrophages. However, it attenuates macrophage inflammatory responses, thus contributing to an anti-inflammatory cell phenotype.

  11. Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.

    PubMed

    Falchi, Mario; Varricchio, Lilian; Martelli, Fabrizio; Masiello, Francesca; Federici, Giulia; Zingariello, Maria; Girelli, Gabriella; Whitsett, Carolyn; Petricoin, Emanuel F; Moestrup, Søren Kragh; Zeuner, Ann; Migliaccio, Anna Rita

    2015-02-01

    Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages. Copyright© Ferrata Storti Foundation.

  12. RP105 facilitates macrophage activation by Mycobacterium tuberculosis lipoproteins.

    PubMed

    Blumenthal, Antje; Kobayashi, Toshihiko; Pierini, Lynda M; Banaei, Niaz; Ernst, Joel D; Miyake, Kensuke; Ehrt, Sabine

    2009-01-22

    RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

  13. Refractory ceramic fibers activate alveolar macrophage eicosanoid and cytokine release.

    PubMed

    Leikauf, G D; Fink, S P; Miller, M L; Lockey, J E; Driscoll, K E

    1995-01-01

    Refractory ceramic fiber has been developed for industrial processes requiring materials with high thermal and mechanical stability. To evaluate the biological activity of this fiber, rat alveolar macrophages were exposed for < or = 24 h to 0-1,000 micrograms/ml of refractory ceramic fiber, crocidolite asbestos, silica (fibrogenic particles), or titanium dioxide (a nonfibrogenic particle), and eicosanoid, tumor necrosis factor-alpha (TNF), and lactate dehydrogenase release were measured. Particle dimensions were determined by electron microscopy. Radioactivity coeluting with leukotriene B4 (LTB4) and immunoreactive LTB4 and TNF release increased after refractory ceramic fiber and were similar in magnitude after asbestos but less than after silica. For example, the total [3H]eicosanoid release increased 3.9-fold after refractory ceramic fiber, 4.6-fold after asbestos, and 8.7-fold after silica. Refractory ceramic fiber and asbestos also have similar particle dimensions (diameter, length, and surface area). Inasmuch as macrophage-derived LTB4 and TNF are potent mediators in inflammatory events, including migration and activation of neutrophils, these findings suggest that refractory ceramic fiber can activate macrophages in vitro to release mediators relevant to in vivo findings of inflammation and fibrotic lung disease in laboratory animals.

  14. A Triple Co-Culture Model of the Human Respiratory Tract to Study Immune-Modulatory Effects of Liposomes and Virosomes

    PubMed Central

    Krempaská, Kristína; Schaerer, Olivier; van Dijk, R. Maarten; Amacker, Mario; Moser, Christian; Hall, Sean R. R.; von Garnier, Christophe; Blank, Fabian

    2016-01-01

    The respiratory tract with its ease of access, vast surface area and dense network of antigen-presenting cells (APCs) represents an ideal target for immune-modulation. Bio-mimetic nanocarriers such as virosomes may provide immunomodulatory properties to treat diseases such as allergic asthma. In our study we employed a triple co-culture model of epithelial cells, macrophages and dendritic cells to simulate the human airway barrier. The epithelial cell line 16HBE was grown on inserts and supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) for exposure to influenza virosomes and liposomes. Additionally, primary human nasal epithelial cells (PHNEC) and EpCAM+ epithelial progenitor cell mono-cultures were utilized to simulate epithelium from large and smaller airways, respectively. To assess particle uptake and phenotype change, cell cultures were analyzed by flow cytometry and pro-inflammatory cytokine concentrations were measured by ELISA. All cell types internalized virosomes more efficiently than liposomes in both mono- and co-cultures. APCs like MDMs and MDDCs showed the highest uptake capacity. Virosome and liposome treatment caused a moderate degree of activation in MDDCs from mono-cultures and induced an increased cytokine production in co-cultures. In epithelial cells, virosome uptake was increased compared to liposomes in both mono- and co-cultures with EpCAM+ epithelial progenitor cells showing highest uptake capacity. In conclusion, all cell types successfully internalized both nanocarriers with virosomes being taken up by a higher proportion of cells and at a higher rate inducing limited activation of MDDCs. Thus virosomes may represent ideal carrier antigen systems to modulate mucosal immune responses in the respiratory tract without causing excessive inflammatory changes. PMID:27685460

  15. Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages.

    PubMed

    Barberà-Cremades, Maria; Baroja-Mazo, Alberto; Pelegrín, Pablo

    2016-02-01

    Macrophages represent a highly heterogenic cell population of the innate immune system, with important roles in the initiation and resolution of the inflammatory response. Purinergic signaling regulates both M1 and M2 macrophage function at different levels by controlling the secretion of cytokines, phagocytosis, and the production of reactive oxygen species. We found that extracellular nucleotides arrest macrophage differentiation from bone marrow precursors via adenosine and P2 receptors. This results in a mature macrophage with increased expression of M2, but not M1, genes. Similar to adenosine and ATP, macrophage growth arrested with LPS treatment resulted in an increase of the M2-related marker Ym1. Recombinant Ym1 was able to affect macrophage proliferation and could, potentially, be involved in the arrest of macrophage growth during hematopoiesis. © Society for Leukocyte Biology.

  16. Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia.

    PubMed

    Gonzalez, Angel; Restrepo, Angela; Cano, Luz E

    2007-01-01

    Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50 U/mL of IFN-gamma or treated with 35 microM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS04. The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

  17. By Homing to the Kidney, Activated Macrophages Potently Exacerbate Renal Injury

    PubMed Central

    Wang, Ying; Wang, Yiping; Cai, Qi; Zheng, Guoping; Lee, Vincent W.S.; Zheng, Dong; Li, Xiaomei; Kui Tan, Thian; Harris, David C.H.

    2008-01-01

    Macrophages are important mediators of injury in most types of human kidney diseases; however, the pathogenic importance of both macrophage number and activation status is unknown. To examine this question, severe-combined immunodeficient mice with adriamycin nephrosis, an experimental model of human focal segmental glomerulosclerosis, were treated intravenously with either resting (1 × 106 to 5 × 106) or activated (1 × 103 to 1 × 106) macrophages on day 6 postadriamycin administration, and the effects on kidney injury were examined. On day 28, renal injury was worse in the group that received activated macrophages at doses as low as 1 × 104 macrophages per mouse compared with control adriamycin nephrotic mice. However, treatment with resting macrophages at doses as high as 5 × 106 macrophages per mouse had no significant effect on either renal histology or function. The transferred activated macrophages homed to inflamed kidneys during the middle-to-late stages of the disease, but such homing was not observed for resting macrophages. This study of in vivo cell adoptive transfer supports the importance of macrophage activation status over macrophage number in causing renal injury. These data suggest that therapeutic strategies for treating progressive kidney diseases should target activated macrophages. PMID:18467704

  18. Progastrin Represses the Alternative Activation of Human Macrophages and Modulates Their Influence on Colon Cancer Epithelial Cells

    PubMed Central

    Hernández, Carlos; Barrachina, María Dolores; Cosín-Roger, Jesús; Ortiz-Masiá, Dolores; Álvarez, Ángeles; Terrádez, Liria; Nicolau, María Jesús; Alós, Rafael; Esplugues, Juan Vicente; Calatayud, Sara

    2014-01-01

    Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization. PMID:24901518

  19. Progastrin represses the alternative activation of human macrophages and modulates their influence on colon cancer epithelial cells.

    PubMed

    Hernández, Carlos; Barrachina, María Dolores; Cosín-Roger, Jesús; Ortiz-Masiá, Dolores; Álvarez, Ángeles; Terrádez, Liria; Nicolau, María Jesús; Alós, Rafael; Esplugues, Juan Vicente; Calatayud, Sara

    2014-01-01

    Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization.

  20. Co-culture in cartilage tissue engineering.

    PubMed

    Hendriks, Jeanine; Riesle, Jens; van Blitterswijk, Clemens A

    2007-01-01

    For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to observe, measure or manipulate cell behaviour and copying the in situ environment of that cell. Most tissues in the body consist of more than one cell type. The organization of the cells in the tissue is essential for the tissue's normal development, homeostasis and repair reaction. In a co-culture system, two or more cell types brought together in the same culture environment very likely interact and communicate. Co-culture has proved to be a powerful in vitro tool in unravelling the importance of cellular interactions during normal physiology, homeostasis, repair and regeneration. The first co-culture studies focused mainly on the influence of cellular interactions on oocytes maturation to a pre-implantation blastocyst. Therefore, a brief overview of these studies is given here. Later on in the history of co-culture studies, it was applied to study cell-cell communication, after which, almost immediately as the field of tissue engineering was recognized, it was introduced in tissue engineering to study cellular interactions and their influence on tissue formation. This review discusses the introduction and applications of co-culture systems in cell biology research, with the emphasis on tissue engineering and its possible application for studying cartilage regeneration.

  1. Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.

    PubMed

    de Leve, Simone; Wirsdörfer, Florian; Cappuccini, Federica; Schütze, Alexandra; Meyer, Alina V; Röck, Katharina; Thompson, Linda F; Fischer, Jens W; Stuschke, Martin; Jendrossek, Verena

    2017-07-01

    While radiotherapy is a mainstay for cancer therapy, pneumonitis and fibrosis constitute dose-limiting side effects of thorax and whole body irradiation. So far, the contribution of immune cells to disease progression is largely unknown. Here we studied the role of ecto-5'-nucelotidase (CD73)/adenosine-induced changes in the myeloid compartment in radiation-induced lung fibrosis. C57BL/6 wild-type or CD73(-/-) mice received a single dose of whole thorax irradiation (WTI, 15 Gy). Myeloid cells were characterized in flow cytometric, histologic, and immunohistochemical analyses as well as RNA analyses. WTI induced a pronounced reduction of alveolar macrophages in both strains that recovered within 6 wk. Fibrosis development in wild-type mice was associated with a time-dependent deposition of hyaluronic acid (HA) and increased expression of markers for alternative activation on alveolar macrophages. These include the antiinflammatory macrophage mannose receptor and arginase-1. Further, macrophages accumulated in organized clusters and expressed profibrotic mediators at ≥25 wk after irradiation (fibrotic phase). Irradiated CD73(-/-) mice showed an altered regulation of components of the HA system and no clusters of alternatively activated macrophages. We speculate that accumulation of alternatively activated macrophages in organized clusters represents the origins of fibrotic foci after WTI and is promoted by a cross-talk between HA, CD73/adenosine signaling, and other profibrotic mediators.-De Leve, S., Wirsdörfer, F., Cappuccini, F., Schütze, A., Meyer, A. V., Röck, K., Thompson, L. F., Fischer, J. W., Stuschke, M., Jendrossek, V. Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs. © FASEB.

  2. A Systematic Approach to Identify Markers of Distinctly Activated Human Macrophages

    PubMed Central

    Sudan, Bayan; Wacker, Mark A.; Wilson, Mary E.; Graff, Joel W.

    2015-01-01

    Polarization has been a useful concept for describing activated macrophage phenotypes and gene expression profiles. However, macrophage activation status within tumors and other settings are often inferred based on only a few markers. Complicating matters for relevance to human biology, many macrophage activation markers have been best characterized in mice and sometimes are not similarly regulated in human macrophages. To identify novel markers of activated human macrophages, gene expression profiles for human macrophages of a single donor subjected to 33 distinct activating conditions were obtained and a set of putative activation markers were subsequently evaluated in macrophages from multiple donors using integrated fluidic circuit (IFC)-based RT-PCR. Using unsupervised hierarchical clustering of the microarray screen, highly altered transcripts (>4-fold change in expression) sorted the macrophage transcription profiles into two major and 13 minor clusters. Among the 1874 highly altered transcripts, over 100 were uniquely altered in one major or two related minor clusters. IFC PCR-derived data confirmed the microarray results and determined the kinetics of expression of potential macrophage activation markers. Transcripts encoding chemokines, cytokines, and cell surface were prominent in our analyses. The activation markers identified by this study could be used to better characterize tumor-associated macrophages from biopsies as well as other macrophage populations collected from human clinical samples. PMID:26074920

  3. Delineation of Diverse Macrophage Activation Programs in Response to Intracellular Parasites and Cytokines

    PubMed Central

    Zhang, Shuyi; Kim, Charles C.; Batra, Sajeev; McKerrow, James H.; Loke, P'ng

    2010-01-01

    Background The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease) and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis). Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated. Methodology/Principal Findings To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome-wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites T. cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS), and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen L. mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. T. cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines. Conclusions/Significance This study provides global gene expression data for a diverse set of biologically significant pathogens and

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

    PubMed

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

    2015-04-01

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

  5. LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress.

    PubMed

    de la Haba, Carlos; Morros, Antoni; Martínez, Paz; Palacio, José R

    2016-12-01

    Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.

  6. Intracellular water motion decreases in apoptotic macrophages after caspase activation.

    PubMed

    Hortelano, S; García-Martín, M L; Cerdán, S; Castrillo, A; Alvarez, A M; Boscá, L

    2001-10-01

    Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.

  7. Multimodality PET/MR imaging agents targeted to activated macrophages

    PubMed Central

    Tu, Chuqiao; Ng, Thomas S. C.; Jacobs, Russell E.; Louie, Angelique Y.

    2013-01-01

    The recent emergence of multimodality imaging, particularly the combination of PET and MRI, has led to excitement over the prospect of improving detection of disease. Iron oxide nanoparticles (IONPs) have become a popular platform for the fabrication of PET/MRI probes due to their advantages of high MRI detection sensitivity, biocompatibility, and biodegradability. In this paper, we report the synthesis of dextran coated iron oxide nanoparticles labeled with the positron emitter 64Cu to generate a PET/MRI probe, and modified with small molecular maleic anhydride to increase negative surface charge. The modified nanoparticulate PET/MRI probe (MDIO-64Cu-DOTA) bears repetitive anionic charges on the surface that facilitate recognition by scavenger receptor type A (SR-A), a ligand-receptor found on activated macrophages but not on normal vessel walls. MDIO-64Cu-DOTA has an average iron oxide core size of 7-8 nm, an average hydrodynamic diameter of 62.7 nm, an r1 relaxivity of 16.8 mM−1·s−1, and an r2 relaxivity of 83.9 mM−1·s−1 (37 °C, 1.4 T). Cell studies confirmed that the probe was nontoxic and was specifically taken up by macrophages via SR-A. In comparison with the non-modified analog, the accumulation of maleylated DIO in macrophages was substantially improved. These characteristics demonstrate the promise of MDIO-64Cu-DOTA for identification of vulnerable atherosclerotic plaques (VAP) via the targeting of macrophages. PMID:24166283

  8. Lectin coated MgO nanoparticle: its toxicity, antileishmanial activity, and macrophage activation.

    PubMed

    Jebali, Ali; Hekmatimoghaddam, Seyedhossein; Kazemi, Bahram; Allaveisie, Azra; Masoudi, Alireza; Daliri, Karim; Sedighi, Najme; Ranjbari, Javad

    2014-10-01

    The purpose of this research was to evaluate toxicity of uncoated magnesium oxide nanoparticles (MgO NPs), MgO NPs coated with Peanut agglutinin (PNA) lectin, and PNA alone on the promastigotes of Leishmania major (L. major) and macrophages of BALB/c mice. On the other hand, antileishmanial property of uncoated MgO NPs, lectin coated MgO NPs, and PNA lectin alone was evaluated, and also macrophage activation was investigated after treatment with these materials by measurement of nitrite, H2O2, and some interleukins. This study showed that PNA lectin and lectin coated MgO NPs had approximately no toxicity on L. major and macrophages, but some toxic effects were observed for uncoated MgO NPs, especially at concentration of 500 µg/mL. Interestingly, lectin coated MgO NPs had the highest antileishmanial activity and macrophage activation, compared with uncoated MgO NPs and PNA lectin.

  9. New insights into the multidimensional concept of macrophage ontogeny, activation and function.

    PubMed

    Ginhoux, Florent; Schultze, Joachim L; Murray, Peter J; Ochando, Jordi; Biswas, Subhra K

    2016-01-01

    Macrophages have protective roles in immunity to pathogens, tissue development, homeostasis and repair following damage. Maladaptive immunity and inflammation provoke changes in macrophage function that are causative of disease. Despite a historical wealth of knowledge about macrophages, recent advances have revealed unknown aspects of their development and function. Following development, macrophages are activated by diverse signals. Such tissue microenvironmental signals together with epigenetic changes influence macrophage development, activation and functional diversity, with consequences in disease and homeostasis. We discuss here how recent discoveries in these areas have led to a multidimensional concept of macrophage ontogeny, activation and function. In connection with this, we also discuss how technical advances facilitate a new roadmap for the isolation and analysis of macrophages at high resolution.

  10. [Hepatic manifestation of a macrophage activation syndrome (MAS)].

    PubMed

    Nagel, Michael; Schwarting, Andreas; Straub, Beate K; Galle, Peter R; Zimmermann, Tim

    2017-04-04

    Background Elevated liver values are the most common pathological laboratory result in Germany. Frequent findings, especially in younger patients, are nutritive- or medicamentous- toxic reasons, viral or autoimmune hepatitis. A macrophage activation syndrome (MAS) may manifest like a viral infectious disease with fever, hepatosplenomegaly and pancytopenia and is associated with a high mortality. It is based on an enhanced activation of macrophages with increased cytokine release, leading to organ damage and multi-organ failure. In addition to genetic causes, MAS is commonly associated with infections and rheumatic diseases. We report the case of a 26-year-old female patient suffering from MAS as a rare cause of elevated liver enzymes. Methods Patient characteristics, laboratory values, liver histology, bone marrow and radiological imaging were documented and analyzed. Case Report After an ordinary upper airway infection with bronchitis, a rheumatic arthritis appeared and was treated with leflunomide und methotrexate. In the further course of the disease, the patient developed an acute hepatitis with fever, pancytopenia and massive hyperferritinemia. Immunohistochemistry of the liver biopsy revealed hemophagocytosis and activation of CD68-positive macrophages. In the radiological and histological diagnostics of the liver and bone marrow, an MAS was diagnosed as underlying disease of the acute hepatitis. Under therapy with prednisolone, the fever disappeared and transaminases and ferritin rapidly normalized. Conclusion Aside from the frequent causes of elevated liver values in younger patients, such as nutritive toxic, drug induced liver injury, viral or autoimmune hepatitis, especially in case of massive hyperferritinemia, a MAS should be considered as a rare cause of acute liver disease.

  11. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages

    PubMed Central

    Moreira-Souza, Aline Cristina Abreu; Marinho, Ygor; Correa, Gladys; Santoro, Giani França; Coutinho, Claudia Mara Lara Melo; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

    2015-01-01

    Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection. PMID:26192447

  12. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

    PubMed

    Moreira-Souza, Aline Cristina Abreu; Marinho, Ygor; Correa, Gladys; Santoro, Giani França; Coutinho, Claudia Mara Lara Melo; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

    2015-01-01

    Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

  13. Three-Dimensional Co-Culture Process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor)

    1997-01-01

    By the process of the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and had some of the characteristics of in vivo tissue. The process provides enhanced 3-dimensional tissue which creates a multicellular organoid differentiation model.

  14. Asbestos-activated peritoneal macrophages release a factors(s) which inhibits lymphocyte mitogenesis

    SciTech Connect

    Donaldson, K.; Davis, J.M.G.; James, K.

    1984-10-01

    Intraperitoneal asbestos injection in mice has previously been reported to elicit an activated macrophage population. In the present study supernatants from such macrophages were tested for their effect on thymocyte mitogenesis in response to concanavalin A; control supernantants were obtained from saline- and latex-elicited macrophages. Supernatants from asbestos-elicited macrophages were significantly inhibitory to thymocyte mitogenesis while saline- and latex-elicited macrophages did not release significant amounts of such activity. Asbestos-activated macrophage supernatants were inhibitory in a dose-dependent way and the activity was not secreted by macrophages from mice which had received asbestos in the long term. The inhibitory activity was partially dialysable. Supernatants prepared by treating macrophages in vitro with a lethal dose of asbestos were not inhibitory suggesting that the inhibitory activity in the supernatants of asbestos-activated macrophages did not leak from dead or dying cells. The asbestos macrophage supernatant was also significantly inhibitory to mature T-cell-enriched spleen cells but had no effect on fibroblasts, suggesting that the inhibitory effect could be lymphoid cell specific.

  15. Three-dimensional co-culture process

    NASA Technical Reports Server (NTRS)

    Wolf, David A. (Inventor); Goodwin, Thomas J. (Inventor)

    1992-01-01

    The present invention relates to a 3-dimensional co-culture process, more particularly to methods or co-culturing at least two types of cells in a culture environment, either in space or in unit gravity, with minimum shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region to form 3-dimensional tissue-like structures. Several examples of multicellular 3-dimensional experiences are included. The protocol and procedure are also set forth. The process allows simultaneous culture of multiple cell types and supporting substrates in a manner which does not disrupt the 3-dimensional spatial orientation of these components. The co-cultured cells cause a mutual induction effect which mimics the natural hormonal signals and cell interactions found in the intact organism. This causes the tissues to differentiate and form higher 3-dimensional structures such as glands, junctional complexes polypoid geometries, and microvilli which represent the corresponding in-vitro structures to a greater degree than when the cell types are cultured individually or by conventional processes. This process was clearly demonstrated for the case of two epithelial derived colon cancer lines, each co-cultured with normal human fibroblasts and with microcarrier bead substrates. The results clearly demonstrate increased 3-dimensional tissue-like structure and biochemical evidence of an increased differentiation state. With the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and has some of the characteristics of in-vitro tissue. The process provides enhanced 3-dimensional tissue which create a multicellular organoid differentiation model.

  16. Control of macrophage metabolism and activation by mTOR and Akt signaling

    PubMed Central

    Covarrubias, Anthony J.; Aksoylar, H. Ibrahim; Horng, Tiffany

    2015-01-01

    Macrophages are pleiotropic cells that assume a variety of functions depending on their tissue of residence and tissue state. They maintain homeostasis as well as coordinate responses to stresses such as infection and metabolic challenge. The ability of macrophages to acquire diverse, context-dependent activities requires their activation (or polarization) to distinct functional states. While macrophage activation is well understood at the level of signal transduction and transcriptional regulation, the metabolic underpinnings are poorly understood. Importantly, emerging studies indicate that metabolic shifts play a pivotal role in control of macrophage activation and acquisition of context-dependent effector activities. The signals that drive macrophage activation impinge on metabolic pathways, allowing for coordinate control of macrophage activation and metabolism. Here we discuss how mTOR and Akt, major metabolic regulators and targets of such activation signals, control macrophage metabolism and activation. Dysregulated macrophage activities contribute to many diseases, including infectious, inflammatory, and metabolic diseases and cancer, thus a better understanding of metabolic control of macrophage activation could pave the way to the development of new therapeutic strategies. PMID:26360589

  17. Control of macrophage metabolism and activation by mTOR and Akt signaling.

    PubMed

    Covarrubias, Anthony J; Aksoylar, H Ibrahim; Horng, Tiffany

    2015-08-01

    Macrophages are pleiotropic cells that assume a variety of functions depending on their tissue of residence and tissue state. They maintain homeostasis as well as coordinate responses to stresses such as infection and metabolic challenge. The ability of macrophages to acquire diverse, context-dependent activities requires their activation (or polarization) to distinct functional states. While macrophage activation is well understood at the level of signal transduction and transcriptional regulation, the metabolic underpinnings are poorly understood. Importantly, emerging studies indicate that metabolic shifts play a pivotal role in control of macrophage activation and acquisition of context-dependent effector activities. The signals that drive macrophage activation impinge on metabolic pathways, allowing for coordinate control of macrophage activation and metabolism. Here we discuss how mTOR and Akt, major metabolic regulators and targets of such activation signals, control macrophage metabolism and activation. Dysregulated macrophage activities contribute to many diseases, including infectious, inflammatory, and metabolic diseases and cancer, thus a better understanding of metabolic control of macrophage activation could pave the way to the development of new therapeutic strategies.

  18. Effect of low-level laser therapy on the modulation of the mitochondrial activity of macrophages

    PubMed Central

    Souza, Nadhia H. C.; Ferrari, Raquel A. M.; Silva, Daniela F. T.; Nunes, Fabio D.; Bussadori, Sandra K.; Fernandes, Kristianne P. S.

    2014-01-01

    BACKGROUND: Macrophages play a major role among the inflammatory cells that invade muscle tissue following an injury. Low-level laser therapy (LLLT) has long been used in clinical practice to accelerate the muscle repair process. However, little is known regarding its effect on macrophages. OBJECTIVE: This study evaluated the effect of LLLT on the mitochondrial activity (MA) of macrophages. METHOD: J774 macrophages were treated with lipopolysaccharide (LPS) and interferon - gamma (IFN-γ) (activation) for 24 h to simulate an inflammatory process, then irradiated with LLLT using two sets of parameters (780 nm; 70 mW; 3 J/cm2 and 660 nm; 15 mW; 7.5 J/cm2). Non-activated/non-irradiated cells composed the control group. MA was evaluated by the cell mitochondrial activity (MTT) assay (after 1, 3 and 5 days) in three independent experiments. The data were analyzed statistically. RESULTS: After 1 day of culture, activated and 780 nm irradiated macrophages showed lower MA than activated macrophages, but activated and 660 nm irradiated macrophages showed MA similar to activated cells. After 3 days, activated and irradiated (660 nm and 780 nm) macrophages showed greater MA than activated macrophages, and after 5 days, the activated and irradiated (660 nm and 780 nm) macrophages showed similar MA to the activated macrophages. CONCLUSIONS: These results show that 660 nm and 780 nm LLLT can modulate the cellular activation status of macrophages in inflammation, highlighting the importance of this resource and of the correct determination of its parameters in the repair process of skeletal muscle. PMID:25076002

  19. Effect of low-level laser therapy on the modulation of the mitochondrial activity of macrophages.

    PubMed

    Souza, Nadhia H C; Ferrari, Raquel A M; Silva, Daniela F T; Nunes, Fabio D; Bussadori, Sandra K; Fernandes, Kristianne P S

    2014-01-01

    Macrophages play a major role among the inflammatory cells that invade muscle tissue following an injury. Low-level laser therapy (LLLT) has long been used in clinical practice to accelerate the muscle repair process. However, little is known regarding its effect on macrophages. This study evaluated the effect of LLLT on the mitochondrial activity (MA) of macrophages. J774 macrophages were treated with lipopolysaccharide (LPS) and interferon - gamma (IFN-γ) (activation) for 24 h to simulate an inflammatory process, then irradiated with LLLT using two sets of parameters (780 nm; 70 mW; 3 J/cm2 and 660 nm; 15 mW; 7.5 J/cm2). Non-activated/non-irradiated cells composed the control group. MA was evaluated by the cell mitochondrial activity (MTT) assay (after 1, 3 and 5 days) in three independent experiments. The data were analyzed statistically. After 1 day of culture, activated and 780 nm irradiated macrophages showed lower MA than activated macrophages, but activated and 660 nm irradiated macrophages showed MA similar to activated cells. After 3 days, activated and irradiated (660 nm and 780 nm) macrophages showed greater MA than activated macrophages, and after 5 days, the activated and irradiated (660 nm and 780 nm) macrophages showed similar MA to the activated macrophages. These results show that 660 nm and 780 nm LLLT can modulate the cellular activation status of macrophages in inflammation, highlighting the importance of this resource and of the correct determination of its parameters in the repair process of skeletal muscle.

  20. Posttranscriptional control of NLRP3 inflammasome activation in colonic macrophages.

    PubMed

    Filardy, A A; He, J; Bennink, J; Yewdell, J; Kelsall, B L

    2016-07-01

    Colonic macrophages (cMPs) are important for intestinal homeostasis as they kill microbes and yet produce regulatory cytokines. Activity of the NLRP3 (nucleotide-binding leucine-rich repeat-containing pyrin receptor 3) inflammasome, a major sensor of stress and microorganisms that results in pro-inflammatory cytokine production and cell death, must be tightly controlled in the intestine. We demonstrate that resident cMPs are hyporesponsive to NLRP3 inflammasome activation owing to a remarkable level of posttranscriptional control of NLRP3 and pro-interleukin-1β (proIL-1β) protein expression, which was also seen for tumor necrosis factor-α and IL-6, but lost during experimental colitis. Resident cMPs rapidly degraded NLRP3 and proIL-1β proteins by the ubiquitin/proteasome system. Finally, blocking IL-10R-signaling in vivo enhanced NLRP3 and proIL-1β protein but not mRNA levels in resident cMPs, implicating a role for IL-10 in environmental conditioning of cMPs. These data are the first to show dramatic posttranscriptional control of inflammatory cytokine production by a relevant tissue-derived macrophage population and proteasomal degradation of proIL-1β and NLRP3 as a mechanism to control inflammasome activation, findings which have broad implications for our understanding of intestinal and systemic inflammatory diseases.

  1. LL-37 Immunomodulatory Activity during Mycobacterium tuberculosis Infection in Macrophages

    PubMed Central

    Torres-Juarez, Flor; Cardenas-Vargas, Albertina; Montoya-Rosales, Alejandra; González-Curiel, Irma; Garcia-Hernandez, Mariana H.; Enciso-Moreno, Jose A.; Hancock, Robert E. W.

    2015-01-01

    Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines. PMID:26351280

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

    PubMed

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

    2017-09-01

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

  3. Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages

    PubMed Central

    Shahraz, Anahita; Kopatz, Jens; Mathy, Rene; Kappler, Joachim; Winter, Dominic; Kapoor, Shoba; Schütza, Vlad; Scheper, Thomas; Gieselmann, Volkmar; Neumann, Harald

    2015-01-01

    Oligosialic and polysialic acid (oligoSia and polySia) of the glycocalyx of neural and immune cells are linear chains, in which the sialic acid monomers are α2.8-glycosidically linked. Sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC-11) is a primate-lineage specific receptor of human tissue macrophages and microglia that binds to α2.8-linked oligoSia. Here, we show that soluble low molecular weight polySia with an average degree of polymerization 20 (avDP20) interacts with SIGLEC-11 and acts anti-inflammatory on human THP1 macrophages involving the SIGLEC-11 receptor. Soluble polySia avDP20 inhibited the lipopolysaccharide (LPS)-induced gene transcription and protein expression of tumor necrosis factor-α (Tumor Necrosis Factor Superfamily Member 2, TNFSF2). In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis. Moreover, polySia avDP20 prevented the oxidative burst of human macrophages triggered by neural debris or fibrillary amyloid-β1–42. In a human macrophage-neuron co-culture system, polySia avDP20 also reduced loss of neurites triggered by fibrillary amyloid-β1–42. Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages. PMID:26582367

  4. NF-kappaB Activity in Macrophages Determines Metastatic Potential of Breast Tumor Cells

    DTIC Science & Technology

    2010-08-01

    macrophages inhibit mammary to lung metastasis? BODY Task 1. Determine effects of increased NF-κB activity in macrophages on mammary to lung...metastasis: Determine effects of increased NF-κB activity in macrophages on lung tumor development using a tail vein metastasis model. These...suggested that activation of NF-κB would have pro-tumor effects resulting in greater numbers of lung metastases. We were therefore surprised when our

  5. A co-culture-based model of human blood-brain barrier: application to active transport of indinavir and in vivo-in vitro correlation.

    PubMed

    Megard, Isabelle; Garrigues, Alexia; Orlowski, Stéphane; Jorajuria, Sylvie; Clayette, Pascal; Ezan, Eric; Mabondzo, Aloïse

    2002-02-15

    The growing array of in vitro models of the blood-brain barrier (BBB) which have been used makes it difficult to draw firm conclusions concerning the BBB penetration of HIV-1 protease inhibitors. What is needed is a combined in vivo and in vitro study on biological models that mimic as closely as possible the normal human BBB, to establish whether and how indinavir crosses the BBB. We developed a new human BBB model using primary endothelial cells and astrocytes. The biological relevance of this model was checked with respect on the one hand, to the close relationship between the log of drug permeability coefficient normalized to molecular weight and the log of the 1-octanol/water partition coefficient, and on the other hand to the functional P-glycoprotein (P-gp) expression. We employed this model to perform transport studies with indinavir and showed that the rate of in vitro indinavir transport from the basal to apical compartment was higher than the rate of apical to basal transport. Pretreatment of the BBB model with the P-gp inhibitor, quinidine, significantly increased apical to basal transport. Intracellular indinavir accumulation was increased in BBB as a result of inhibition of active transport. These data were correlated with the indinavir-mediated P-gp ATPase modulation showing that indinavir specifically interacted with a binding site on P-gp. Moreover, the activation of P-gp ATPase by indinavir was inhibited by quinidine. In addition, the in vivo brain to plasma concentration ratio of indinavir into mice showed that indinavir concentration was up to five times higher in the brain of mdr1a(-/-) mice than in the brain of mdr1a(+/+) mice. All these results confirm the role of P-gp in preventing the passage of indinavir across BBB and thus its entry into the central nervous system (CNS). Our human BBB model represents a useful tool for the evaluation of drug penetration into the CNS.

  6. Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation

    PubMed Central

    2014-01-01

    Background In neuroinflammatory diseases, macrophages can play a dual role in the process of tissue damage, depending on their activation status (M1 / M2). M1 macrophages are considered to exert damaging effects to neurons, whereas M2 macrophages are reported to aid regeneration and repair of neurons. Their migration within the central nervous system may be of critical importance in the final outcome of neurodegeneration in neuroinflammatory diseases e.g. multiple sclerosis (MS). To provide insight into this process, we examined the migratory capacity of human monocyte-derived M1 and M2 polarised macrophages towards chemoattractants, relevant for neuroinflammatory diseases like MS. Methods Primary cultures of human monocyte-derived macrophages were exposed to interferon gamma and lipopolysaccharide (LPS) to evoke proinflammatory (M1) activation or IL-4 to evoke anti-inflammatory (M2) activation. In a TAXIScan assay, migration of M0, M1 and M2 towards chemoattractants was measured and quantified. Furthermore the adhesion capacity and the expression levels of integrins as well as chemokine receptors of M0, M1 and M2 were assessed. Alterations in cell morphology were analysed using fluorescent labelling of the cytoskeleton. Results Significant differences were observed between M1 and M2 macrophages in the migration towards chemoattractants. We show that M2 macrophages migrated over longer distances towards CCL2, CCL5, CXCL10, CXCL12 and C1q compared to non-activated (M0) and M1 macrophages. No differences were observed in the adhesion of M0, M1 and M2 macrophages to multiple matrix components, nor in the expression of integrins and chemokine receptors. Significant changes were observed in the cytoskeleton organization upon stimulation with CCL2, M0, M1 and M2 macrophages adopt a spherical morphology and the cytoskeleton is rapidly rearranged. M0 and M2 macrophages are able to form filopodia, whereas M1 macrophages only adapt a spherical morphology. Conclusions

  7. Macrophage immunomodulatory activity of polysaccharides isolated from Opuntia polyacantha

    PubMed Central

    Schepetkin, Igor A.; Xie, Gang; Kirpotina, Liliya N.; Klein, Robyn A.; Jutila, Mark A.; Quinn, Mark T.

    2008-01-01

    Opuntia polyacantha (prickly pear cactus) has been used extensively for its nutritional properties; however, less is known regarding medicinal properties of Opuntia tissues. In the present study, we extracted polysaccharides from O. polyacantha and used size-exclusion chromatography to fractionate the crude polysaccharides into four polysaccharide fractions (designated as Opuntia polysaccharides C-I to C-IV). The average Mr of fractions C-I through C-IV was estimated to be 733, 550, 310, and 168 kDa, respectively, and sugar composition analysis revealed that Opuntia polysaccharides consisted primarily of galactose, galacturonic acid, xylose, arabinose, and rhamnose. Analysis of the effects of Opuntia polysaccharides on human and murine macrophages demonstrated that all four fractions had potent immunomodulatory activity, inducing production of reactive oxygen species, nitric oxide, tumor necrosis factor α, and interleukin 6. Furthermore, modulation of macrophage function by Opuntia polysaccharides was mediated, at least in part, through activation of nuclear factor κB. Together, our results provide a molecular basis to explain a portion of the beneficial therapeutic properties of extracts from O. polyacantha and support the concept of using Opuntia polysaccharides as an immunotherapeutic adjuvant. PMID:18597716

  8. Macrophage activation syndrome in the course of monogenic autoinflammatory disorders.

    PubMed

    Rigante, Donato; Emmi, Giacomo; Fastiggi, Michele; Silvestri, Elena; Cantarini, Luca

    2015-08-01

    An overwhelming activation of cytotoxic T cells and well-differentiated macrophages leading to systemic overload of inflammatory mediators characterizes the so-called macrophage activation syndrome (MAS); this potentially life-threatening clinical entity may derive from several genetic defects involved in granule-mediated cytotoxicity but has been largely observed in patients with juvenile idiopathic arthritis, many rheumatologic diseases, infections, and malignancies. The occurrence of MAS in the natural history or as the revealing clue of monogenic autoinflammatory disorders (AIDs), rare conditions caused by disrupted innate immunity pathways with overblown release of proinflammatory cytokines, has been only reported in few isolated patients with cryopyrin-associated periodic syndrome, mevalonate kinase deficiency, familial Mediterranean fever, and tumor necrosis factor receptor-associated periodic syndrome since 2001. All these patients displayed various clinical, laboratory, and histopathologic features of MAS and have often required intensive care support. Only one patient has died due to MAS. Defective cytotoxic cell function was documented in a minority of patients. Corticosteroids were the first-line treatment, but anakinra was clinically effective in three refractory cases. Even if MAS and AIDs share multiple clinical features as well as heterogeneous pathogenetic scenes and a potential response to anti-interleukin-1 targeted therapies, MAS requires a prompt specific recognition in the course of AIDs due to its profound severity and high mortality rate.

  9. Macrophage immunomodulatory activity of polysaccharides isolated from Opuntia polyacantha.

    PubMed

    Schepetkin, Igor A; Xie, Gang; Kirpotina, Liliya N; Klein, Robyn A; Jutila, Mark A; Quinn, Mark T

    2008-10-01

    Opuntia polyacantha (prickly pear cactus) has been used extensively for its nutritional properties; however, less is known regarding medicinal properties of Opuntia tissues. In the present study, we extracted polysaccharides from O. polyacantha and used size-exclusion chromatography to fractionate the crude polysaccharides into four polysaccharide fractions (designated as Opuntia polysaccharides C-I to C-IV). The average M(r) of fractions C-I through C-IV was estimated to be 733, 550, 310, and 168 kDa, respectively, and sugar composition analysis revealed that Opuntia polysaccharides consisted primarily of galactose, galacturonic acid, xylose, arabinose, and rhamnose. Analysis of the effects of Opuntia polysaccharides on human and murine macrophages demonstrated that all four fractions had potent immunomodulatory activity, inducing production of reactive oxygen species, nitric oxide, tumor necrosis factor alpha, and interleukin 6. Furthermore, modulation of macrophage function by Opuntia polysaccharides was mediated, at least in part, through activation of nuclear factor kappaB. Together, our results provide a molecular basis to explain a portion of the beneficial therapeutic properties of extracts from O. polyacantha and support the concept of using Opuntia polysaccharides as an immunotherapeutic adjuvant.

  10. Prostaglandin D2-loaded microspheres effectively activate macrophage effector functions.

    PubMed

    Pereira, Priscilla Aparecida Tartari; Bitencourt, Claudia da Silva; dos Santos, Daiane Fernanda; Nicolete, Roberto; Gelfuso, Guilherme Martins; Faccioli, Lúcia Helena

    2015-10-12

    Biodegradable lactic-co-glycolic acid (PLGA) microspheres (MS) improve the stability of biomolecules stability and allow enable their sustained release. Lipid mediators represent a strategy for improving host defense; however, most of these mediators, such as prostaglandin D2 (PGD2), have low water solubility and are unstable. The present study aimed to develop and characterize MS loaded with PGD2 (PGD2-MS) to obtain an innovative tool to activate macrophages. PGD2-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process, and the size, zeta potential, surface morphology and encapsulation efficiency were determined. It was also evaluated in vitro the phagocytic index, NF-κB activation, as well as nitric oxide and cytokine production by alveolar macrophages (AMs) in response to PGD2-MS. PGD2-MS were spherical with a diameter of 5.0±3.3 μm and regular surface, zeta potential of -13.4±5.6 mV, and 36% of encapsulation efficiency, with 16-26% release of entrapped PGD2 at 4 and 48 h, respectively. PGD2-MS were more efficiently internalized by AMs than unloaded-MS, and activated NF-κB more than free PGD2. Moreover, PGD2-MS stimulated the production of nitric oxide, TNF-α, IL-1β, and TGF-β, more than free PGD2, indicating that microencapsulation increased the activating effect of PGD2 on cells. In LPS-pre-treated AMs, PGD2-MS decreased the release of IL-6 but increased the production of nitric oxide and IL-1β. These results show that the morphological characteristics of PGD2-MS facilitated interaction with, and activation of phagocytic cells; moreover, PGD2-MS retained the biological activities of PGD2 to trigger effector mechanisms in AMs. It is suggested that PGD2-MS represent a strategy for therapeutic intervention in the lungs of immunocompromised subjects.

  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.

  12. STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

    PubMed

    Leopold Wager, Chrissy M; Hole, Camaron R; Wozniak, Karen L; Olszewski, Michal A; Mueller, Mathias; Wormley, Floyd L

    2015-12-01

    Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO.

  13. Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications

    PubMed Central

    Hardbower, Dana M.; Asim, Mohammad; Luis, Paula B.; Singh, Kshipra; Barry, Daniel P.; Yang, Chunying; Steeves, Meredith A.; Cleveland, John L.; Schneider, Claus; Piazuelo, M. Blanca; Gobert, Alain P.; Wilson, Keith T.

    2017-01-01

    Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms. PMID:28096401

  14. Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

    PubMed

    Ren, Jiafa; Li, Jianzhong; Feng, Ye; Shu, Bingyan; Gui, Yuan; Wei, Wei; He, Weichun; Yang, Junwei; Dai, Chunsun

    2017-08-01

    Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor(-/-) macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor(+/+) macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  15. Interaction of human leukocytes and Entamoeba histolytica. Killing of virulent amebae by the activated macrophage.

    PubMed Central

    Salata, R A; Pearson, R D; Ravdin, J I

    1985-01-01

    Capable effector mechanisms in the human immune response against the cytolytic, protozoan parasite Entamoeba histolytica have not been described. To identify a competent human effector cell, we studied the in vitro interactions of normal human polymorphonuclear neutrophils, peripheral blood mononuclear cells (PBMC), monocytes (MC), and MC-derived macrophages with virulent axenic amebae (strain HMI-IMSS). Amebae killed neutrophils, PBMC, MC, and MC-derived macrophages (P less than 0.001), without loss of parasite viability. The addition of heat-inactivated immune serum did not enable leukocytes to kill amebae, nor did it protect these host cells from amebae. MC-derived macrophages, activated with lymphokine elicited by the mitogens conconavalin A, phytohemagglutinin, or an amebic soluble protein preparation (strain HK9), killed 55% of amebae by 3 h in a trypan blue exclusion assay (P less than 0.001); during this time, 40% of the activated macrophages died. Lysis of amebae was confirmed using 111Indium oxine radiolabeled parasites and was antibody independent. Macrophage death appeared to be due to the deleterious effect of lysed amebae rather than the contact-dependent effector mechanisms of E. histolytica. Adherence between activated macrophages and amebae was greater than that between other leukocytes and amebae (P less than 0.001). Microscopic observations, kinetic analysis of the killing of amebae by activated macrophages, and suspension of amebae with adherent activated macrophages in a 10% dextran solution indicated that contact by activated macrophages was necessary to initiate the killing of amebae. Catalase but not superoxide dismutase inhibited the amebicidal capacity of activated macrophages (P less than 0.001). However, activated macrophages from an individual with chronic granulomatous disease were able to kill amebae, but not as effectively as normal cells (P less than 0.01). In summary, activated MC-derived macrophages killed virulent E. histolytica

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

    PubMed

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

    2015-02-01

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

  17. Low-power laser irradiation enhance macrophage phagocytic capacity through Src activation

    NASA Astrophysics Data System (ADS)

    Wu, Shengnan; Zhou, Feifan; Xing, Da

    2012-03-01

    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 organic functions. In this study, we focused our attention on the effects of He-Ne laser on the phagocytic activity of macrophages, the regulation mechanism of phagocytosis was also discussed. Our results indicated that Low-power laser irradiation can enhance the phagocytosis of macrophage through activation of Src.

  18. Photobiomodulation with 670 nm light ameliorates Müller cell-mediated activation of microglia and macrophages in retinal degeneration.

    PubMed

    Lu, Yen-Zhen; Natoli, Riccardo; Madigan, Michele; Fernando, Nilisha; Saxena, Kartik; Aggio-Bruce, Riemke; Jiao, Haihan; Provis, Jan; Valter, Krisztina

    2017-09-06

    Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm(2)) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1β was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Müller cells. Following PD, 670 nm light-treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1β and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Müller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated

  19. Iron modulates the replication of virulent Mycobacterium bovis in resting and activated bovine and possum macrophages.

    PubMed

    Denis, Michel; Buddle, Bryce M

    2005-09-15

    Bovine and possum macrophages were infected in vitro with a virulent strain of Mycobacterium bovis, and mycobacterial replication was measured in the infected macrophages cultured under a variety of conditions. Virulent M. bovis replicated substantially in alveolar possum macrophages as well as in bovine blood monocyte-derived macrophages. Addition of recombinant bovine interferon-gamma (IFN-gamma) with low concentrations of lipopolysaccharide (LPS) rendered bovine macrophages significantly more resistant to M. bovis replication. Disruption of iron levels in infected macrophages by addition of apotransferrin or bovine lactoferrin blocked replication of M. bovis in both bovine and possum macrophages. On the other hand, addition of exogenous iron, either in the form of iron citrate or iron-saturated transferrin, rendered macrophages of both species much more permissive for the replication of M. bovis. The impact of iron deprivation/loading on the mycobacteriostatic activity of cells was independent of nitric-oxide release, as well as independent of the generation of oxygen radical species in both possum and bovine macrophages. Exogenous iron was shown to reverse the ability of IFN-gamma/LPS pulsed bovine macrophages to restrict M. bovis replication. When autologous possum lymphocytes from animals vaccinated with M. bovis strain BCG were added to infected macrophages, they rendered the macrophages less permissive for virulent M. bovis replication. Loading the cells with iron prior to this macrophage-lymphocyte interaction, reversed this immune effect induced by sensitized cells. We conclude that, in two important animal species, intracellular iron level plays an important role in M. bovis replication in macrophages, irrespective of their activation status.

  20. Evidence for the opposing roles of different gamma delta T cell subsets in macrophage homeostasis.

    PubMed

    Tramonti, Daniela; Andrew, Elizabeth M; Rhodes, Kate; Newton, Darren J; Carding, Simon R

    2006-07-01

    To ensure invading pathogens are eliminated with minimal damage to host tissues it is essential that macrophage activation be tightly regulated. Previously we demonstrated that a subset of gammadelta T cells (Vgamma1(+)) contributes to resolving pathogen-induced immune responses by killing activated macrophages. However, the exaggerated macrophage response seen in infected Vgamma1(+) T cell-deficient mice suggests that gammadelta T cells play a broader role in macrophage homeostasis and other subsets might promote macrophage activation. Using a macrophage:gammadelta T cell co-culture system we have shown that gammadelta T cells increase the activity of macrophages activated in vivo by Listeria monocytogenes infection. In a dose-dependent manner, gammadelta T cells up-regulated production of cytokines (TNF-alpha, IL-6, IL-10) and chemokines (MIP-1alpha, MIP-1beta) by Listeria-elicited macrophages. The ability to increase macrophage cytokine production was prominent among Vgamma4(+) gammadelta T cells. Reciprocally, Vgamma4(+) gammadelta T cells were activated by Listeria-elicited macrophages, resulting in production of the anti-inflammatory cytokine, IL-10. gammadelta T cell adoptive transfer experiments showed that Vgamma4(+) T cells protected TCRdelta(-/-) mice against Listeria-induced liver injury and necrosis. These findings identify distinct and non-overlapping roles for gammadelta T cell subsets in regulating macrophage function during pathogen-induced immune responses.

  1. Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution

    PubMed Central

    Luo, Bangwei; Wang, Jinsong; Liu, Zongwei; Shen, Zigang; Shi, Rongchen; Liu, Yu-Qi; Liu, Yu; Jiang, Man; Wu, Yuzhang; Zhang, Zhiren

    2016-01-01

    Inflammation resolution is an active process, the failure of which causes uncontrolled inflammation which underlies many chronic diseases. Therefore, endogenous pathways that regulate inflammation resolution are fundamental and of wide interest. Here, we demonstrate that phagocyte respiratory burst-induced hypoxia activates macrophage erythropoietin signalling to promote acute inflammation resolution. This signalling is activated following acute but not chronic inflammation. Pharmacological or genetical inhibition of the respiratory burst suppresses hypoxia and macrophage erythropoietin signalling. Macrophage-specific erythropoietin receptor-deficient mice and chronic granulomatous disease (CGD) mice, which lack the capacity for respiratory burst, display impaired inflammation resolution, and exogenous erythropoietin enhances this resolution in WT and CGD mice. Mechanistically, erythropoietin increases macrophage engulfment of apoptotic neutrophils via PPARγ, promotes macrophage removal of debris and enhances macrophage migration to draining lymph nodes. Together, our results provide evidences of an endogenous pathway that regulates inflammation resolution, with important implications for treating inflammatory conditions. PMID:27397585

  2. Cytokine production by co-cultures exposed to monodisperse amorphous silica nanoparticles: the role of size and surface area.

    PubMed

    Napierska, Dorota; Thomassen, Leen C J; Vanaudenaerde, Bart; Luyts, Katrien; Lison, Dominique; Martens, Johan A; Nemery, Benoit; Hoet, Peter H M

    2012-06-01

    The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104 nm) at concentrations of 5 μg/cm² cell culture SA or 10 cm² particle SA/cm². A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-α, IL-6, IL-8 and MIP-1α were measured. Significant secretion of all cytokines was observed for the 2 nm particles at both concentrations and in both compartments. Larger particles of 60 nm induced significant cytokine secretion at the dose of 10 cm² particle SA/cm². The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Interleukin-25 fails to activate STAT6 and induce alternatively activated macrophages.

    PubMed

    Stolfi, Carmine; Caruso, Roberta; Franzè, Eleonora; Sarra, Massimiliano; De Nitto, Daniela; Rizzo, Angelamaria; Pallone, Francesco; Monteleone, Giovanni

    2011-01-01

    Interleukin-25 (IL-25), a T helper type 2 (Th2) -related factor, inhibits the production of inflammatory cytokines by monocytes/macrophages. Since Th2 cytokines antagonize classically activated monocytes/macrophages by inducing alternatively activated macrophages (AAMs), we here assessed the effect of IL-25 on the alternative activation of human monocytes/macrophages. The interleukins IL-25, IL-4 and IL-13 were effective in reducing the expression of inflammatory chemokines in monocytes. This effect was paralleled by induction of AAMs in cultures added with IL-4 or IL-13 but not with IL-25, regardless of whether cells were stimulated with lipopolysaccharide or interferon-γ. Moreover, pre-incubation of cells with IL-25 did not alter the ability of both IL-4 and IL-13 to induce AAMs. Both IL-4 and IL-13 activated signal transducer and activator of transcription 6 (STAT6), and silencing of this transcription factor markedly reduced the IL-4/IL-13-driven induction of AAMs. In contrast, IL-25 failed to trigger STAT6 activation. Among Th2 cytokines, only IL-25 and IL-10 were able to activate p38 mitogen-activated protein kinase. These results collectively indicate that IL-25 fails to induce AAMs and that Th2-type cytokines suppress inflammatory responses in human monocytes by activating different intracellular signalling pathways.

  4. Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.

    PubMed

    Wegiel, Barbara; Larsen, Rasmus; Gallo, David; Chin, Beek Yoke; Harris, Clair; Mannam, Praveen; Kaczmarek, Elzbieta; Lee, Patty J; Zuckerbraun, Brian S; Flavell, Richard; Soares, Miguel P; Otterbein, Leo E

    2014-11-01

    Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1-deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1-deficient mice. IL-1β cleavage and secretion were impaired in HO-1-deficient macrophages, and CO-dependent processing of IL-1β required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1β inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes.

  5. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    SciTech Connect

    Kim, Sun-Ah; Lee, Eun Kyung; Kuh, Hyo-Jeong

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  6. rPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB.

    PubMed

    Valim, Clarissa Xavier Resende; da Silva, Elaine Zayas Marcelino; Assis, Mariana Aprigio; Fernandes, Fabricio Freitas; Coelho, Paulo Sergio Rodrigues; Oliver, Constance; Jamur, Maria Célia

    2015-01-01

    The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients. Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1. The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for additional studies on the pathogenesis of P. brasiliensis.

  7. rPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB

    PubMed Central

    Valim, Clarissa Xavier Resende; da Silva, Elaine Zayas Marcelino; Assis, Mariana Aprigio; Fernandes, Fabricio Freitas; Coelho, Paulo Sergio Rodrigues; Oliver, Constance; Jamur, Maria Célia

    2015-01-01

    Background The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients. Methodology/Principal Findings Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1. Conclusions/Significance The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for

  8. Macrophage activation syndrome in the era of biologic therapy.

    PubMed

    Grom, Alexei A; Horne, AnnaCarin; De Benedetti, Fabrizio

    2016-05-01

    Macrophage activation syndrome (MAS) refers to acute overwhelming inflammation caused by a 'cytokine storm'. Although increasingly recognized as a life-threatening complication of various rheumatic diseases, clinically, MAS is strikingly similar to primary and secondary forms of haemophagocytic lymphohistiocytosis (HLH). Not surprisingly, many rheumatologists prefer the term secondary HLH rather than MAS to describe this condition, and efforts to change the nomenclature are in progress. The pathophysiology of MAS remains elusive, but observations in animal models, as well as data on the effects of new anticytokine therapies on rates and clinical presentations of MAS in patients with systemic juvenile idiopathic arthritis (sJIA), provide clues to the understanding of this perplexing clinical phenomenon. In this Review, we explore the latest available evidence and discuss potential diagnostic challenges in the era of increasing use of biologic therapies.

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

    PubMed Central

    2011-01-01

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

  10. CKIP-1 regulates macrophage proliferation by inhibiting TRAF6-mediated Akt activation

    PubMed Central

    Zhang, Luo; Wang, Yiwu; Xiao, Fengjun; Wang, Shaoxia; Xing, Guichun; Li, Yang; Yin, Xiushan; Lu, Kefeng; Wei, Rongfei; Fan, Jiao; Chen, Yuhan; Li, Tao; Xie, Ping; Yuan, Lin; Song, Lei; Ma, Lanzhi; Ding, Lujing; He, Fuchu; Zhang, Lingqiang

    2014-01-01

    Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3β, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3β, leading to the stabilization of CKIP-1 and β-catenin proteins. β-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1−/− mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation. PMID:24777252

  11. Liver X Receptor (LXR) activation negatively regulates visfatin expression in macrophages

    SciTech Connect

    Mayi, Therese Hervee; Rigamonti, Elena; Pattou, Francois; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2011-01-07

    Research highlights: {yields} Synthetic LXR ligands decreased visfatin expression in human macrophages. {yields} LXR activation leads to a modest and transient decrease of NAD{sup +} concentration. {yields} LXR activation decreased PPAR{gamma}-induced visfatin in human macrophages. -- Abstract: Adipose tissue macrophages (ATM) are the major source of visfatin, a visceral fat adipokine upregulated during obesity. Also known to play a role in B cell differentiation (pre-B cell colony-enhancing factor (PBEF)) and NAD biosynthesis (nicotinamide phosphoribosyl transferase (NAMPT)), visfatin has been suggested to play a role in inflammation. Liver X Receptor (LXR) and Peroxisome Proliferator-Activated Receptor (PPAR){gamma} are nuclear receptors expressed in macrophages controlling the inflammatory response. Recently, we reported visfatin as a PPAR{gamma} target gene in human macrophages. In this study, we examined whether LXR regulates macrophage visfatin expression. Synthetic LXR ligands decreased visfatin gene expression in a LXR-dependent manner in human and murine macrophages. The decrease of visfatin mRNA was paralleled by a decrease of protein secretion. Consequently, a modest and transient decrease of NAD{sup +} concentration was observed. Interestingly, LXR activation decreased the PPAR{gamma}-induced visfatin gene and protein secretion in human macrophages. Our results identify visfatin as a gene oppositely regulated by the LXR and PPAR{gamma} pathways in human macrophages.

  12. Lipid homeostasis and inflammatory activation are disturbed in classically activated macrophages with peroxisomal β-oxidation deficiency.

    PubMed

    Geric, Ivana; Tyurina, Yulia Y; Krysko, Olga; Krysko, Dmitri V; De Schryver, Evelyn; Kagan, Valerian E; Van Veldhoven, Paul P; Baes, Myriam; Verheijden, Simon

    2017-09-22

    Macrophage activation is characterized by pronounced metabolic adaptation. Classically activated macrophages show decreased rates of mitochondrial fatty acid oxidation and oxidative phosphorylation and acquire a glycolytic state together with their pro-inflammatory phenotype. In contrast, alternatively activated macrophages require oxidative phosphorylation and mitochondrial fatty acid oxidation for their anti-inflammatory function. Although it is evident that mitochondrial metabolism is regulated during macrophage polarization and essential for macrophage function, little is known on the regulation and role of peroxisomal β-oxidation during macrophage activation. In this study, we show that peroxisomal β-oxidation is strongly decreased in classically activated bone marrow derived macrophages (BMDM) and mildly induced in alternatively activated BMDM. To examine the role of peroxisomal β-oxidation in macrophages, we used Mfp2(-/-) BMDM lacking the key enzyme of this pathway. Impairment of peroxisomal β-oxidation in Mfp2(-/-) BMDM did not cause lipid accumulation but rather an altered distribution of lipid species with very long chain fatty acids accumulating in the triglyceride and phospholipid fraction. These lipid alterations in Mfp2(-/-) macrophages led to decreased inflammatory activation of Mfp2(-/-) BMDM and peritoneal macrophages evidenced by impaired production of several inflammatory cytokines and chemokines, but did not affect anti-inflammatory polarization. The disturbed inflammatory responses of Mfp2(-/-) macrophages did not affect immune cell infiltration, as mice with selective elimination of MFP2 from myeloid cells showed normal monocyte and neutrophil influx upon challenge with zymosan. Together, these data demonstrate that peroxisomal β-oxidation is involved in fine-tuning the phenotype of macrophages, likely by influencing the dynamic lipid profile during macrophage polarization. This article is protected by copyright. All rights reserved

  13. Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone attenuates postincisional pain by regulating macrophage polarization

    SciTech Connect

    Hasegawa-Moriyama, Maiko; Ohnou, Tetsuya; Godai, Kohei; Kurimoto, Tae; Nakama, Mayo; Kanmura, Yuichi

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Rosiglitazone attenuated postincisional pain. Black-Right-Pointing-Pointer Rosiglitazone alters macrophage polarization to F4/80{sup +}CD206{sup +} M2 macrophages at the incisional sites. Black-Right-Pointing-Pointer Transplantation of rosiglitazone-treated macrophages produced analgesic effects. -- Abstract: Acute inflammation triggered by macrophage infiltration to injured tissue promotes wound repair and may induce pain hypersensitivity. Peroxisome proliferator-activated receptor {gamma} (PPAR){gamma} signaling is known to regulate heterogeneity of macrophages, which are often referred to as classically activated (M1) and alternatively activated (M2) macrophages. M1 macrophages have considerable antimicrobial activity and produce a wide variety of proinflammatory cytokines. In contrast, M2 macrophages are involved in anti-inflammatory and homeostatic functions linked to wound healing and tissue repair. Although it has been suggested that PPAR{gamma} agonists attenuate pain hypersensitivity, the molecular mechanism of macrophage-mediated effects of PPAR{gamma} signaling on pain development has not been explored. In this study, we investigated the link between the phenotype switching of macrophage polarization induced by PPAR{gamma} signaling and the development of acute pain hypersensitivity. Local administration of rosiglitazone significantly ameliorated hypersensitivity to heat and mechanical stimuli, and paw swelling. Consistent with the down-regulation of nuclear factor {kappa}B (NF{kappa}B) phosphorylation by rosiglitazone at the incisional sites, the number of F4/80{sup +}iNOS{sup +} M1 macrophages was decreased whereas numbers of F4/80{sup +}CD206{sup +} M2 macrophages were increased in rosiglitazone-treated incisional sites 24 h after the procedure. In addition, gene induction of anti-inflammatory M2-macrophage-associated markers such as arginase1, FIZZ1 and interleukin (IL)-10 were significantly increased, whereas

  14. Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway.

    PubMed Central

    Ashany, D; Song, X; Lacy, E; Nikolic-Zugic, J; Friedman, S M; Elkon, K B

    1995-01-01

    The Fas/APO-1 cytotoxic pathway plays an important role in the regulation of peripheral immunity. Recent evidence indicates that this regulatory function operates through deletion of activated T and B lymphocytes by CD4+ T cells expressing the Fas ligand. Because macrophages play a key role in peripheral immunity, we asked whether Fas was involved in T-cell-macrophage interactions. Two-color flow cytometry revealed that Fas receptor (FasR) was expressed on resting murine peritoneal macrophages. FasR expression was upregulated after activation of macrophages with cytokines or lipopolysaccharide, although only tumor necrosis factor-alpha rendered macrophages sensitive to anti-FasR antibody-mediated death. To determine the consequence of antigen presentation by macrophages to CD4+ T cells, macrophages were pulsed with antigen and then incubated with either Th1 or Th2 cell lines or clones. Th1, but not Th2, T cells induced lysis of 60-80% of normal macrophages, whereas macrophages obtained from mice with mutations in the FasR were totally resistant to Th1-mediated cytotoxicity. Macrophage cytotoxicity depended upon specific antigen recognition by T cells and was major histocompatibility complex restricted. These findings indicate that, in addition to deletion of activated lymphocytes, Fas plays an important role in deletion of activated macrophages after antigen presentation to Th1 CD4+ T cells. Failure to delete macrophages that constitutively present self-antigens may contribute to the expression of autoimmunity in mice deficient in FasR (lpr) or Fas ligand (gld). PMID:7479970

  15. Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway.

    PubMed

    Ashany, D; Song, X; Lacy, E; Nikolic-Zugic, J; Friedman, S M; Elkon, K B

    1995-11-21

    The Fas/APO-1 cytotoxic pathway plays an important role in the regulation of peripheral immunity. Recent evidence indicates that this regulatory function operates through deletion of activated T and B lymphocytes by CD4+ T cells expressing the Fas ligand. Because macrophages play a key role in peripheral immunity, we asked whether Fas was involved in T-cell-macrophage interactions. Two-color flow cytometry revealed that Fas receptor (FasR) was expressed on resting murine peritoneal macrophages. FasR expression was upregulated after activation of macrophages with cytokines or lipopolysaccharide, although only tumor necrosis factor-alpha rendered macrophages sensitive to anti-FasR antibody-mediated death. To determine the consequence of antigen presentation by macrophages to CD4+ T cells, macrophages were pulsed with antigen and then incubated with either Th1 or Th2 cell lines or clones. Th1, but not Th2, T cells induced lysis of 60-80% of normal macrophages, whereas macrophages obtained from mice with mutations in the FasR were totally resistant to Th1-mediated cytotoxicity. Macrophage cytotoxicity depended upon specific antigen recognition by T cells and was major histocompatibility complex restricted. These findings indicate that, in addition to deletion of activated lymphocytes, Fas plays an important role in deletion of activated macrophages after antigen presentation to Th1 CD4+ T cells. Failure to delete macrophages that constitutively present self-antigens may contribute to the expression of autoimmunity in mice deficient in FasR (lpr) or Fas ligand (gld).

  16. Chronic hepatitis C infection–induced liver fibrogenesis is associated with M2 macrophage activation

    PubMed Central

    Bility, Moses T.; Nio, Kouki; Li, Feng; McGivern, David R.; Lemon, Stanley M.; Feeney, Eoin R.; Chung, Raymond T.; Su, Lishan

    2016-01-01

    The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV–induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV–induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis. PMID:28000758

  17. Chronic hepatitis C infection-induced liver fibrogenesis is associated with M2 macrophage activation.

    PubMed

    Bility, Moses T; Nio, Kouki; Li, Feng; McGivern, David R; Lemon, Stanley M; Feeney, Eoin R; Chung, Raymond T; Su, Lishan

    2016-12-21

    The immuno-pathogenic mechanisms of chronic hepatitis C virus (HCV) infection remain to be elucidated and pose a major hurdle in treating or preventing chronic HCV-induced advanced liver diseases such as cirrhosis. Macrophages are a major component of the inflammatory milieu in chronic HCV-induced liver disease, and are generally derived from circulating inflammatory monocytes; however very little is known about their role in liver diseases. To investigate the activation and role of macrophages in chronic HCV-induced liver fibrosis, we utilized a recently developed humanized mouse model with autologous human immune and liver cells, human liver and blood samples and cell culture models of monocyte/macrophage and/or hepatic stellate cell activation. We showed that M2 macrophage activation was associated with liver fibrosis during chronic HCV infection in the livers of both humanized mice and patients, and direct-acting antiviral therapy attenuated M2 macrophage activation and associated liver fibrosis. We demonstrated that supernatant from HCV-infected liver cells activated human monocytes/macrophages with M2-like phenotypes. Importantly, HCV-activated monocytes/macrophages promoted hepatic stellate cell activation. These results suggest a critical role for M2 macrophage induction in chronic HCV-associated immune dysregulation and liver fibrosis.

  18. α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming.

    PubMed

    Liu, Pu-Ste; Wang, Haiping; Li, Xiaoyun; Chao, Tung; Teav, Tony; Christen, Stefan; Di Conza, Giusy; Cheng, Wan-Chen; Chou, Chih-Hung; Vavakova, Magdalena; Muret, Charlotte; Debackere, Koen; Mazzone, Massimiliano; Huang, Hsien-Da; Fendt, Sarah-Maria; Ivanisevic, Julijana; Ho, Ping-Chih

    2017-09-01

    Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming.

  19. TIPE2 regulates tumor-associated macrophages in skin squamous cell carcinoma.

    PubMed

    Li, Xin

    2016-04-01

    Tumor-associated macrophages (TAMs) play an essential role in the immunology, growth, invasion, and metastases of skin squamous cell carcinoma (SCC). However, the molecular mechanisms underlying the activation and regulation of TAMs by SCC are not completely understood. Here, in a Transwell co-culture system, we found that SCC cells induced polarization of macrophages to a M2 phenotype, evident by expression of surface markers CD163, CD206, and CD301, as well as reduction of cellular iNOS levels and augmentation of cellular arginase levels. Moreover, tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2) was induced in macrophages by co-culturing with SCC cells. Depletion of TIPE2 in macrophages abolished the effects of co-cultured SCC cells on phenotypic modification of macrophages. Furthermore, patients with SCC were divided into two groups based on TIPE2 levels in TAMs at the time of tumor resection. We found that patients with high-TIPE2 TAMs had an overall poor 5-year survival. Together, our data suggest a previously unappreciated role of TIPE2 in the crosstalk between skin SCC and TAMs and highlight TIPE2 as a promising novel target for skin SCC treatment.

  20. Roles of alternatively activated M2 macrophages in allergic contact dermatitis.

    PubMed

    Suzuki, Kotaro; Meguro, Kazuyuki; Nakagomi, Daiki; Nakajima, Hiroshi

    2017-03-17

    Alternatively activated macrophages (M2 macrophages) play key roles in the suppression of Th1 cell responses and the orchestration of tissue repair. However, recent studies have shown that M2 macrophages have potentials to produce high levels of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, suggesting that M2 macrophages may exacerbate inflammation in some settings. In this regard, we have recently shown that large numbers of M2 macrophages accumulate in the sites of hapten-induced contact hypersensitivity (CHS), an animal model of allergic contact dermatitis, and that M2 macrophages exacerbate hapten-induced CHS by producing matrix metalloproteinase 12 (MMP12). We have also shown that suppressor of cytokine signaling-3 (SOCS3), a member of SOCS family proteins that are cytokine-inducible negative regulators of the JAK/STAT signaling pathways, is highly and preferentially expressed in M2 macrophages in hapten-induced CHS and that SOCS3 expressed in M2 macrophages is involved in the attenuation of CHS by suppressing MMP12 production. These findings underscore the importance of M2 macrophage-derived MMP12 in the development of CHS, and suggest that inhibition of M2 macrophages or MMP12 could be a potential therapeutic strategy for the treatment of allergic contact dermatitis.

  1. Jund is a determinant of macrophage activation and is associated with glomerulonephritis susceptibility

    PubMed Central

    Behmoaras, Jacques; Bhangal, Gurjeet; Smith, Jennifer; McDonald, Kylie; Mutch, Brenda; Lai, Ping Chin; Domin, Jan; Game, Laurence; Salama, Alan; Foxwell, Brian M; Pusey, Charles D; Cook, H Terence; Aitman, Timothy J

    2009-01-01

    Crescentic glomerulonephritis is an important cause of human kidney failure for which the underlying molecular basis is largely unknown. In previous studies, we mapped several susceptibility loci, Crgn1–Crgn7, for crescentic glomerulonephritis in the Wistar Kyoto (WKY) rat1. Here we show by combined congenic, linkage and microarray studies that the activator protein-1 (AP-1) transcription factor JunD is a major determinant of macrophage activity and is associated with glomerulonephritis susceptibility. Introgression of Crgn2 from the nonsusceptible Lewis strain onto the WKY background leads to significant reductions in crescent formation, macrophage infiltration, Fc receptor–mediated macrophage activation and cytokine production. Haplotype analysis restricted the Crgn2 linkage interval to a 430-kb interval containing Jund, which is markedly overexpressed in WKY macrophages and glomeruli. Jund knockdown in rat and human primary macrophages led to significantly reduced macrophage activity and cytokine secretion, indicating conservation of JunD function in macrophage activation in rats and humans and suggesting in vivo inhibition of Jund as a possible new therapeutic strategy for diseases characterized by inflammation and macrophage activation. PMID:18443593

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

    PubMed

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

    2015-04-16

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

  3. Candida albicans Chitin Increases Arginase-1 Activity in Human Macrophages, with an Impact on Macrophage Antimicrobial Functions

    PubMed Central

    MacCallum, Donna M.; Brown, Gordon D.

    2017-01-01

    ABSTRACT   The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host’s arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival. PMID:28119468

  4. Macrophages overexpressing Aire induce CD4+Foxp3+ T cells.

    PubMed

    Sun, Jitong; Fu, Haiying; Wu, Jing; Zhu, Wufei; Li, Yi; Yang, Wei

    2013-01-01

    Aire plays an important role in central immune tolerance by regulating the transcription of thousands of genes. However, the role of Aire in the peripheral immune system is poorly understood. Regulatory T (Treg) cells are considered essential for the maintenance of peripheral tolerance, but the effect of Aire on Treg cells in the peripheral immune system is currently unknown. In this study, we investigated the effects of macrophages overexpressing Aire on CD4+Foxp3+ Treg cells by co-culturing Aire-overexpressing RAW264.7 cells or their supernatant with splenocytes. The results show that macrophages overexpressing Aire enhanced the expression of Foxp3 mRNA and induced different subsets of Treg cells in splenocytes through cell-cell contact or a co-culture supernatants. TGF-β is a key molecule in the increases of CD4+CD45RA+Foxp3hi T cell and activating Treg (aTreg) levels observed following cell‑supernatant co-culturing. Subsets of Treg cells were induced by Aire-overexpressing macrophages, and the manipulation of Treg cells by the targeting of Aire may provide a method for the treatment of inflammatory or autoimmune diseases.

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

    NASA Astrophysics Data System (ADS)

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

    1990-07-01

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

  6. Macrophages Contribute to the Cyclic Activation of Adult Hair Follicle Stem Cells

    PubMed Central

    Castellana, Donatello; Paus, Ralf; Perez-Moreno, Mirna

    2014-01-01

    Skin epithelial stem cells operate within a complex signaling milieu that orchestrates their lifetime regenerative properties. The question of whether and how immune cells impact on these stem cells within their niche is not well understood. Here we show that skin-resident macrophages decrease in number because of apoptosis before the onset of epithelial hair follicle stem cell activation during the murine hair cycle. This process is linked to distinct gene expression, including Wnt transcription. Interestingly, by mimicking this event through the selective induction of macrophage apoptosis in early telogen, we identify a novel involvement of macrophages in stem cell activation in vivo. Importantly, the macrophage-specific pharmacological inhibition of Wnt production delays hair follicle growth. Thus, perifollicular macrophages contribute to the activation of skin epithelial stem cells as a novel, additional cue that regulates their regenerative activity. This finding may have translational implications for skin repair, inflammatory skin diseases and cancer. PMID:25536657

  7. Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

    PubMed

    Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

    2013-07-01

    The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

  8. Hybrid-Actuating Macrophage-Based Microrobots for Active Cancer Therapy

    PubMed Central

    Han, Jiwon; Zhen, Jin; Du Nguyen, Van; Go, Gwangjun; Choi, Youngjin; Ko, Seong Young; Park, Jong-Oh; Park, Sukho

    2016-01-01

    Using macrophage recruitment in tumors, we develop active, transportable, cancer theragnostic macrophage-based microrobots as vector to deliver therapeutic agents to tumor regions. The macrophage-based microrobots contain docetaxel (DTX)-loaded poly-lactic-co-glycolic-acid (PLGA) nanoparticles (NPs) for chemotherapy and Fe3O4 magnetic NPs (MNPs) for active targeting using an electromagnetic actuation (EMA) system. And, the macrophage-based microrobots are synthesized through the phagocytosis of the drug NPs and MNPs in the macrophages. The anticancer effects of the microrobots on tumor cell lines (CT-26 and 4T1) are evaluated in vitro by cytotoxic assay. In addition, the active tumor targeting by the EMA system and macrophage recruitment, and the chemotherapeutic effect of the microrobots are evaluated using three-dimensional (3D) tumor spheroids. The microrobots exhibited clear cytotoxicity toward tumor cells, with a low survivability rate (<50%). The 3D tumor spheroid assay showed that the microrobots demonstrated hybrid actuation through active tumor targeting by the EMA system and infiltration into the tumor spheroid by macrophage recruitment, resulting in tumor cell death caused by the delivered antitumor drug. Thus, the active, transportable, macrophage-based theragnostic microrobots can be considered to be biocompatible vectors for cancer therapy. PMID:27346486

  9. Adipogenic role of alternatively activated macrophages in β-adrenergic remodeling of white adipose tissue.

    PubMed

    Lee, Yun-Hee; Kim, Sang-Nam; Kwon, Hyun-Jung; Maddipati, Krishna Rao; Granneman, James G

    2016-01-01

    De novo brown adipogenesis involves the proliferation and differentiation of progenitors, yet the mechanisms that guide these events in vivo are poorly understood. We previously demonstrated that treatment with a β3-adrenergic receptor (ADRB3) agonist triggers brown/beige adipogenesis in gonadal white adipose tissue following adipocyte death and clearance by tissue macrophages. The close physical relationship between adipocyte progenitors and tissue macrophages suggested that the macrophages that clear dying adipocytes might generate proadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243 identified a subpopulation that contained elevated lipid and expressed CD44. Lipidomic analysis of fluorescence-activated cell sorting-isolated macrophages demonstrated that CD44+ macrophages contained four- to five-fold higher levels of the endogenous peroxisome-proliferator activated receptor gamma (PPARγ) ligands 9-hydroxyoctadecadienoic acid (HODE), and 13-HODE compared with CD44- macrophages. Gene expression profiling and immunohistochemistry demonstrated that ADRB3 agonist treatment upregulated expression of ALOX15, the lipoxygenase responsible for generating 9-HODE and 13-HODE. Using an in vitro model of adipocyte efferocytosis, we found that IL-4-primed tissue macrophages accumulated lipid from dying fat cells and upregulated expression of Alox15. Furthermore, treatment of differentiating adipocytes with 9-HODE and 13-HODE potentiated brown/beige adipogenesis. Collectively, these data indicate that noninflammatory removal of adipocyte remnants and coordinated generation of PPARγ ligands by M2 macrophages provides localized adipogenic signals to support de novo brown/beige adipogenesis.

  10. Modulatory effect of plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) on macrophage functions in BALB/c mice. I. Potentiation of macrophage bactericidal activity.

    PubMed

    Abdul, K M; Ramchender, R P

    1995-09-01

    The modulatory ability of plumbagin, a natural product from Plumbago zeylanica, was studied on peritoneal macrophages of BALB/c mice. The macrophage functions evaluated were bactericidal activity, hydrogen peroxide and superoxide anion release. The bactericidal capacity of in vivo plumbagin-treated mouse macrophages was estimated against Staphylococcus aureus. In low doses plumbagin exerted a constant increase in bactericidal activity throughout the study period whereas with a high dose a higher response was observed up to six weeks. But in the next two weeks a considerable decline in the bactericidal activity was noticed compared to low dose. Plumbagin was also seen to exert a similar response on oxygen radical release by macrophages in vivo showing a clear correlation between oxygen radical release and the bactericidal activity. The data indicate that plumbagin augments the macrophage bactericidal activity by potentiating the oxyradical release at low concentration whereas at the higher concentration it has inhibitory activity.

  11. Neural Transplantation Model Using Integration Co-culture Chamber

    NASA Astrophysics Data System (ADS)

    Shimba, Kenta; Saito, Atsushi; Takeuchi, Akimasa; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

    Regenerative medicine is a promising therapy for injuries and diseases of the central nervous system (CNS). Implantation of stem cell-derived neurons into the recipient tissue is one of the key processes of the therapy. How the implanted cells establish functional connections with the intact neurons, and whether the established connections are maintained stably for a long time, remain unknown. Here, we report a novel co-culture device for visualizing interconnections between primary and differentiated neuronal cultures, and long-term monitoring of neuronal activity. A circular micro-chamber surrounded by another chamber is aligned on a microelectrode array (MEA). These chambers are interconnected through 36 micro-tunnels. Stem cell-derived neurons were cultured in the inner circular chamber, and primary neurons taken from mouse cortices were cultured in the surrounding chamber. Neurites outgrew into the micro-tunnels from both primary and differentiated neurons. The immunofluorescence images indicate that synaptic connections are formed between them. Propagation of electrical activity was observed 6 days after starting co-culture. More than half of the spontaneous activity was initiated from primary neurons, and probability of activity propagation to the stem cell-derived neurons gradually increased with culture days. These results suggest that our device is feasible for long-term monitoring of interaction between stem cell-derived cells and the recipient tissue.

  12. Activated macrophages containing tumor marker in colon carcinoma: immunohistochemical proof of a concept.

    PubMed

    Faber, T J E; Japink, D; Leers, M P G; Sosef, M N; von Meyenfeldt, M F; Nap, M

    2012-04-01

    The presence of carcinoembryonic antigen (CEA)-containing activated macrophages has been demonstrated in peripheral blood from patients with colorectal carcinoma. Macrophages migrate from the circulation into the tissue, phagocytose debris, and return to the bloodstream. Hence it seems likely that activated macrophages containing tumor debris, i.e., tumor marker, are present in the stroma of colorectal carcinoma. After phagocytosis, they could follow a hematogenic or lymphogenic route to the peripheral blood. The aim of this study is to assess the presence of tumor marker-containing activated macrophages in the stroma of colon carcinoma and in regional lymph nodes. From 10 cases of colon carcinoma, samples of tumor tissue and metastasis-free lymph nodes were cut in serial sections and stained for CD68 to identify macrophages and for CEA, cytokeratin, or M30 presence. Slides were digitalised and visually inspected using two monitors, comparing the CD68 stain to the tumor marker stain to evaluate the presence of tumor marker-positive macrophages. Macrophages containing tumor marker could be identified in tumor stroma and in metastasis-free regional lymph nodes. The distribution varied for the different markers, CEA-positive macrophages being most abundant. The presence of macrophages containing tumor marker in the tumor stroma and lymph nodes from patients with colon carcinoma could be confirmed in this series using serial immunohistochemistry. This finding supports the concept of activated macrophages, after phagocytosing cell debris, being transported or migrating through the lymphatic system. These results support the potential of tumor marker-containing macrophages to serve as a marker for diagnosis and follow-up of colon cancer patients.

  13. Membrane-Tethered MUC1 Mucin Counter-Regulates the Phagocytic Activity of Macrophages.

    PubMed

    Kato, Kosuke; Uchino, Reina; Lillehoj, Erik P; Knox, Kenneth; Lin, Yong; Kim, K Chul

    2016-04-01

    MUC1 (MUC in human; Muc in animals) is a transmembrane mucin glycoprotein expressed in mucosal epithelial cells and hematopoietic cells. MUC1 is involved in the resolution of inflammation during airway Pseudomonas aeruginosa (Pa) infection by suppressing Toll-like receptor signaling in airway epithelial cells. Although alveolar macrophages are recognized as critical mediators of cell-mediated immunity against microorganisms inhaled into the airways, the role of MUC1 in regulating their response is unknown. The aims of this study were to determine whether macrophages express MUC1, and, if so, whether MUC1 expression might be associated with macrophage M0/M1/M2 differentiation or phagocytic activity. Human and mouse MUC1/Muc1 expression was drastically up-regulated in classically activated (M1) macrophages compared with nonactivated (M0) and alternatively activated (M2) macrophages. M1 polarization and Pa stimulation each increased MUC1 ectodomain shedding from the macrophage surface in a TNF-α-converting enzyme-dependent manner. MUC1/Muc1 deficiency in M0 macrophages increased adhesion and phagocytosis of Pa and Escherichia coli compared with MUC1/Muc1-expressing cells, and attenuation of phagocytosis by MUC1 was augmented after polarization into M1 macrophages compared with M0 macrophages. Finally, MUC1/Muc1 deficiency in macrophages increased reactive oxygen species production and TNF-α release in response to Pa compared with MUC1/Muc1-sufficient cells. These results indicate that MUC1/Muc1 expression by macrophages is predominantly in the M1 subtype, and that MUC1/Muc1 expression in these cells decreases their phagocytic activity in an antiinflammatory manner.

  14. Magnetic stromal layers for enhanced and unbiased recovery of co-cultured hematopoietic cells.

    PubMed

    Savvateeva, Maria V; Demin, Alexander M; Krasnov, Victor P; Belyavsky, Alexander V

    2016-09-15

    Cell co-culture systems have a long history of application in hematology and hold promise for successful hematopoietic stem and progenitor cell expansion. Here we report that various types of stromal cells used in such co-cultures can be rapidly and efficiently labeled with l-lysine-modified Fe3O4 magnetic nanoparticles. Hematopoiesis-supporting activity does not seem to be compromised after magnetic labeling of stromal cells, and the loss of the label by stromal layers during extended culturing is negligible. Magnetic labeling allows for simple and efficient removal of stromal component, yielding unbiased hematopoietic cell populations. When Lin(-) bone mouse marrow fraction was co-cultured with magnetic stromal layers and resulting cell populations were harvested by trypsinization, the yields of total nucleated cells, colony forming cells, and phenotypically primitive Lin(-)Sca-1(+)c-kit(+) subset were substantially higher as compared with nonadherent cell fractions harvested after conventional stromal co-culture. The advantage offered by the magnetic stroma approach over the traditional one was even more significant after a second round of co-culture and was more dramatic for more primitive hematopoietic cells. We conclude that magnetic stromal layers represent a simple, efficient, and convenient tool for co-culturing and subsequent recovery of sufficiently pure unbiased populations of hematopoietic cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Interactions between Naïve and Infected Macrophages Reduce Mycobacterium tuberculosis Viability

    PubMed Central

    Hartman, Michelle L.; Kornfeld, Hardy

    2011-01-01

    A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which

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

    PubMed

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

    2003-01-01

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

  17. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  18. Mechanistic study of macrophage activation by LPS stimulation using fluorescence imaging techinques

    NASA Astrophysics Data System (ADS)

    Lu, Cuixia; Zhou, Feifan; Chen, Wei R.; Xing, Da

    2012-03-01

    Lipopolysaccharide (LPS), a structural component of the outer membrane of gram negative bacteria, has been suggested that stimulates macrophages secrete a wide variety of inflammatory mediators, such as nitric oxide (NO). However, the cellular mechanisms of NO generation in macrophage by LPS stimulation are not well known. In this study, LPS stimulated NO generation in macrophage was determined by measuring fluorescence changes with a NO specific probe DAF-FM DA. Using the fluorescence resonance energy transfer (FRET) techniques, we found an increase of protein kinase C (PKC) activation was dynamically monitored in macrophages treated with LPS. Nuclear factor kappa B (NF-κB) translocated from the cytoplasm to the nucleus in macrophage was measured by confocal laser scanning microscopy. Moreover, the PKC inhibitor GÖ6983 inhibited LPS-stimulated NF-κB activation and NO production. These results indicated that LPS stimulated NF-κB mediated NO production by activating PKC.

  19. Mechanistic study of macrophage activation by LPS stimulation using fluorescence imaging techinques

    NASA Astrophysics Data System (ADS)

    Lu, Cuixia; Zhou, Feifan; Chen, Wei R.; Xing, Da

    2011-11-01

    Lipopolysaccharide (LPS), a structural component of the outer membrane of gram negative bacteria, has been suggested that stimulates macrophages secrete a wide variety of inflammatory mediators, such as nitric oxide (NO). However, the cellular mechanisms of NO generation in macrophage by LPS stimulation are not well known. In this study, LPS stimulated NO generation in macrophage was determined by measuring fluorescence changes with a NO specific probe DAF-FM DA. Using the fluorescence resonance energy transfer (FRET) techniques, we found an increase of protein kinase C (PKC) activation was dynamically monitored in macrophages treated with LPS. Nuclear factor kappa B (NF-κB) translocated from the cytoplasm to the nucleus in macrophage was measured by confocal laser scanning microscopy. Moreover, the PKC inhibitor GÖ6983 inhibited LPS-stimulated NF-κB activation and NO production. These results indicated that LPS stimulated NF-κB mediated NO production by activating PKC.

  20. Macrophage activation and its role in repair and pathology after spinal cord injury.

    PubMed

    Gensel, John C; Zhang, Bei

    2015-09-04

    The injured spinal cord does not heal properly. In contrast, tissue repair and functional recovery occur after skin or muscle injuries. The reason for this dichotomy in wound repair is unclear but inflammation, and specifically macrophage activation, likely plays a key role. Macrophages have the ability to promote the repair of injured tissue by regulating transitions through different phase of the healing response. In the current review we compare and contrast the healing and inflammatory responses between spinal cord injuries and tissues that undergo complete wound resolution. Through this comparison, we identify key macrophage phenotypes that are inaptly triggered or absent after spinal cord injury and discuss spinal cord stimuli that contribute to this maladaptive response. Sequential activation of classic, pro-inflammatory, M1 macrophages and alternatively activated, M2a, M2b, and M2c macrophages occurs during normal healing and facilitates transitions through the inflammatory, proliferative, and remodeling phases of repair. In contrast, in the injured spinal cord, pro-inflammatory macrophages potentiate a prolonged inflammatory phase and remodeling is not properly initiated. The desynchronized macrophage activation after spinal cord injury is reminiscent of the inflammation present in chronic, non-healing wounds. By refining the role macrophages play in spinal cord injury repair we bring to light important areas for future neuroinflammation and neurotrauma research. This article is part of a Special Issue entitled SI: Spinal cord injury.

  1. Down-regulation of Stathmin Is Required for the Phenotypic Changes and Classical Activation of Macrophages.

    PubMed

    Xu, Kewei; Harrison, Rene E

    2015-07-31

    Macrophages are important cells of innate immunity with specialized capacity for recognition and elimination of pathogens and presentation of antigens to lymphocytes for adaptive immunity. Macrophages become activated upon exposure to pro-inflammatory cytokines and pathogenic stimuli. Classical activation of macrophages with interferon-γ (IFNγ) and lipopolysaccharide (LPS) triggers a wide range of signaling events and morphological changes to induce the immune response. Our previous microtubule (MT) proteomic work revealed that the stathmin association with MTs is considerably reduced in activated macrophages, which contain significantly more stabilized MTs. Here, we show that there is a global decrease in stathmin levels, an MT catastrophe protein, in activated macrophages using both immunoblotting and immunofluorescent microscopy. This is an LPS-specific response that induces proteasome-mediated degradation of stathmin. We explored the functions of stathmin down-regulation in activated macrophages by generating a stable cell line overexpressing stathmin-GFP. We show that stathmin-GFP overexpression impacts MT stability, impairs cell spreading, and reduces activation-associated phenotypes. Furthermore, overexpressing stathmin reduces complement receptor 3-mediated phagocytosis and cellular activation, implicating a pivotal inhibitory role for stathmin in classically activated macrophages.

  2. Down-regulation of Stathmin Is Required for the Phenotypic Changes and Classical Activation of Macrophages*

    PubMed Central

    Xu, Kewei; Harrison, Rene E.

    2015-01-01

    Macrophages are important cells of innate immunity with specialized capacity for recognition and elimination of pathogens and presentation of antigens to lymphocytes for adaptive immunity. Macrophages become activated upon exposure to pro-inflammatory cytokines and pathogenic stimuli. Classical activation of macrophages with interferon-γ (IFNγ) and lipopolysaccharide (LPS) triggers a wide range of signaling events and morphological changes to induce the immune response. Our previous microtubule (MT) proteomic work revealed that the stathmin association with MTs is considerably reduced in activated macrophages, which contain significantly more stabilized MTs. Here, we show that there is a global decrease in stathmin levels, an MT catastrophe protein, in activated macrophages using both immunoblotting and immunofluorescent microscopy. This is an LPS-specific response that induces proteasome-mediated degradation of stathmin. We explored the functions of stathmin down-regulation in activated macrophages by generating a stable cell line overexpressing stathmin-GFP. We show that stathmin-GFP overexpression impacts MT stability, impairs cell spreading, and reduces activation-associated phenotypes. Furthermore, overexpressing stathmin reduces complement receptor 3-mediated phagocytosis and cellular activation, implicating a pivotal inhibitory role for stathmin in classically activated macrophages. PMID:26082487

  3. High salt reduces the activation of IL-4– and IL-13–stimulated macrophages

    PubMed Central

    Binger, Katrina J.; Gebhardt, Matthias; Heinig, Matthias; Rintisch, Carola; Schroeder, Agnes; Neuhofer, Wolfgang; Hilgers, Karl; Manzel, Arndt; Schwartz, Christian; Kleinewietfeld, Markus; Voelkl, Jakob; Schatz, Valentin; Linker, Ralf A.; Lang, Florian; Voehringer, David; Wright, Mark D.; Hubner, Norbert; Dechend, Ralf; Jantsch, Jonathan; Titze, Jens; Müller, Dominik N.

    2015-01-01

    A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt–induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis. PMID:26485286

  4. Plutonium behavior after pulmonary administration according to solubility properties, and consequences on alveolar macrophage activation.

    PubMed

    Van der Meeren, Anne; Gremy, Olivier; Renault, Daniel; Miroux, Amandine; Bruel, Sylvie; Griffiths, Nina; Tourdes, Françoise

    2012-01-01

    The physico-chemical form in which plutonium enters the body influences the lung distribution and the transfer rate from lungs to blood. In the present study, we evaluated the early lung damage and macrophage activation after pulmonary contamination of plutonium of various preparation modes which produce different solubility and distribution patterns. Whatever the solubility properties of the contaminant, macrophages represent a major retention compartment in lungs, with 42 to 67% of the activity from broncho-alveolar lavages being associated with macrophages 14 days post-contamination. Lung changes were observed 2 and 6 weeks post-contamination, showing inflammatory lesions and accumulation of activated macrophages (CD68 positive) in plutonium-contaminated rats, although no increased proliferation of pneumocytes II (TTF-1 positive cells) was found. In addition, acid phosphatase activity in macrophages from contaminated rats was enhanced 2 weeks post-contamination as compared to sham groups, as well as inflammatory mediator levels (TNF-α, MCP-1, MIP-2 and CINC-1) in macrophage culture supernatants. Correlating with the decrease in activity remaining in macrophages after plutonium contamination, inflammatory mediator production returned to basal levels 6 weeks post-exposure. The production of chemokines by macrophages was evaluated after contamination with Pu of increasing solubility. No correlation was found between the solubility properties of Pu and the activation level of macrophages. In summary, our data indicate that, despite the higher solubility of plutonium citrate or nitrate as compared to preformed colloids or oxides, macrophages remain the main lung target after plutonium contamination and may participate in the early pulmonary damage.

  5. Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.

    PubMed

    Tarique, Abdullah A; Logan, Jayden; Thomas, Emma; Holt, Patrick G; Sly, Peter D; Fantino, Emmanuelle

    2015-11-01

    Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. M1 and M2 were identified as CD64(+)CD80(+) and CD11b(+)CD209(+), respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-γ, IL-8, TNF-α, IL-1β, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b(+)CD209(+) and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli.

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

    SciTech Connect

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

    2011-06-15

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

  7. β-glucans from Coriolus versicolor protect mice against S. typhimurium challenge by activation of macrophages.

    PubMed

    Shi, Shao-Hua; Yang, Wen-Tao; Huang, Ke-Yan; Jiang, Yan-Long; Yang, Gui-Lian; Wang, Chun-Feng; Li, Yu

    2016-05-01

    The effects of β-glucans from Coriolus versicolor (CVP), which are extracted from a well-known immune stimulator C. versicolor, have been demonstrated extensively in vitro and in vivo. However, until now, the phagocytic activity has not been elucidated. Hence, the objective of the present study was to identify the antibacterial activity of CVP or CVP-treated macrophages by an analysis of cell cytotoxicity, phagocytic activity, intracellular bacterial survival, macrophage activation, production of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) in CVP-treated macrophages using flow cytometry, RT-PCR, a gentamicin protection assay, a Nitric oxide assay and an iNOS enzymatic activity assay. The results indicate that CVP-treated macrophages can phagocytize and kill bacteria, probably due to the production of NO and iNOS. More importantly, CVP-treated macrophages are effective at protecting mice against the challenge of Salmonella typhimurium. The results of this study suggest that the antibacterial effects of CVP are probably caused by the activation of innate immune cells, especially macrophages, because the activated macrophage produces NO, which kills bacteria. These phenomena indicate the possibility of CVP as a potential alternative for antibiotics against resistant bacteria.

  8. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

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

    PubMed Central

    1986-01-01

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

  10. Kaurane diterpenes protect against apoptosis and inhibition of phagocytosis in activated macrophages.

    PubMed

    de las Heras, B; Hortelano, S; Girón, N; Bermejo, P; Rodríguez, B; Boscá, L

    2007-09-01

    The kaurane diterpenes foliol and linearol are inhibitors of the activation of nuclear factor kappaB, a transcription factor involved in the inflammatory response. Effects of these diterpenes on apoptosis and phagocytosis have been analysed in cultured peritoneal macrophages and in the mouse macrophage cell line, RAW 264.7. Macrophages were maintained in culture and activated with pro-inflammatory stimuli in the absence or presence of diterpenes. Apoptosis and the phagocytosis in these cells under these conditions were determined. Incubation of macrophages with a mixture of bacterial lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) induced apoptosis through a NO-dependent pathway, an effect significantly inhibited by foliol and linearol in the low muM range, without cytotoxic effects. Apoptosis in macrophages induced by NO donors was also inhibited. The diterpenes prevented apoptosis through a mechanism compatible with the inhibition of caspase-3 activation, release of cytochrome c to the cytosol and p53 overexpression, as well as an alteration in the levels of proteins of the Bcl-2 family, in particular, the levels of Bax. Cleavage of poly(ADP-ribose) polymerase, a well-established caspase substrate, was reduced by these diterpenes. Treatment of cells with foliol and linearol decreased phagocytosis of zymosan bioparticles by RAW 264.7 cells and to a greater extent by peritoneal macrophages. Both diterpenes protected macrophages from apoptosis and inhibited phagocytosis, resulting in a paradoxical control of macrophage function, as viability was prolonged but inflammatory and phagocytic functions were impaired.

  11. Cathepsin Activity-Based Probes and Inhibitor for Preclinical Atherosclerosis Imaging and Macrophage Depletion

    PubMed Central

    Abd-Elrahman, Ihab; Kosuge, Hisanori; Wises Sadan, Tommy; Ben-Nun, Yael; Meir, Karen; Rubinstein, Chen; Bogyo, Matthew; McConnell, Michael V.

    2016-01-01

    Background and Purpose Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered “vulnerable” while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease. Methods We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for in vivo imaging using fluorescent molecular tomography (FMT), as well as ex vivo fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy. Results We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques. Conclusions Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation. PMID:27532109

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-08-01

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

  14. Sustained nitric oxide delivery delays nitric oxide-dependent apoptosis in macrophages: contribution to the physiological function of activated macrophages.

    PubMed

    Hortelano, Sonsoles; Través, Paqui G; Zeini, Miriam; Alvarez, Alberto M; Boscá, Lisardo

    2003-12-01

    Treatment of the macrophage cell line RAW 264.7 with the short-lived NO donor S-nitrosoglutathione triggers apoptosis through the release of mitochondrial mediators. However, continuous supply of NO by long-lived NO donors protected cells from apoptosis through mechanisms that involved the maintenance or an increase in the levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, cIAP-2, and xIAP and decreases in the accumulation of p53 and in the levels and targeting of Bax to the mitochondria. As a result of these changes, the activation of caspases 9 and 3 was notably delayed, expanding the time of viability of the macrophages. Moreover, inhibition of NO synthase 2 activity after 8 h of stimulation of RAW 264.7 cells with LPS and IFN-gamma accelerated apoptosis via an increase in the processing and activation of caspases. These data suggest that NO exerts an important role in the autoregulation of apoptosis in macrophages.

  15. Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages.

    PubMed

    Shen, Ruizhong; Meng, Gang; Ochsenbauer, Christina; Clapham, Paul R; Grams, Jayleen; Novak, Lea; Kappes, John C; Smythies, Lesley E; Smith, Phillip D

    2011-05-01

    Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.

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

    PubMed Central

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

    2016-01-01

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

  17. Alternative activation deprives macrophages of a coordinated defense program to Mycobacterium tuberculosis.

    PubMed

    Kahnert, Antje; Seiler, Peter; Stein, Maik; Bandermann, Silke; Hahnke, Karin; Mollenkopf, Hans; Kaufmann, Stefan H E

    2006-03-01

    A potent Th1 immune response is critical to the control of tuberculosis. The impact of an additive Th2 response on the course of disease has so far been insufficiently characterized, despite increased morbidity after co-infection with Mycobacterium tuberculosis and Th2-eliciting helminths and possible involvement of Th2 polarization in reactivation of latent tuberculosis. Here, we describe the gene expression profile of murine bone marrow-derived macrophages alternatively activated by IL-4 in response to infection with M. tuberculosis. Comparison of transcriptional profiles of infected IL-4- and IFN-gamma-activated macrophages revealed delayed and partially diminished responses to intracellular bacteria in alternatively activated macrophages, characterized by reduced exposure to nitrosative stress and increased iron availability, respectively. Alternative activation of host macrophages correlated with elevated expression of the M. tuberculosis iron storage protein bacterioferritin as well as reduced expression of the mycobactin synthesis genes mbtI and mbtJ. The extracellular matrix-remodeling enzyme matrix metalloproteinase (MMP)-12 was induced in alternatively activated macrophages in vitro, and MMP-12-expressing macrophages were abundant at late, but not early, stages of tuberculosis in murine lungs. Our findings emphasize that alternative activation deprives macrophages of control mechanisms that limit mycobacterial growth in vivo, thus supporting intracellular persistence of M. tuberculosis.

  18. Depolymerization of macrophage microfilaments prevents induction and inhibits activity of nitric oxide synthase.

    PubMed

    Fernandes, P D; Araujo, H M; Riveros-Moreno, V; Assreuy, J

    1996-12-01

    We have investigated the relationship between peritoneal murine macrophage cytoskeleton and nitric oxide (NO) synthase (NOS). Activation of the cells with lipopolysaccharide plus interferon-gamma (LI) induced iNOS, detected by nitrite or by labeled L-citrulline production and by a specific antibody against macrophage iNOS. Addition of cytochalasin B (a microfilament-depolymerizing agent) caused a dose-dependent inhibition in NO production by macrophages, whereas colchicine (a microtubule depolymerizing agent) inhibited it only by 20% and not dose-dependently. Addition of cytochalasin B together with LI abolished nitrite and L-citrulline accumulation as well as the amount of iNOS antigen in activated macrophage. Moreover, addition of cytochalasin B 6 or 12 h after stimulus, also decreased the nitrite and L-citrulline production by macrophages although iNOS antigen content by Western blot was the same in the presence or in the absence of cytochalasin B added 12 h after activation. Since cytochalasin B failed to inhibit iNOS activity directly, its inhibitory effects on NO production by macrophages is likely to be indirect, through microfilament network in central regions of cells, but not in filaments seen at pseudopodia or edging processes. Our findings demonstrate that disruption of microfilaments but not of microtubules prevents the iNOS induction process and inhibits its enzymatic activity in activated macrophages.

  19. DHA Suppresses Primary Macrophage Inflammatory Responses via Notch 1/ Jagged 1 Signaling

    PubMed Central

    Ali, Mehboob; Heyob, Kathryn; Rogers, Lynette K.

    2016-01-01

    Persistent macrophages were observed in the lungs of murine offspring exposed to maternal LPS and neonatal hyperoxia. Maternal docosahexaenoic acid (DHA) supplementation prevented the accumulation of macrophages and improved lung development. We hypothesized that these macrophages are responsible for pathologies observed in this model and the effects of DHA supplementation. Primary macrophages were isolated from adult mice fed standard chow, control diets, or DHA supplemented diets. Macrophages were exposed to hyperoxia (O2) for 24 h and LPS for 6 h or 24 h. Our data demonstrate significant attenuation of Notch 1 and Jagged 1 protein levels in response to DHA supplementation in vivo but similar results were not evident in macrophages isolated from mice fed standard chow and supplemented with DHA in vitro. Co-culture of activated macrophages with MLE12 epithelial cells resulted in the release of high mobility group box 1 and leukotriene B4 from the epithelial cells and this release was attenuated by DHA supplementation. Collectively, our data indicate that long term supplementation with DHA as observed in vivo, resulted in deceased Notch 1/Jagged 1 protein expression however, DHA supplementation in vitro was sufficient to suppress release LTB4 and to protect epithelial cells in co-culture. PMID:26940787

  20. DHA Suppresses Primary Macrophage Inflammatory Responses via Notch 1/ Jagged 1 Signaling.

    PubMed

    Ali, Mehboob; Heyob, Kathryn; Rogers, Lynette K

    2016-03-04

    Persistent macrophages were observed in the lungs of murine offspring exposed to maternal LPS and neonatal hyperoxia. Maternal docosahexaenoic acid (DHA) supplementation prevented the accumulation of macrophages and improved lung development. We hypothesized that these macrophages are responsible for pathologies observed in this model and the effects of DHA supplementation. Primary macrophages were isolated from adult mice fed standard chow, control diets, or DHA supplemented diets. Macrophages were exposed to hyperoxia (O2) for 24 h and LPS for 6 h or 24 h. Our data demonstrate significant attenuation of Notch 1 and Jagged 1 protein levels in response to DHA supplementation in vivo but similar results were not evident in macrophages isolated from mice fed standard chow and supplemented with DHA in vitro. Co-culture of activated macrophages with MLE12 epithelial cells resulted in the release of high mobility group box 1 and leukotriene B4 from the epithelial cells and this release was attenuated by DHA supplementation. Collectively, our data indicate that long term supplementation with DHA as observed in vivo, resulted in deceased Notch 1/Jagged 1 protein expression however, DHA supplementation in vitro was sufficient to suppress release LTB4 and to protect epithelial cells in co-culture.

  1. Mechanisms of particle-induced activation of alveolar macrophages.

    PubMed

    Gercken, G; Berg, I; Dörger, M; Schlüter, T

    1996-11-01

    Bovine alveolar macrophages were exposed in vitro to quartz dusts, metal-containing dusts or silica particles coated with a single metal oxide. The release of reactive oxygen intermediates (ROI) was measured in short-term incubations (90 min). The secretion of both ROI was markedly enhanced by silica particles coated with vanadium oxide and lowered by copper oxide-coated particles. The particle-induced ROI release was significantly decreased by the inhibition of protein kinase C (PKC) as well as phospholipase A2, suggesting the involvement of both enzymes in the NADPH oxidase activation. Quartz dusts induced a transient increase of free cytosolic calcium ion concentration, slight intracellular acidification, and depolarization of the plasma membrane. In the presence of EGTA or verapamil the rise of [Ca2+]i was diminished, suggesting an influx of extracellular calcium ions. The PKC inhibitor GF 109203X did not inhibit the quartz-induced calcium rise, while both the cytosolic acidification and depolarization were prevented. BSA-coating of the quartz particles abolished the calcium influx as well as the decrease of pHi, and possibly hyperpolarized the plasma membrane.

  2. Macrophage activation syndrome: why and what should a gastroenterologist know.

    PubMed

    Jayakar, Bijal A; Hashkes, Philip J

    2011-03-01

    We recently treated a patient with adult-onset Still's disease who developed macrophage activation syndrome (MAS) secondary to disseminated histoplasmosis while being treated with adalimumab. The gastroenterology service was consulted early, before diagnosis, as the patient presented with elevated liver enzymes and disseminated intravascular coagulation. MAS is an exaggerated immune response that can develop as a primary condition or secondary to infections, drugs and various diseases, resulting in liver dysfunction, encephalopathy, pancytopenia and disseminated intravascular coagulation. The development of MAS has also been reported in patients with inflammatory bowel disease and post-liver transplantation and has been triggered by medications used by gastroenterologists, particularly sulfasalazine and anti-tumor necrosis factor biologic modifiers. Therefore, we present a review on etiology, pathogenesis, clinical and laboratory features, and treatment of MAS with a focus on gastrointestinal aspects and presentations. MAS is a life threatening condition with a high mortality rate if untreated. Therefore it is important to recognize this condition early. As these patients may occasionally present to gastroenterologists we hope this review will increase awareness of this rare, but serious syndrome.

  3. Biological response of tissues with macrophagic activity to titanium dioxide.

    PubMed

    Olmedo, Daniel G; Tasat, Deborah R; Evelson, Pablo; Guglielmotti, María B; Cabrini, Rómulo L

    2008-03-15

    The titanium dioxide layer is composed mainly of anatase and rutile. This layer is prone to break, releasing particles to the milieu. Therefore, corrosion may cause implant failure and body contamination. We have previously shown that commercial anatase-titanium dioxide (TiO(2)-anatase) is deposited in organs with macrophagic activity, transported in the blood by phagocytic-mononuclear cells, and induces an increase in the production of reactive oxygen species (ROS). In this study, we evaluated the effects of rutile-titanium dioxide (TiO(2)-rutile). Male Wistar rats were injected i.p. with a suspension of TiO(2)-rutile powder at a dose of 1.60 g/100 g b.w. Six months postinjection, the presence of Ti was assessed in serum, blood cells, liver, spleen, and lung. Titanium was found in phagocytic mononuclear cells, serum, and in the parenchyma of all the organs tested. TiO(2)-rutile generated a rise in the percentage of reactive cells, which was smaller than that observed when TiO(2)-anatase was employed in a previous study. Although TiO(2)-rutile provoked an augmentation of ROS, it failed to induce damage to membrane lipids, possibly due to an adaptive response. The present study reveals that TiO(2)-rutile is less bioreactive than TiO(2)-anatase.

  4. A defined co-culture of Geobacter sulfurreducens and Escherichia coli in a membrane-less microbial fuel cell.

    PubMed

    Bourdakos, Nicholas; Marsili, Enrico; Mahadevan, Radhakrishnan

    2014-04-01

    Wastewater-fed microbial fuel cells (MFCs) are a promising technology to treat low-organic carbon wastewater and recover part of the chemical energy in wastewater as electrical power. However, the interactions between electrochemically active and fermentative microorganisms cannot be easily studied in wastewater-fed MFCs because of their complex microbial communities. Defined co-culture MFCs provide a detailed understanding of such interactions. In this study, we characterize the extracellular metabolites in laboratory-scale membrane-less MFCs inoculated with Geobacter sulfurreducens and Escherichia coli co-culture and compare them with pure culture MFCs. G. sulfurreducens MFCs are sparged to maintain anaerobic conditions, while co-culture MFCs rely on E. coli for oxygen removal. G. sulfurreducens MFCs have a power output of 128 mW m(-2) , compared to 63 mW m(-2) from the co-culture MFCs. Analysis of metabolites shows that succinate production in co-culture MFCs decreases current production by G. sulfurreducens and that the removal of succinate is responsible for the increased current density in the late co-culture MFCs. Interestingly, pH adjustment is not required for co-culture MFCs but a base addition is necessary for E. coli MFCs and cultures in vials. Our results show that defined co-culture MFCs provide clear insights into metabolic interactions among bacteria while maintaining a low operational complexity.

  5. Critical role of p38 MAPK in IL-4-induced alternative activation of peritoneal macrophages.

    PubMed

    Jiménez-Garcia, Lidia; Herránz, Sandra; Luque, Alfonso; Hortelano, Sonsoles

    2015-01-01

    Alternative activation of macrophages plays an important role in a range of physiological and pathological processes. This alternative phenotype, also known as M2 macrophages, is induced by type 2 cytokines such as IL-4. The binding of IL-4 to its receptor leads to activation of two major signaling pathways: STAT-6 and PI3K. However, recent studies have described that p38 MAPK might play a role in IL-4-dependent signaling in some cells, although its role in macrophages is still controversial. In this study, we investigated whether p38 MAPK plays a role in the polarization of macrophages in mice. Our results reveal that IL-4 induces phosphorylation of p38 MAPK in thioglycollate-elicited murine peritoneal macrophages, in addition to STAT-6 and PI3K activation. Furthermore, p38 MAPK inactivation, by gene silencing or pharmacological inhibition, suppressed IL-4-induced typical M2 markers, indicating the involvement of p38 MAPK in the signaling of IL-4 leading to M2-macrophage polarization. Moreover, p38 MAPK inhibition blocked phosphorylation of STAT-6 and Akt, suggesting that p38 MAPK is upstream of these signaling pathways. Finally, we show that in an in vivo model of chitin-induced M2 polarization, p38 MAPK inhibition also diminished activation of M2 markers. Taken together, our data establish a new role for p38 MAPK during IL-4-induced alternative activation of macrophages. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The potency of immunomodulatory herbs may be primarily dependent upon macrophage activation.

    PubMed

    Groom, S N; Johns, T; Oldfield, P R

    2007-03-01

    Standardized extracts of Echinacea, cat's claw, and saw palmetto were each evaluated for ability to activate macrophage and natural killer cells, in vitro, using two independent measures of activation for each immune cell population. A standard series of exposure concentrations were tested for each herbal extract in a panel of four assays that evaluated macrophage phagocytosis, macrophage synthesis of interleukin-12, natural killer (NK) cell cytocidal activity (synthesis of granzyme B), and NK cell synthesis of interferon-gamma. Macrophage phagocytosis was stimulated by all three herbs tested: saw palmetto (up to 2.3-fold, P < .05), Echinacea (up to 3.6-fold, P < .01), and cat's claw (up to 4.7-fold, P < .01). Additionally, NK cell synthesis of interferon-gamma was stimulated by saw palmetto (up to 6.3-fold, P < .01) and Echinacea (up to 8.1 fold, P < .01) but not by exposure to cat's claw. None of the three herbs stimulated macrophage synthesis of interleukin-12 or NK cell synthesis of granzyme B. Comparison of the in vitro data with our earlier observations that cat's claw and Echinacea (but not saw palmetto) were each effective in reducing B16/F10 lung tumor colony formation in C57BL/6J mice suggests macrophage activation is the primary means by which these herbs modulate the immune system. Thus, macrophage activation (phagocytosis) may provide a potentially higher throughput method to identify herbal extracts with in vivo stimulatory effects.

  7. TRPV4 channels augment macrophage activation and ventilator-induced lung injury

    PubMed Central

    Hamanaka, Kazutoshi; Jian, Ming-Yuan; Townsley, Mary I.; King, Judy A.; Liedtke, Wolfgang; Weber, David S.; Eyal, Fabien G.; Clapp, Mary M.

    2010-01-01

    We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4−/− and TRPV4+/+ mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (Kf) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH2O PIP did not significantly increase in lungs from TRPV4−/− mice but increased >2.2-fold in TRPV4+/+ lungs, TRPV4+/+ lungs instilled with TRPV4−/− macrophages, and TRPV4−/− lungs instilled with TRPV4+/+ macrophages after ventilation with 35 cmH2O PIP. Activation of TRPV4 with 4-α-phorbol didecanoate (4αPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4+/+ macrophages but not TRPV4−/− macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4+/+ macrophages compared with TRPV4−/− macrophages after 4αPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4+/+ lungs compared with low PIP ventilated TRPV4+/+ or high PIP ventilated TRPV4−/− lungs. Thus TRPV4+/+ macrophages restored susceptibility of TRPV4−/− lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4+/+ macrophages but not TRPV4−/− macrophages. Kf increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production. PMID:20562229

  8. Macrophage Polarization.

    PubMed

    Murray, Peter J

    2017-02-10

    Macrophage polarization refers to how macrophages have been activated at a given point in space and time. Polarization is not fixed, as macrophages are sufficiently plastic to integrate multiple signals, such as those from microbes, damaged tissues, and the normal tissue environment. Three broad pathways control polarization: epigenetic and cell survival pathways that prolong or shorten macrophage development and viability, the tissue microenvironment, and extrinsic factors, such as microbial products and cytokines released in inflammation. A plethora of advances have provided a framework for rationally purifying, describing, and manipulating macrophage polarization. Here, I assess the current state of knowledge about macrophage polarization and enumerate the major questions about how activated macrophages regulate the physiology of normal and damaged tissues.

  9. Tim-3 fosters HCC development by enhancing TGF-β-mediated alternative activation of macrophages.

    PubMed

    Yan, Wenjiang; Liu, Xiao; Ma, Hongxin; Zhang, Hualin; Song, Xiaojia; Gao, Lifen; Liang, Xiaohong; Ma, Chunhong

    2015-10-01

    Tumour-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Tim-3 is highly expressed on macrophages and regulates macrophage functions in several conditions. However, whether Tim-3 is involved in the activation and the function of TAMs has not been reported. Tim-3 expression in HCC samples was evaluated by flow cytometry, immunohistochemistry and confocal analysis. We analysed the effects of Tim-3 knockdown on macrophages in growth of H22 tumour homografts in BALB/c mice. Tim-3 interference was performed by neutralising antibody, small interfering RNA or short hairpin RNA-expressing lentivirus. Cytokine production was evaluated by reverse transcription PCR, ELISA or Cytometric Bead Array. The effects of Tim-3 interference in macrophages were examined with regard to alternative activation of macrophages and proliferation and migration of Hepa1-6 cells. Cell growth curve, colony formation and transwell assays were involved to estimate cell proliferation and migration. Tim-3 expression was significantly increased in both peripheral blood monocytes and TAMs in patients with HCC. The Tim-3 expression in monocytes/TAMs strongly correlated with higher tumour grades and the poor survival of patients with HCC. Consistently, HCC conditioned medium or transforming growth factor-β fostered Tim-3 expression and the alternative activation of macrophages. Moreover, Tim-3 interference in macrophages significantly inhibited the alternative activation of macrophages and suppressed HCC cell growth both in vitro and in vivo. Blocking interleukin 6 reversed the Tim-3-mediated effects on HCC cell growth in vitro. Tim-3 displays critical roles in microenvironment-induced activation and protumoral effects of TAMs in HCC. Interference of Tim-3 might be great potential in HCC therapy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go

  10. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages

    PubMed Central

    1992-01-01

    Tuberculosis remains one of the major infectious causes of morbidity and mortality in the world, yet the mechanisms by which macrophages defend against Mycobacterium tuberculosis have remained obscure. Results from this study show that murine macrophages, activated by interferon gamma, and lipopolysaccharide or tumor necrosis factor alpha, both growth inhibit and kill M. tuberculosis. This antimycobacterial effect, demonstrable both in murine macrophage cell lines and in peritoneal macrophages of BALB/c mice, is independent of the macrophage capacity to generate reactive oxygen intermediates (ROI). Both the ROI-deficient murine macrophage cell line D9, and its ROI-generating, parental line J774.16, expressed comparable antimycobacterial activity upon activation. In addition, the oxygen radical scavengers superoxide dismutase (SOD), catalase, mannitol, and diazabicyclooctane had no effect on the antimycobacterial activity of macrophages. These findings, together with the results showing the relative resistance of M. tuberculosis to enzymatically generated H2O2, suggest that ROI are unlikely to be significantly involved in killing M. tuberculosis. In contrast, the antimycobacterial activity of these macrophages strongly correlates with the induction of the L-arginine- dependent generation of reactive nitrogen intermediates (RNI). The effector molecule(s) that could participate in mediating this antimycobacterial function are toxic RNI, including NO, NO2, and HNO2, as demonstrated by the mycobacteriocidal effect of acidified NO2. The oxygen radical scavenger SOD adventitiously perturbs RNI production, and cannot be used to discriminate between cytocidal mechanisms involving ROI and RNI. Overall, our results provide support for the view that the L-arginine-dependent production of RNI is the principal effector mechanism in activated murine macrophages responsible for killing and growth inhibiting virulent M. tuberculosis. PMID:1552282

  11. Effect of lipopolysaccharide on thymidine salvage as related to macrophage activation.

    PubMed Central

    Harada, Y; Nagao, S; Nakamura, M; Okada, F; Tanigawa, Y

    1995-01-01

    Lipopolysaccharide (LPS), known as one of the potent activators of macrophages, has inhibitory effects on the proliferation of normal macrophages and macrophage-like cell lines. We report here that LPS dose- and time-dependently suppressed the tritiated thymidine ([3H]TdR) incorporation into the acid-insoluble fraction with a significant inverse correlation to the tumour necrosis factor-alpha (TNF) production in the J774.1 macrophage cell line. Among the three tested enzymes involved in DNA synthesis, only thymidine kinase (TK) activity decreased progressively in parallel with the decline in [3H]TdR incorporation, reaching 97% inhibition within 12 hr of LPS treatment, while changes in the activities of other two enzymes, DNA polymerase alpha and thymidylate synthase (TS), were less significant. On the other hand, LPS inhibited the cell proliferation only incompletely, as judged by 62% inhibition of cell growth at 36 hr. Even in the experiments done in a TdR-free medium, cell growth was inhibited by LPS to the same extent, suggesting that TK was not directly involved in the proliferation of J774 cells. LPS also inhibited the conversion of TdR to thymidine monophosphate (TMP) in murine peritoneal exudate macrophages (PEM). Thus LPS-induced suppression of TdR salvage related to TNF production is common in both normal and neoplastic macrophages, and therefore may be of potential importance in the process of macrophage activation. PMID:7751001

  12. Peritonitis-induced antitumor activity of peritoneal macrophages from uremic patients.

    PubMed

    Turyna, Bohdan; Jurek, Aleksandra; Gotfryd, Kamil; Siaśkiewicz, Agnieszka; Kubit, Piotr; Klein, Andrzej

    2004-01-01

    The macrophages belong to the effector cells of both nonspecific and specific immune response. These cells generally express little cytotoxicity unless activated. The present work was intended to determine if peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during episodes of peritonitis were active against human tumor cell lines without further in vitro stimulation. We also compared macrophage antitumor potential with effectiveness of drugs used in cancer therapy (taxol and suramin). Conditioned medium (CM) of macrophages collected during inflammation-free periods did not exhibit cytostatic and cytotoxic activity against both tumor (A549 and HTB44) and non-transformed (BEAS-2B and CRL2190) cells. Exposure of tumor cells to CM of macrophages harvested during peritonitis resulted in significant suppression of proliferation, impairment of viability and induction of apoptosis, in contrast to non-transformed cells, which remained unaffected. The efficacy of CM of inflammatory macrophages as an antitumor agent appeared to be comparable to cytostatic and cytotoxic potency of taxol and suramin or, in the case of HTB44 cells, even higher. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.

  13. Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines.

    PubMed

    Refai, Ali K; Textor, Marcus; Brunette, Donald M; Waterfield, J Douglas

    2004-08-01

    The macrophage has a major role in normal wound healing and the reparative process around implants. Murine macrophage-like cells RAW 264.7 were used to investigate the effect of titanium surfaces on macrophage activation and secretion of proinflammatory cytokines [interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha] and chemokines (monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha). Four topographies were used: those produced by mechanically polishing, coarse sand blasting, acid etching, and sandblasting and acid etching (SLA). Macrophages were plated on the four titanium surfaces at a population density of 5 x 10(5) cells/mL/well. Tissue culture plastic and tissue culture plastic plus lipopolysaccharide (LPS) served as negative and positive control, respectively. In addition, all surfaces were tested for their effects on macrophages in the presence of LPS. Supernatants were collected for assays after 6, 24, and 48 h and the numbers of macrophages attached to the surfaces were quantified using the DAPI (4,6-di-amidino-2-phenylindole) assay. Cytokine and chemokine levels were measured with sandwich enzyme-linked immunosorbent assays. Statistical comparison between the surfaces and the controls was determined by using the two-way analysis of variance including interaction effect (two tailed and p < or = 0.05). Unstimulated macrophages increased their secretion of the proinflammatory cytokine (TNF-alpha) when attached to rough surfaces (acid etching and SLA, p < or = 0.05). In macrophages stimulated with LPS, the roughest surface SLA produced higher levels of IL-1 beta, IL-6, and TNF-alpha at 24 and 48 h than all other surfaces (p < or = 0.05). Surface topography also modulated the secretion of the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha by macrophages. Unstimulated macrophages attached to the SLA surface down-regulated their production of chemokines (p < or = 0.05) whereas

  14. A Role for Activated Macrophages in Resistance to Infection with Toxoplasma

    PubMed Central

    Remington, Jack S.; Krahenbuhl, James L.; Mendenhall, Joy W.

    1972-01-01

    Activated macrophages from mice which were chronically infected with Toxoplasma gondii or Besnoitia jellisoni, or which had received Freund complete adjuvant, had an enhanced capacity to to kill intracellular Toxoplasma. Enhanced killing by activated macrophages was demonstrated by decreased incorporation of isotopically labeled uridine by intracellular Toxoplasma and by inhibition of plaque formation. The latter resulted from lack of proliferation of the intracellular Toxoplasma which is normally followed by destruction of the host cell (macrophage) and secondary invasion and destruction of fibroblast monolayers. Images PMID:4637298

  15. Distinctive role of activated tumor-associated macrophages in photosensitizer accumulation

    NASA Astrophysics Data System (ADS)

    Korbelik, Mladen; Krosl, Gorazd

    1995-05-01

    Cells dissociated from tumors (carcinomas and sarcomas) growing subcutaneously in mice that have been administered Photofrin or other photosensitizers were analyzed by flow cytometry. Monoclonal antibodies were used for identification of major cellular populations contained in these tumors. The results demonstrate that a subpopulation of tumor-associated macrophages (TAMs) is unique among tumor cell populations in that it excels in the accumulation of very high levels of photosensitizers. These macrophages showed an increased expression of interleukin 2 receptor, which is indicative of their activated state. since macrophages were reported to concentrate in the periphery of human neoplasms, it is suggested that activates TAMs are the determinants of tumor-localized photosensitizer fluorescence.

  16. Platelets Mediate Host Defense against Staphylococcus aureus through Direct Bactericidal Activity and by Enhancing Macrophage Activities.

    PubMed

    Ali, Ramadan A; Wuescher, Leah M; Dona, Keith R; Worth, Randall G

    2017-01-01

    Platelets are the chief effector cells in hemostasis. However, recent evidence suggests they have multiple roles in host defense against infection. Reports by us and others showed that platelets functionally contribute to protection against Staphylococcus aureus infection. In the current study, the capacity of mouse platelets to participate in host defense against S. aureus infection was determined by assessing two possibilities. First, we determined the ability of platelets to kill S. aureus directly; and, second, we tested the possibility that platelets enhance macrophage phagocytosis and intracellular killing of S. aureus In this study we report evidence in support of both mechanisms. Platelets effectively killed two different strains of S. aureus. A clinical isolate of methicillin-resistant S. aureus was killed by platelets (>40% killing in 2 h) in a thrombin-dependent manner whereas a methicillin-sensitive strain was killed to equal extent but did not require thrombin. Interestingly, thrombin-stimulated platelets also significantly enhanced peritoneal macrophage phagocytosis of both methicillin-resistant S. aureus and methicillin-sensitive S. aureus by >70%, and restricted intracellular growth by >40%. Enhancement of macrophage anti-S. aureus activities is independent of contact with platelets but is mediated through releasable products, namely IL-1β. These data confirm our hypothesis that platelets participate in host defense against S. aureus both through direct killing of S. aureus and enhancing the antimicrobial function of macrophages in protection against S. aureus infection. Copyright © 2016 by The American Association of Immunologists, Inc.

  17. Cot/tpl2 participates in the activation of macrophages by adiponectin

    PubMed Central

    Sanz-Garcia, Carlos; Nagy, Laura E.; Lasunción, Miguel A.; Fernandez, Margarita; Alemany, Susana

    2014-01-01

    Whereas the main function of APN is to enhance insulin activity, it is also involved in modulating the macrophage phenotype. Here, we demonstrate that at physiological concentrations, APN activates Erk1/2 via the IKKβ-p105/NF-κΒ1-Cot/tpl2 intracellular signal transduction cassette in macrophages. In peritoneal macrophages stimulated with APN, Cot/tpl2 influences the ability to phagocytose beads. However, Cot/tpl2 did not modulate the known capacity of APN to decrease lipid content in peritoneal macrophages in response to treatment with oxLDL or acLDL. A microarray analysis of gene-expression profiles in BMDMs exposed to APN revealed that APN modulated the expression of ∼3300 genes; the most significantly affected biological functions were the inflammatory and the infectious disease responses. qRT-PCR analysis of WT and Cot/tpl2 KO macrophages stimulated with APN for 0, 3, and 18 h revealed that Cot/tpl2 participated in the up-regulation of APN target inflammatory mediators included in the cytokine–cytokine receptor interaction pathway (KEGG ID 4060). In accordance with these data, macrophages stimulated with APN increased secretion of cytokines and chemokines, including IL-1β, IL-1α, TNF-α, IL-10, IL-12, IL-6, and CCL2. Moreover, Cot/tpl2 also played an important role in the production of these inflammatory mediators upon stimulation of macrophages with APN. It has been reported that different types of signals that stimulate TLRs, IL-1R, TNFR, FcγR, and proteinase-activated receptor-1 activate Cot/tpl2. Here, we demonstrate that APN is a new signal that activates the IKKβ-p105/NF-κΒ1-Cot/tpl2-MKK1/2-Erk1/2 axis in macrophages. Furthermore, this signaling cassette modulates the biological functions triggered by APN in macrophages. PMID:24532642

  18. Carbon nanotubes activate macrophages into a M1/M2 mixed status: recruiting naïve macrophages and supporting angiogenesis.

    PubMed

    Meng, Jie; Li, Xiaojin; Wang, Chuan; Guo, Hua; Liu, Jian; Xu, Haiyan

    2015-02-11

    The potential of carbon nanotubes (CNTs) in medical applications has been attracting constant research interest as well as raising concerns related to toxicity. The immune system serves as the first line of defense against invasion. In this work, interactions of oxidized multiwalled carbon nanotubes (MWCNT) with macrophages were investigated to unravel the activation profile of macrophages, using cytokine array, ELISA assay, transwell assay, confocal microscopy, and reactive oxygen species examination. Results show that MWCNT initiate phagocytosis of macrophages and upregulate CD14, CD11b, TLR-4/MD2, and CD206, which does not alter the MHCII expression of the macrophages. The macrophages engulfing MWCNT (MWCNT-RAW) secrete a large amount of MIP-1α and MIP-2 to recruit naïve macrophages and produce angiogenesis-related cytokines MMP-9 and VEGF, while inducing much lower levels of proinflammatory cytokines than those activated by LPS. In conclusion, MWCNT activate macrophages into a M1/M2 mixed status, which allows the cells to recruit naïve macrophages and support angiogenesis.

  19. Pulmonary Chlamydia muridarum challenge activates lung interstitial macrophages which correlate with IFN-γ production and infection control in mice.

    PubMed

    Gracey, Eric; Baglaenko, Yuriy; Prayitno, Nadia; Van Rooijen, Nico; Akram, Ali; Lin, Aifeng; Chiu, Basil; Inman, Robert D

    2015-12-01

    Protective immunity to the pathogen Chlamydia is dependent on a robust IFN-γ response generated by innate and adaptive lymphocytes. Here we assess the role of the macrophage in orchestrating a protective response in vivo to the murine pathogen, Chlamydia muridarum. During acute pulmonary and peritoneal infection, resident macrophages in both sites are infected with C. muridarum and adopt an inflammatory phenotype. In the lung, this activation is restricted to interstitial macrophages, which harbor higher levels of C. muridarum 16sRNA than alveolar macrophages. We examined innate and adaptive lymphocyte activation in the peritoneal cavity with macrophage depletion and with adoptive transfer of infected macrophages. These experiments demonstrate macrophage activation correlates with a protective IFN-γ response and effective control of C. muridarum. These studies suggest that a quantitative or qualitative alteration in macrophages may play a key role in the development of Chlamydia-associated diseases.

  20. Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

    PubMed Central

    Wegiel, Barbara; Larsen, Rasmus; Gallo, David; Chin, Beek Yoke; Harris, Clair; Mannam, Praveen; Kaczmarek, Elzbieta; Lee, Patty J.; Zuckerbraun, Brian S.; Flavell, Richard; Soares, Miguel P.; Otterbein, Leo E.

    2014-01-01

    Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1–deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1–deficient mice. IL-1β cleavage and secretion were impaired in HO-1–deficient macrophages, and CO-dependent processing of IL-1β required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1β inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes. PMID:25295542

  1. In vitro evaluation of the cytotoxicity of two root canal sealers on macrophage activity.

    PubMed

    de Oliveira Mendes, Sônia Teresa; Ribeiro Sobrinho, Antônio Paulino; de Carvalho, André Teixeira; de Souza Côrtes, Maria Ilma; Vieira, Leda Quercia

    2003-02-01

    Although some studies have been concerned with the cytotoxicity of endodontic sealers and their components, few have approached the effects of endodontic sealers on macrophage viability and activity. In this study the effect of two zinc oxide-eugenol-based sealers, freshly prepared or after setting for 24 h, was determined on macrophage activity in vitro. Sealers were placed inside a glass capillary tube and added to mouse-elicited macrophage cultures. Sealers did not affect macrophage viability; however, adherence to glass and phagocytosis were impaired. Moreover, nitric oxide production in response to activation with interferon-gamma was diminished, but interleukin-12 production in response to Listeria monocytogenes was not altered. Interestingly, freshly mixed and solid test samples had similar inhibitory activities. In conclusion, the tested sealers did not affect a pro-inflammatory response (interleukin-12 production) but had an inhibitory effect on the effector responses measured (phagocytosis and nitric oxide production).

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

    PubMed

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

    2015-08-01

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

  3. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    PubMed

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

  4. Fra-1 protooncogene regulates IL-6 expression in macrophages and promotes the generation of M2d macrophages.

    PubMed

    Wang, Qingshan; Ni, Hong; Lan, Lan; Wei, Xiaoli; Xiang, Rong; Wang, Yue

    2010-06-01

    The tumor microenvironment (TME) plays a prominent role in the growth of tumor cells. As the major inflammatory component of the TME, M2d macrophages are educated by the TME such that they adopt an immunosuppressive role that promotes tumor metastasis and progression. Fra-1 forms activator protein-1 heterodimers with Jun partners and drives gene transcription. Fra-1 is thought to drastically induce tumorigenesis and progression. However, the functional role of Fra-1 in the generation of M2d macrophages is poorly understood to date. Here, we demonstrate that 4T1 mammary carcinoma cells, when co-cultured with RAW264.7 macrophage cells, skew the RAW264.7 macrophage cell differentiation into M2d macrophages. The 4T1 cells stimulate de novo overexpression of Fra-1 in RAW264.7 cells, and then Fra-1 binds to the interleukin 6 (IL-6) promoter to increase the production of the cytokine IL-6 in RAW264.7 cells. IL-6 acts in an autocrine fashion to skew RAW264.7 macrophage cell differentiation into M2d macrophages. These findings open new insights into how to reverse M2d macrophage-induced immune tolerance to improve the efficacy of immunotherapeutic approaches.

  5. Perifusion of co-cultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity in vitro.

    PubMed

    Gebhardt, R; Wegner, H; Alber, J

    1996-04-01

    The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 micromol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including

  6. Translational Regulation of Specific mRNAs Controls Feedback Inhibition and Survival during Macrophage Activation

    PubMed Central

    Schott, Johanna; Reitter, Sonja; Philipp, Janine; Haneke, Katharina; Schäfer, Heiner; Stoecklin, Georg

    2014-01-01

    For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the

  7. Hyponatremia, hypophosphatemia, and hypouricemia in a girl with macrophage activation syndrome.

    PubMed

    Yamazawa, Kazuki; Kodo, Kazuki; Maeda, Jun; Omori, Sayu; Hida, Mariko; Mori, Tetsuya; Awazu, Midori

    2006-12-01

    Macrophage activation syndrome, a life-threatening complication of rheumatic disorders, is accompanied by the overproduction of cytokines. We describe a girl with macrophage activation syndrome complicating systemic-onset juvenile arthritis who developed hyponatremia, hypophosphatemia, and hypouricemia associated with a high level of serum tumor necrosis factor alpha. Renal proximal tubule dysfunction was considered to be the cause, which may be attributable to tumor necrosis factor alpha.

  8. Cyclophilin A Aggravates Collagen-Induced Arthritis via Promoting Classically Activated Macrophages.

    PubMed

    Dongsheng, Zhai; Zhiguang, Fu; Junfeng, Jia; Zifan, Lu; Li, Wang

    2017-07-29

    Activated macrophages exhibiting diverse phenotypes and various functions contribute to the pathogenesis or amelioration of different diseases like cancer, inflammation, and infectious and autoimmune diseases. However, the mechanisms of macrophage polarization in inflamed joint and its effects on rheumatoid arthritis (RA) are still not clarified. This study is designed to explore the effects of cyclophilin A (CypA) on macrophage polarization and describe the underlying mechanisms. Collagen-induced arthritis (CIA) was employed to address the pro-arthritic effects of CypA. Flow cytometry was performed to investigate the populations of M1 and M2 macrophages in synovial tissues of the mice. Knockdown or overexpression of CypA macrophage cells was used to study the functions of CypA on macrophage polarization. Western blot was carried out to examine the potential signaling pathways. We found that CypA aggravated the severity of CIA in mice, as assessed by the increase of clinical score of inflammation, cartilage damage, and bone erosion. Moreover, the level of cytokines, such as IL-6, IL-1β, and IL-17, and the number of pro-inflammatory macrophages in synovial fluid were significantly elevated. In accordance with our observation, CypA dysregulation could actually influence the M1 macrophages polarization and pro-inflammatory cytokines production. Further mechanism study disclosed that CypA could regulate the transcriptional activity of NF-κB, the pivotal transcriptional factor regulating M1 polarization, dependent of its PPIase activity. Our findings provide evidence that PPIase CypA promoted macrophages polarization toward pro-inflammatory M1 phenotype via transcriptional activating NF-κB, thus leading to aggravated arthritis.

  9. Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture

    PubMed Central

    Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

    2017-01-01

    The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation. PMID:28783133

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

    PubMed

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

    2011-07-01

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

  11. Potential of fungal co-culturing for accelerated biodegradation of petroleum hydrocarbons in soil.

    PubMed

    Yanto, Dede Heri Yuli; Tachibana, Sanro

    2014-08-15

    The potential of fungal co-culture of the filamentous Pestalotiopsis sp. NG007 with four different basidiomycetes--Trametes versicolor U97, Pleurotus ostreatus PL1, Cerena sp. F0607, and Polyporus sp. S133--for accelerating biodegradation of petroleum hydrocarbons (PHCs) was studied using three different physicochemical characteristic PHCs in soil. All the combinations showed a mutual intermingling mycelial interaction on the agar plates. However, only NG007/S133 (50/50) exhibited an optimum growth rate and enzymatic activities that supported the degradation of asphalt in soil. The co-culture also degraded all fractions at even higher concentrations of the different PHCs. In addition, asphaltene, which is a difficult fraction for a single microorganism to degrade, was markedly degraded by the co-culture, which indicated that the simultaneous biodegradation of aliphatic, aromatic, resin, and asphaltene fractions had occurred in the co-culture. An examination of in-vitro degradation by the crude enzymes and the retrieval fungal culture from the soil after the experiment confirmed the accelerated biodegradation due to enhanced enzyme activities in the co-culture. The addition of piperonyl butoxide or AgNO3 inhibited biodegradation by 81-99%, which demonstrated the important role of P450 monooxygenases and/or dioxygenases in the initial degradation of the aliphatic and aromatic fractions in PHCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Garlic exerts allelopathic effects on pepper physiology in a hydroponic co-culture system

    PubMed Central

    Ding, Haiyan; Liu, Menglong; Hayat, Sikandar; Feng, Han

    2016-01-01

    ABSTRACT A hydroponic co-culture system was adopted to determine the allelopathic potential of garlic on the growth of pepper plants. Different numbers of garlic plants (0, 2, 4, 8 and 12) were hydroponically co-cultured with two pepper plants to investigate allelopathic effects on the growth attributes and antioxidative defense system of the test pepper plants. The responses of the pepper plants depended on the number of garlic plants included in the co-culture system, indicating an association of pepper growth with the garlic root exudate concentration. When grown at a pepper/garlic ratio of 1:1 or 1:2, the pepper plant height, chlorophyll content, and peroxidase (POD), catalase (CAT) and phenylalanine ammonia-lyase (PAL) activities were significantly increased after 30 days of co-culture; in contrast, reduction in methane dicarboxylic aldehyde (MDA) content was observed. However, when the pepper/garlic ratio was 1:4 or higher, these morphological indices and protective enzyme activities were significantly inhibited, whereas MDA levels in the pepper leaves were significantly increased due to severe membrane lipid peroxidation. The results indicate that although low concentrations of garlic root exudates appear to induce protective enzyme systems and promote pepper growth, high concentrations have deleterious effects. These findings suggest that further investigations should optimize the co-culture pepper/garlic ratio to reduce continuous cropping obstacles in pepper production. PMID:27095440

  13. The Dipeptidyl Peptidases 4, 8, and 9 in Mouse Monocytes and Macrophages: DPP8/9 Inhibition Attenuates M1 Macrophage Activation in Mice.

    PubMed

    Waumans, Yannick; Vliegen, Gwendolyn; Maes, Lynn; Rombouts, Miche; Declerck, Ken; Van Der Veken, Pieter; Vanden Berghe, Wim; De Meyer, Guido R Y; Schrijvers, Dorien; De Meester, Ingrid

    2016-02-01

    Atherosclerosis remains the leading cause of death in Western countries. Dipeptidyl peptidase (DPP) 4 has emerged as a novel target for the prevention and treatment of atherosclerosis. Family members DPP8 and 9 are abundantly present in macrophage-rich regions of atherosclerotic plaques, and DPP9 inhibition attenuates activation of human M1 macrophages in vitro. Studying this family in a mouse model for atherosclerosis would greatly advance our knowledge regarding their potential as therapeutic targets. We found that DPP4 is downregulated during mouse monocyte-to-macrophage differentiation. DPP8 and 9 expression seems relatively low in mouse monocytes and macrophages. Viability of primary mouse macrophages is unaffected by DPP4 or DPP8/9 inhibition. Importantly, DPP8/9 inhibition attenuates macrophage activation as IL-6 secretion is significantly decreased. Mouse macrophages respond similarly to DPP inhibition, compared to human macrophages. This shows that the mouse could become a valid model species for the study of DPPs as therapeutic targets in atherosclerosis.

  14. Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-α and NO production in macrophages.

    PubMed

    Liu, Qiang; Zheng, Jin; Yin, Dan-Dan; Xiang, Jie; He, Fei; Wang, Yao-Chun; Liang, Liang; Qin, Hong-Yan; Liu, Li; Liang, Ying-Min; Han, Hua

    2012-05-01

    Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP-MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.

  15. Berberine augments ATP-induced inflammasome activation in macrophages by enhancing AMPK signaling

    PubMed Central

    Xu, Li-Hui; Liang, Yi-Dan; Wei, Hong-Xia; Hu, Bo; Pan, Hao; Zha, Qing-Bing; Ouyang, Dong-Yun; He, Xian-Hui

    2017-01-01

    The isoquinoline alkaloid berberine possesses many pharmacological activities including antibacterial infection. Although the direct bactericidal effect of berberine has been documented, its influence on the antibacterial functions of macrophages is largely unknown. As inflammasome activation in macrophages is important for the defense against bacterial infection, we aimed to investigate the influence of berberine on inflammasome activation in murine macrophages. Our results showed that berberine significantly increased ATP-induced inflammasome activation as reflected by enhanced pyroptosis as well as increased release of caspase-1p10 and mature interleukin-1β (IL-1β) in macrophages. Such effects of berberine could be suppressed by AMP-activated protein kinase (AMPK) inhibitor compound C or by knockdown of AMPKα expression, indicating the involvement of AMPK signaling in this process. In line with increased IL-1β release, the ability of macrophages to kill engulfed bacteria was also intensified by berberine. This was corroborated by the in vivo finding that the peritoneal live bacterial load was decreased by berberine treatment. Moreover, berberine administration significantly improved survival of bacterial infected mice, concomitant with increased IL-1β levels and elevated neutrophil recruitment in the peritoneal cavity. Collectively, these data suggested that berberine could enhance bacterial killing by augmenting inflammasome activation in macrophages through AMPK signaling. PMID:27980220

  16. High salt primes a specific activation state of macrophages, M(Na).

    PubMed

    Zhang, Wu-Chang; Zheng, Xiao-Jun; Du, Lin-Juan; Sun, Jian-Yong; Shen, Zhu-Xia; Shi, Chaoji; Sun, Shuyang; Zhang, Zhiyuan; Chen, Xiao-Qing; Qin, Mu; Liu, Xu; Tao, Jun; Jia, Lijun; Fan, Heng-Yu; Zhou, Bin; Yu, Ying; Ying, Hao; Hui, Lijian; Liu, Xiaolong; Yi, Xianghua; Liu, Xiaojing; Zhang, Lanjing; Duan, Sheng-Zhong

    2015-08-01

    High salt is positively associated with the risk of many diseases. However, little is known about the mechanisms. Here we showed that high salt increased proinflammatory molecules, while decreased anti-inflammatory and proendocytic molecules in both human and mouse macrophages. High salt also potentiated lipopolysaccharide-induced macrophage activation and suppressed interleukin 4-induced macrophage activation. High salt induced the proinflammatory aspects by activating p38/cFos and/or Erk1/2/cFos pathways, while inhibited the anti-inflammatory and proendocytic aspects by Erk1/2/signal transducer and activator of transcription 6 pathway. Consistent with the in vitro results, high-salt diet increased proinflammatory gene expression of mouse alveolar macrophages. In mouse models of acute lung injury, high-salt diet aggravated lipopolysaccharide-induced pulmonary macrophage activation and inflammation in lungs. These results identify a novel macrophage activation state, M(Na), and high salt as a potential environmental risk factor for lung inflammation through the induction of M(Na).

  17. High salt primes a specific activation state of macrophages, M(Na)

    PubMed Central

    Zhang, Wu-Chang; Zheng, Xiao-Jun; Du, Lin-Juan; Sun, Jian-Yong; Shen, Zhu-Xia; Shi, Chaoji; Sun, Shuyang; Zhang, Zhiyuan; Chen, Xiao-qing; Qin, Mu; Liu, Xu; Tao, Jun; Jia, Lijun; Fan, Heng-yu; Zhou, Bin; Yu, Ying; Ying, Hao; Hui, Lijian; Liu, Xiaolong; Yi, Xianghua; Liu, Xiaojing; Zhang, Lanjing; Duan, Sheng-Zhong

    2015-01-01

    High salt is positively associated with the risk of many diseases. However, little is known about the mechanisms. Here we showed that high salt increased proinflammatory molecules, while decreased anti-inflammatory and proendocytic molecules in both human and mouse macrophages. High salt also potentiated lipopolysaccharide-induced macrophage activation and suppressed interleukin 4-induced macrophage activation. High salt induced the proinflammatory aspects by activating p38/cFos and/or Erk1/2/cFos pathways, while inhibited the anti-inflammatory and proendocytic aspects by Erk1/2/signal transducer and activator of transcription 6 pathway. Consistent with the in vitro results, high-salt diet increased proinflammatory gene expression of mouse alveolar macrophages. In mouse models of acute lung injury, high-salt diet aggravated lipopolysaccharide-induced pulmonary macrophage activation and inflammation in lungs. These results identify a novel macrophage activation state, M(Na), and high salt as a potential environmental risk factor for lung inflammation through the induction of M(Na). PMID:26206316

  18. Model-Based Characterization of Inflammatory Gene Expression Patterns of Activated Macrophages

    PubMed Central

    Ehlting, Christian; Thomas, Maria; Zanger, Ulrich M.; Sawodny, Oliver; Häussinger, Dieter; Bode, Johannes G.

    2016-01-01

    Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization. PMID:27464342

  19. The mycotoxin deoxynivalenol inhibits the cell surface expression of activation markers in human macrophages.

    PubMed

    Waché, Yann J; Hbabi-Haddioui, Laila; Guzylack-Piriou, Laurence; Belkhelfa, Haouaria; Roques, Christine; Oswald, Isabelle P

    2009-08-21

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-gamma, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-gamma upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5muM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed.

  20. Macrophages from disease resistant B2 haplotype chickens activate T lymphocytes more effectively than macrophages from disease susceptible B19 birds.

    PubMed

    Collisson, Ellen; Griggs, Lisa; Drechsler, Yvonne

    2017-02-01

    Resistance to respiratory pathogens, including coronavirus-induced infection and clinical illness in chickens has been correlated with the B (MHC) complex and differential ex vivo macrophage responses. In the current study, in vitro T lymphocyte activation measured by IFNγ release was significantly higher in B2 versus B19 haplotypes. AIV infection of macrophages was required to activate T lymphocytes and prior in vivo exposure of chickens to NP AIV plasmid enhanced responses to infected macrophages. This study suggests that the demonstrated T lymphocyte activation is in part due to antigen presentation by the macrophages as well as cytokine release by the infected macrophages, with B2 haplotypes showing stronger activation. These responses were present both in CD4 and CD8 T lymphocytes. In contrast, T lymphocytes stimulated by ConA showed greater IFNγ release of B19 haplotype cells, further indicating the greater responses in B2 haplotypes to infection is due to macrophages, but not T cells. In summary, resistance of B2 haplotype chickens appears to be directly linked to a more vigorous innate immune response and the role macrophages play in activating adaptive immunity.

  1. Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability

    PubMed Central

    Chávez-Galán, Leslie; Vesin, Dominique; Martinvalet, Denis

    2016-01-01

    Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies. PMID:27833923

  2. Activation of TLR3/interferon signaling pathway by bluetongue virus results in HIV inhibition in macrophages.

    PubMed

    Dai, Ming; Wang, Xu; Li, Jie-Liang; Zhou, Yu; Sang, Ming; Liu, Jin-Biao; Wu, Jian-Guo; Ho, Wen-Zhe

    2015-12-01

    Bluetongue virus (BTV), a nonenveloped double-stranded RNA virus, is a potent inducer of type Ι interferons in multiple cell systems. In this study, we report that BTV16 treatment of primary human macrophages induced both type I and III IFN expression, resulting in the production of multiple antiviral factors, including myxovirus resistance protein A, 2',5'-oligoadenylate synthetase, and the IFN-stimulated gene 56. Additionally, BTV-treated macrophages expressed increased HIV restriction factors (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3 G/F/H) and CC chemokines (macrophage inflammatory protein 1-α, macrophage inflammatory protein 1-β, regulated on activation of normal T cell expressed and secreted), the ligands for HIV entry coreceptor CC chemokine receptor type 5. BTV16 also induced the expression of tetherin, which restricts HIV release from infected cells. Furthermore, TLR3 signaling of macrophages by BTV16 resulted in the induction of several anti-HIV microRNAs (miRNA-28, -29a, -125b, -150, -223, and -382). More importantly, the induction of antiviral responses by BTV resulted in significant suppression of HIV in macrophages. These findings demonstrate the potential of BTV-mediated TLR3 activation in macrophage innate immunity against HIV.

  3. Enhancement of Anti-Inflammatory Activity of Curcumin Using Phosphatidylserine-Containing Nanoparticles in Cultured Macrophages.

    PubMed

    Wang, Ji; Kang, Yu-Xia; Pan, Wen; Lei, Wan; Feng, Bin; Wang, Xiao-Juan

    2016-06-20

    Macrophages are one kind of innate immune cells, and produce a variety of inflammatory cytokines in response to various stimuli, such as oxidized low density lipoprotein found in the pathogenesis of atherosclerosis. In this study, the effect of phosphatidylserine on anti-inflammatory activity of curcumin-loaded nanostructured lipid carriers was investigated using macrophage cultures. Different amounts of phosphatidylserine were used in the preparation of curcumin nanoparticles, their physicochemical properties and biocompatibilities were then compared. Cellular uptake of the nanoparticles was investigated using a confocal laser scanning microscope and flow cytometry analysis in order to determine the optimal phosphatidylserine concentration. In vitro anti-inflammatory activities were evaluated in macrophages to test whether curcumin and phosphatidylserine have interactive effects on macrophage lipid uptake behavior and anti-inflammatory responses. Here, we showed that macrophage uptake of phosphatidylserine-containing nanostructured lipid carriers increased with increasing amount of phosphatidylserine in the range of 0%-8%, and decreased when the phosphatidylserine molar ratio reached over 12%. curcumin-loaded nanostructured lipid carriers significantly inhibited lipid accumulation and pro-inflammatory factor production in cultured macrophages, and evidently promoted release of anti-inflammatory cytokines, when compared with curcumin or phosphatidylserine alone. These results suggest that the delivery system using PS-based nanoparticles has great potential for efficient delivery of drugs such as curcumin, specifically targeting macrophages and modulation of their anti-inflammatory functions.

  4. Relationship of MMP-14 and TIMP-3 Expression with Macrophage Activation and Human Atherosclerotic Plaque Vulnerability

    PubMed Central

    Johnson, Jason L.; Jenkins, Nicholas P.; Huang, Wei-Chun; Sala-Newby, Graciela B.; Scholtes, Vincent P. W.; Moll, Frans L.; Pasterkamp, Gerard; Newby, Andrew C.

    2014-01-01

    Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14hi  TIMP-3lo rabbit foam cells are more invasive and more prone to apoptosis than MMP-14lo  TIMP-3hi cells. We investigated the implications of these findings for human atherosclerosis. In vitro generated macrophages and foam-cell macrophages, together with atherosclerotic plaques characterised as unstable or stable, were examined for expression of MMP-14, TIMP-3, and inflammatory markers. Proinflammatory stimuli increased MMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages. However, conversion to foam-cells with oxidized LDL increased MMP-14 and decreased TIMP-3 protein, independently of inflammatory mediators and partly through posttranscriptional mechanisms. Within atherosclerotic plaques, MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage markers, whereas TIMP-3 was present in less foamy macrophages and colocalised with CD206. MMP-14 positive macrophages were more abundant whereas TIMP-3 positive macrophages were less abundant in plaques histologically designated as rupture prone. We conclude that foam-cells characterised by high MMP-14 and low TIMP-3 expression are prevalent in rupture-prone atherosclerotic plaques, independent of pro- or anti-inflammatory activation. Therefore reducing MMP-14 activity and increasing that of TIMP-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction. PMID:25301980

  5. Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability.

    PubMed

    Chávez-Galán, Leslie; Vesin, Dominique; Martinvalet, Denis; Garcia, Irene

    2016-01-01

    Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

  6. Activation of Rac1 GTPase by nanoparticulate structures in human macrophages.

    PubMed

    Diesel, Britta; Hoppstädter, Jessica; Hachenthal, Nina; Zarbock, Robert; Cavelius, Christian; Wahl, Birgit; Thewes, Nicolas; Jacobs, Karin; Kraegeloh, Annette; Kiemer, Alexandra K

    2013-06-01

    Inflammatory activation of alveolar macrophages by ambient particles can be facilitated via Toll-like receptors (TLR). The action of TLR agonists and antagonists has been reported to depend on the formation of nanoparticulate structures. Aim of the present study was to identify the signaling pathways induced by nanoparticulate structures in human macrophages, which might be critical for inflammatory cell activation. Studies were performed in primary human alveolar macrophages or in differentiated THP-1 macrophages. Silica nanoparticles were prepared by Stöber synthesis and characterized by dynamic light scattering and scanning electron microscopy. Mycobacterial DNA was isolated from Mycobacterium bovis BCG, and nanoparticle formation was assessed by atomic force microscopy and dynamic light scattering. Actin polymerization was measured by phalloidin-TRITC staining, and cell activation was determined by reverse transcription quantitative PCR analysis, L929 cytotoxicity assay (cytokine induction), and pull-down assays (Rho GTPases). In contrast to immune stimulatory sequence ISS 1018, BCG DNA spontaneously formed nanoparticulate structures and induced actin polymerization as did synthetic silica nanoparticles. Co-incubation with silica nanoparticles amplified the responsiveness of macrophages toward the TLR9 ligand ISS 1018. The activation of Rac1 was induced by silica nanoparticles as well as BCG DNA and is suggested as the critical signaling event inducing both cytoskeleton changes as well as inflammatory cell activation. Nanoparticles can induce signaling pathways, which amplify an inflammatory response in macrophages. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Schistosoma mansoni Hemozoin Modulates Alternative Activation of Macrophages via Specific Suppression of Retnla Expression and Secretion

    PubMed Central

    Truscott, Martha; Evans, D. Andrew; Gunn, Matt

    2013-01-01

    The trematode Schistosoma mansoni is one of the etiological agents of schistosomiasis, a key neglected tropical disease responsible for an estimated annual loss of 70 million disability-adjusted life years. Hematophagy represents the primary nutrient acquisition pathway of this parasite, but digestion of hemoglobin also liberates toxic heme. Schistosomes detoxify heme via crystallization into hemozoin, which is subsequently regurgitated into the host's circulation. Here we demonstrate that during experimental schistosomiasis, hemozoin accumulating in the mouse liver is taken up by phagocytes at a time coincident with the development of the egg-induced T-helper 2 (Th2) granulomatous immune response. Furthermore, the uptake of hemozoin also coincides with the hepatic expression of markers of alternative macrophage activation. Alternatively activated macrophages are a key effector cell population associated with protection against schistosomiasis, making hemozoin well placed to play an important immunomodulatory role in this disease. To systematically explore this hypothesis, S. mansoni hemozoin was purified and added to in vitro bone marrow-derived macrophage cultures concurrently exposed to cytokines chosen to reflect the shifting state of macrophage activation in vivo. Macrophages undergoing interleukin-4 (IL-4)-induced alternative activation in the presence of hemozoin developed a phenotype specifically lacking in Retnla, a characteristic alternatively activated macrophage product associated with regulation of Th2 inflammatory responses. As such, in addition to its important detoxification role during hematophagy, we propose that schistosome hemozoin also provides a potent immunomodulatory function in the coevolved network of host-parasite relationships during schistosomiasis. PMID:23090958

  8. Transplantable Subcutaneous Hepatoma 22a Affects Functional Activity of Resident Tissue Macrophages in Periphery

    PubMed Central

    Kisseleva, Ekaterina P.; Krylov, Andrei V.; Stepanova, Olga I.; Lioudyno, Victoria I.

    2011-01-01

    Tumors spontaneously develop central necroses due to inadequate blood supply. Recent data indicate that dead cells and their products are immunogenic to the host. We hypothesized that macrophage tumor-dependent reactions can be mediated differentially by factors released from live or dead tumor cells. In this study, functional activity of resident peritoneal macrophages was investigated in parallel with tumor morphology during the growth of syngeneic nonimmunogenic hepatoma 22a. Morphometrical analysis of tumor necroses, mitoses and leukocyte infiltration was performed in histological sections. We found that inflammatory potential of peritoneal macrophages in tumor-bearing mice significantly varied depending on the stage of tumor growth and exhibited two peaks of activation as assessed by nitroxide and superoxide anion production, 5′-nucleotidase activity and pinocytosis. Increased inflammatory reactions were not followed by the enhancement of angiogenic potential as assessed by Vascular Endothelial Growth Factor mRNA expression. Phases of macrophage activity corresponded to the stages of tumor growth characterized by high proliferative potential. The appearance and further development of necrotic tissue inside the tumor did not coincide with changes in macrophage behavior and therefore indirectly indicated that activation of macrophages was a reaction mostly to the signals produced by live tumor cells. PMID:21760797

  9. Hyper-Inflammation and Skin Destruction Mediated by Rosiglitazone Activation of Macrophages in IL-6 Deficiency

    PubMed Central

    Das, Lopa M; Rosenjack, Julie; Au, Liemin; Galle, Pia S; Hansen, Morten B; Cathcart, Martha K; McCormick, Thomas S; Cooper, Kevin D; Silverstein, Roy L; Lu, Kurt Q

    2015-01-01

    Injury initiates recruitment of macrophages to support tissue repair; however, excessive macrophage activity may exacerbate tissue damage causing further destruction and subsequent delay in wound repair. Here we show that the peroxisome proliferation–activated receptor-γ agonist, rosiglitazone (Rosi), a medication recently reintroduced as a drug to treat diabetes and with known anti-inflammatory properties, paradoxically generates pro-inflammatory macrophages. This is observed in both IL-6-deficient mice and control wild-type mice experimentally induced to produce high titers of auto-antibodies against IL-6, mimicking IL-6 deficiency in human diseases. IL-6 deficiency when combined with Rosi-mediated upregulation of suppressor of cytokine signaling 3 leads to an altered ratio of nuclear signal transducer and activator of transcription 3/NF-κB that allows hyper-induction of inducible nitric oxide synthase (iNOS). Macrophages activated in this manner cause de novo tissue destruction, recapitulating human chronic wounds, and can be reversed in vivo by recombinant IL-6, blocking macrophage infiltration, or neutralizing iNOS. This study provides insight into an unanticipated paradoxical role of Rosi in mediating hyper-inflammatory macrophage activation significant for diseases associated with IL-6 deficiency. PMID:25184961

  10. Augmentation of macrophage growth-stimulating activity of lipids by their peroxidation

    SciTech Connect

    Yui, S.; Yamazaki, M. )

    1990-02-15

    Previously, we reported that some kinds of lipids (cholesterol esters, triglycerides, and some negatively charged phospholipids) that are constituents of lipoproteins or cell membranes induce growth of peripheral macrophages in vitro. In this paper, we examined the effect of peroxidation of lipids on their macrophage growth-stimulating activity because lipid peroxidation is observed in many pathological states such as inflammation. When phosphatidylserine, one of the phospholipids with growth-stimulating activity, was peroxidized by UV irradiation, its macrophage growth-stimulating activity was augmented in proportion to the extent of its peroxidation. The activity of phosphatidylethanolamine was also increased by UV irradiation. On the other hand, phosphatidylcholine or highly unsaturated free fatty acids, such as arachidonic acid and eicosapentaenoic acid, did not induce macrophage growth irrespective of whether they were peroxidized. The augmented activity of UV-irradiated phosphatidylserine was not affected by the coexistence of an antioxidant, vitamin E or BHT. These results suggest that some phospholipids included in damaged cells or denatured lipoproteins which are scavenged by macrophages in vivo may induce growth of peripheral macrophages more effectively when they are peroxidized by local pathological processes.

  11. The insect peptide CopA3 inhibits lipopolysaccharide-induced macrophage activation.

    PubMed

    Nam, Hyo Jung; Oh, Ah Reum; Nam, Seung Taek; Kang, Jin Ku; Chang, Jong Soo; Kim, Dae Hong; Lee, Ji Hye; Hwang, Jae Sam; Shong, Ko Eun; Park, Mi Jung; Seok, Heon; Kim, Ho

    2012-10-01

    We recently demonstrated that the insect peptide CopA3 (LLCIALRKK), a disulfide-linked dimeric peptide, exerts antimicrobial and anti-inflammatory activities in a mouse colitis model. Here, we examined whether CopA3 inhibited activation of macrophages by LPS. Exposure of an unseparated mouse peritoneal cell population or isolated peritoneal macrophages to LPS markedly increased secretion of IL-6 and TNF-α; these effects were significantly inhibited by CopA3 treatment. The inhibitory effect of CopA3 was also evident in murine macrophage cell line, RAW 264.7. Western blotting revealed that LPS-induced activation of STAT1 and STAT5 in macrophages was significantly inhibited by CopA3. Inhibition of JAK (STAT1/STAT5 kinase) with AG490 markedly reduced the production of IL-6 and TNF-α in macrophages. Collectively, these observations suggest that CopA3 inhibits macrophage activation by inhibiting activating phosphorylations of the transcription factors, STAT1 and STAT5, and blocking subsequent production of IL-6 and TNF-α and indicate that CopA3 may be useful as an immune-modulating agent.

  12. The macrophage chemotactic activity of Edwardsiella tarda extracellular products

    USDA-ARS?s Scientific Manuscript database

    The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from ...

  13. Statin attenuates experimental anti-glomerular basement membrane glomerulonephritis together with the augmentation of alternatively activated macrophages.

    PubMed

    Fujita, Emiko; Shimizu, Akira; Masuda, Yukinari; Kuwahara, Naomi; Arai, Takashi; Nagasaka, Shinya; Aki, Kaoru; Mii, Akiko; Natori, Yasuhiro; Iino, Yasuhiko; Katayama, Yasuo; Fukuda, Yuh

    2010-09-01

    Macrophages are heterogeneous and include classically activated M1 and alternatively activated M2 macrophages, characterized by pro- and anti-inflammatory functions, respectively. Macrophages that express heme oxygenase-1 also exhibit anti-inflammatory effects. We assessed the anti-inflammatory effects of statin in experimental anti-glomerular basement membrane glomerulonephritis and in vitro, focusing on the macrophage heterogeneity. Rats were induced anti-glomerular basement membrane glomerulonephritis and treated with atorvastatin (20 mg/kg/day) or vehicle (control). Control rats showed infiltration of macrophages in the glomeruli at day 3 and developed crescentic glomerulonephritis by day 7, together with increased mRNA levels of the M1 macrophage-associated cytokines, interferon-gamma, tumor necrosis factor-alpha, and interleukin-12. In contrast, statin reduced the level of proteinuria, reduced infiltration of macrophages in glomeruli with suppression of monocyte chemotactic protein-1 expression, and inhibited the formation of necrotizing and crescentic lesions. The number of glomerular ED3-positive macrophages decreased with down-regulation of M1 macrophage-associated cytokines. Furthermore, statin augmented ED2-positive M2 macrophages with up-regulation of the M2 macrophage-associated chemokines and cytokines, chemokine (C-C motif) Iigand-17 and interleukin-10. Statin also increased the glomerular interleukin-10-expressing heme oxygenase-1-positive macrophages. Statin inhibited macrophage development, and suppressed ED3-positive macrophages, but augmented ED2-positive macrophages in M2-associated cytokine environment in vitro. We conclude that the anti-inflammatory effects of statin in glomerulonephritis are mediated through inhibition of macrophage infiltration as well as augmentation of anti-inflammatory macrophages.

  14. Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach.

    PubMed

    Adnani, Navid; Vazquez-Rivera, Emmanuel; Adibhatla, Srikar N; Ellis, Gregory A; Braun, Doug R; Bugni, Tim S

    2015-09-24

    With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.

  15. Multilineage co-culture of adipose-derived stem cells for tissue engineering.

    PubMed

    Zhao, Yimu; Waldman, Stephen D; Flynn, Lauren E

    2015-07-01

    Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue-engineering strategies with adipose-derived stem cells (ASCs), the focus of this study was on developing indirect co-culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co-culture with each of the mature cell populations was shown to successfully induce or enhance lineage-specific differentiation of the ASCs. In general, a more homogeneous but lower-level differentiation response was observed in co-culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non-canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF-1 and DKK-1 on multilineage differentiation in co-culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co-culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach

    PubMed Central

    Adnani, Navid; Vazquez-Rivera, Emmanuel; Adibhatla, Srikar N.; Ellis, Gregory A.; Braun, Doug R.; Bugni, Tim S.

    2015-01-01

    With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes. PMID:26404321

  17. PGC-1β suppresses saturated fatty acid-induced macrophage inflammation by inhibiting TAK1 activation.

    PubMed

    Chen, Hongen; Liu, Yan; Li, Di; Song, Jiayi; Xia, Min

    2016-02-01

    Inflammation of infiltrated macrophages in adipose tissue is a key contributor to the initiation of adipose insulin resistance. These macrophages are exposed to high local concentrations of free fatty acids (FFAs) and can be proinflammatory activated by saturated fatty acids (SFAs). However, the regulatory mechanisms on SFA-induced macrophage inflammation are still elusive. Peroxisome proliferator-activated receptor γ coactivator-1β (PGC-1β) is a member of the PGC-1 family of transcriptional coactivators and has been reported to play a key role in SFAs metabolism and in the regulation of inflammatory signaling. However, it remains unclear whether PGC-1β is involved in SFA-induced macrophage inflammation. In this study, we found that PGC-1β expression was significantly decreased in response to palmitic acid (PA) in macrophages in a dose dependent manner. PGC-1β inhibited PA induced TNFα, MCP-1, and IL-1β mRNA and protein expressions. Furthermore, PGC-1β significantly antagonized PA induced macrophage nuclear factor-κB (NF-κB) p65 and JUN N-terminal kinase activation. Mechanistically, we revealed that TGF-β-activated kinase 1 (TAK1) and its adaptor protein TAK1 binding protein 1 (TAB1) played a dominant role in the regulatory effects of PGC-1β. We confirmed that PGC-1β inhibited downstream inflammatory signals via binding with TAB1 and thus preventing TAB1/TAK1 binding and TAK1 activation. Finally, we showed that PGC-1β overexpression in PA treated macrophages improved adipocytes PI3K-Akt insulin signaling in a paracrine fashion. Collectively, our results uncovered a novel mechanism on how macrophage inflammation induced by SFAs was regulated and suggest a potential target in the treatment of obesity induced insulin resistance.

  18. A New Immunomodulatory Role for Peroxisomes in Macrophages Activated by the TLR4 Ligand Lipopolysaccharide.

    PubMed

    Vijayan, Vijith; Srinu, Tumpara; Karnati, Srikanth; Garikapati, Vannuruswamy; Linke, Monika; Kamalyan, Lilit; Mali, Srihari Reddy; Sudan, Kritika; Kollas, Andreas; Schmid, Tobias; Schulz, Sabine; Spengler, Bernhard; Weichhart, Thomas; Immenschuh, Stephan; Baumgart-Vogt, Eveline

    2017-03-15

    Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene Pex14 or Mfp2 markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation. Copyright © 2017 by The American Association of Immunologists, Inc.

  19. Macrophage antitumor activity induced by the antigenic lymphoma L5178Y/DTIC subline.

    PubMed

    Marelli, O; Mantovani, A; Franco, P; Nicolin, A

    1982-10-31

    Murine leukemic cells, after in vivo treatment with antineoplastic drugs, have been shown to express new antigenic specificities that were not detectable on parental cells and that were heritable after the withdrawal of drug treatment. A study was conducted of macrophage antitumor activity triggered by LY/DTIC cells, a subline of LY murine lymphoma, antigenically altered by the drug DTIC. In vitro non-specific inhibition of tumor cell growth was exhibited by spleen and peritoneal macrophages from mice previously challenged with viable LY/DTIC. Peritoneal macrophages from LY/DTIC immune animals showed moderate, although significant lytic activity against unrelated tumor target cells. Supernatants from mixed lymphocyte-tumor cell cultures, in which LY/DTIC immune lymphocytes and LY/DTIC tumor cells had been cultured, rendered normal macrophages non-specifically growth inhibitory for tumor cells.

  20. METEORIN-LIKE is a cytokine associated with barrier tissues and alternatively activated macrophages

    PubMed Central

    Ushach, Irina; Burkhardt, Amanda M.; Martinez, Cynthia; Hevezi, Peter A.; Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Valle-Rios, Ricardo; Vazquez, Monica I.; Homey, Bernhard; Zlotnik, Albert

    2014-01-01

    Cytokines are involved in many functions of the immune system including initiating, amplifying and resolving immune responses. Through bioinformatics analyses of a comprehensive database of gene expression (BIGE: Body Index of Gene Expression) we observed that a small secreted protein encoded by a poorly characterized gene called meteorin-like (METRNL), is highly expressed in mucosal tissues, skin and activated macrophages. Further studies indicate that Metrnl is produced by Alternatively Activated Macrophages (AAM) and M-CSF cultured bone marrow macrophages (M2-like macrophages). In the skin, METRNL is expressed by resting fibroblasts and IFNγ-treated keratinocytes. A screen of human skin-associated diseases showed significant over-expression of METRNL in psoriasis, prurigo nodularis, actinic keratosis and atopic dermatitis. METRNL is also up-regulated in synovial membranes of human rheumatoid arthritis. Taken together, these results indicate that Metrnl represents a novel cytokine, which is likely involved in both innate and acquired immune responses. PMID:25486603

  1. Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms

    PubMed Central

    Rőszer, Tamás

    2015-01-01

    The alternatively activated or M2 macrophages are immune cells with high phenotypic heterogeneity and are governing functions at the interface of immunity, tissue homeostasis, metabolism, and endocrine signaling. Today the M2 macrophages are identified based on the expression pattern of a set of M2 markers. These markers are transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. This review discusses whether these M2 markers can be reliably used to identify M2 macrophages and define their functional subdivisions. Also, it provides an update on the novel signals of the tissue environment and the neuroendocrine system which shape the M2 activation. The possible evolutionary roots of the M2 macrophage functions are also discussed. PMID:26089604

  2. Functional Activity of Monocytes and Macrophages in HTLV-1 Infected Subjects

    PubMed Central

    Amorim, Camila F.; Souza, Anselmo S.; Diniz, Angela G.; Carvalho, Natália B.; Santos, Silvane B.; Carvalho, Edgar M.

    2014-01-01

    The Human T lymphotropic virus type-1 (HTLV-1) infects predominantly T cells, inducing proliferation and lymphocyte activation. Additionally, HTLV-1 infected subjects are more susceptible to other infections caused by other intracellular agents. Monocytes/macrophages are important cells in the defense against intracellular pathogens. Our aims were to determine the frequency of monocytes subsets, expression of co-stimulatory molecules in these cells and to evaluate microbicidal ability and cytokine and chemokine production by macrophages from HTLV-1 infected subjects. Participants were 23 HTLV-1 carriers (HC), 22 HAM/TSP patients and 22 healthy subjects (HS) not infected with HTLV-1. The frequencies of monocyte subsets and expression of co-stimulatory molecules were determined by flow cytometry. Macrophages were infected with L. braziliensis or stimulated with LPS. Microbicidal activity of macrophages was determined by optic microscopy. Cytokines/chemokines from macrophage supernatants were measured by ELISA. HAM/TSP patients showed an increase frequency of intermediate monocytes, but expression of co-stimulatory molecules was similar between the groups. Macrophages from HTLV-1 infected individuals were infected with L. braziliensis at the same ratio than macrophages from HS, and all the groups had the same ability to kill Leishmania parasites. However, macrophages from HTLV-1 infected subjects produced more CXCL9 and CCL5, and less IL-10 than cells from HS. While there was no correlation between IFN-γ and cytokine/chemokine production by macrophages, there was a correlation between proviral load and TNF and CXCL10. These data showed a dissociation between the inflammatory response and microbicidal ability of macrophages from HTLV-1 infected subjects. While macrophages ability to kill an intracellular pathogen did not differ among HTLV-1 infected subjects, these cells secreted high amount of chemokines even in unstimulated cultures. Moreover the increasing

  3. PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation

    PubMed Central

    Iwata, Hiroshi; Goettsch, Claudia; Sharma, Amitabh; Ricchiuto, Piero; Goh, Wilson Wen Bin; Halu, Arda; Yamada, Iwao; Yoshida, Hideo; Hara, Takuya; Wei, Mei; Inoue, Noriyuki; Fukuda, Daiju; Mojcher, Alexander; Mattson, Peter C.; Barabási, Albert-László; Boothby, Mark; Aikawa, Elena; Singh, Sasha A.; Aikawa, Masanori

    2016-01-01

    Despite the global impact of macrophage activation in vascular disease, the underlying mechanisms remain obscure. Here we show, with global proteomic analysis of macrophage cell lines treated with either IFNγ or IL-4, that PARP9 and PARP14 regulate macrophage activation. In primary macrophages, PARP9 and PARP14 have opposing roles in macrophage activation. PARP14 silencing induces pro-inflammatory genes and STAT1 phosphorylation in M(IFNγ) cells, whereas it suppresses anti-inflammatory gene expression and STAT6 phosphorylation in M(IL-4) cells. PARP9 silencing suppresses pro-inflammatory genes and STAT1 phosphorylation in M(IFNγ) cells. PARP14 induces ADP-ribosylation of STAT1, which is suppressed by PARP9. Mutations at these ADP-ribosylation sites lead to increased phosphorylation. Network analysis links PARP9–PARP14 with human coronary artery disease. PARP14 deficiency in haematopoietic cells accelerates the development and inflammatory burden of acute and chronic arterial lesions in mice. These findings suggest that PARP9 and PARP14 cross-regulate macrophage activation. PMID:27796300

  4. Effect of cinnamon water extract on monocyte-to-macrophage differentiation and scavenger receptor activity

    PubMed Central

    2014-01-01

    Background Water soluble cinnamon extract has been shown to increase insulin sensitivity and modulate macrophage activation, a desirable trait for the management of obesity or atherosclerosis. Our present study investigated whether cinnamon water extract (CWE) may influence the differentiation of monocytes into macrophages and the activity of macrophage scavenger receptors, commonly observed in atherosclerotic lesions. Methods We investigated the effect of CWE on the expression of various surface markers and the uptake of acetylated low density lipoprotein (LDL) in phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cells. The protein levels of PMA or macrophage-colony stimulating factor (M-CSF)-stimulated type 1 macrophage scavenger receptor (SRA) were analyzed. Finally, the role of extracellar signal-related kinase (ERK) 1/2 in SRA synthesis and the effect of CWE on PMA-stimulated ERK1/2 were determined. Results CWE inhibited the differentiation of monocyte by decreasing the expression of CD11b, CD36 and SRA and the uptake of acetyl LDL. CWE suppressed the upregulation of SRA by M-CSF and modulated ERK1/2 activity, which was required for PMA-induced SRA synthesis. Conclusions Our results demonstrate that CWE was able to interfere with monocyte differentiation and macrophage scavenger activity, indicating its potential in preventing the development of atherosclerotic lesions. PMID:24602512

  5. Contribution of alternatively activated macrophages to allergic lung inflammation: a tale of mice and men.

    PubMed

    Dasgupta, Preeta; Keegan, Achsah D

    2012-01-01

    The concept that macrophages play an active role in inflammatory responses began its development in the late 1800s with the now iconic studies by Elie Metchnikoff using starfish larvae and Daphnia [reviewed in Kaufmann SHE: Nat Immunol 2008;9:705-712 and Cavaillon JM: J Leukoc Biol 2011;90:413-424]. Based on his observation of the phagocyte response to a foreign body (rose thorn) and yeast, he proposed that phagocytes acted in host defense and were active participants in the inflammatory process. Flash forward more than 100 years and we find that these basic tenets hold true. However, it is now appreciated that macrophages come in many different flavors and can adopt a variety of nuanced phenotypes depending on the tissue environment in which the macrophage is found. In this brief review, we discuss the role of one type of macrophage termed the alternatively activated macrophage (AAM), also known as the M2 type of macrophage, in regulating allergic lung inflammation and asthma. Recent studies using mouse models of allergic lung inflammation and samples from human asthma patients contribute to the emerging concept that AAMs are not just bystanders of the interleukin (IL)-4- and IL-13-rich environment found in allergic asthma but are also active players in orchestrating allergic lung disease.

  6. ROS-responsive activatable photosensitizing agent for imaging and photodynamic therapy of activated macrophages.

    PubMed

    Kim, Hyunjin; Kim, Youngmi; Kim, In-Hoo; Kim, Kyungtae; Choi, Yongdoo

    2013-01-01

    The optical properties of macrophage-targeted theranostic nanoparticles (MacTNP) prepared from a Chlorin e6 (Ce6)-hyaluronic acid (HA) conjugate can be activated by reactive oxygen species (ROS) in macrophage cells. MacTNP are nonfluorescent and nonphototoxic in their native state. However, when treated with ROS, especially peroxynitrite, they become highly fluorescent and phototoxic. In vitro cell studies show that MacTNP emit near-infrared (NIR) fluorescence inside activated macrophages. The NIR fluorescence is quenched in the extracellular environment. MacTNP are nontoxic in macrophages up to a Ce6 concentration of 10 μM in the absence of light. However, MacTNP become phototoxic upon illumination in a light dose-dependent manner. In particular, significantly higher phototoxic effect is observed in the activated macrophage cells compared to human dermal fibroblasts and non-activated macrophages. The ROS-responsive MacTNP, with their high target-to-background ratio, may have a significant potential in selective NIR fluorescence imaging and in subsequent photodynamic therapy of atherosclerosis with minimum side effects.

  7. Activation of Adipose Tissue Macrophages in Obese Mice does not Require Lymphocytes

    PubMed Central

    Behan, JW; Ehsanipour, EA; Sheng, X; Pramanik, R; Wang, Xingchao; Hsieh, Yao-Te; Kim, Yong-Mi; Mittelman, Steven D.

    2012-01-01

    Macrophages which infiltrate adipose tissue and secrete pro-inflammatory cytokines may be responsible for obesity-induced insulin resistance. However, why macrophages migrate into adipose tissue and become activated remains unknown, though some studies suggest this may be regulated by T and B lymphocytes. In the present study, we test whether T and B lymphocytes and NK cells are necessary for the obesity-induced activation of macrophages in adipose tissue. NOD/SCID/IL2-receptor gamma-chain knockout (NSG) mice, which lack mature T and B lymphocytes and NK cells, were made obese by selectively reducing litters and weaning onto a high-fat diet. Mice were then maintained on the diet for 10-11 weeks. Adipose tissue from obese NSG mice had more activated macrophages than non-obese mice. These macrophages were found in “crown like structures” surrounding adipocytes, and expressed higher levels of the inflammatory cytokine TNFα. However, obesity did not impair glucose tolerance in the NSG mice. These studies demonstrate that T and B lymphocytes and NK cells are not necessary for adipose tissue macrophage activation in obese mice. T and B lymphocytes and/or NK cells may be necessary for the development of obesity-induced impaired glucose tolerance. PMID:23754826

  8. High and low molecular weight hyaluronic acid differentially influence macrophage activation.

    PubMed

    Rayahin, Jamie E; Buhrman, Jason S; Zhang, Yu; Koh, Timothy J; Gemeinhart, Richard A

    2015-07-13

    Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs.

  9. High and low molecular weight hyaluronic acid differentially influence macrophage activation

    PubMed Central

    Rayahin, Jamie E.; Buhrman, Jason S.; Zhang, Yu; Koh, Timothy J.; Gemeinhart, Richard A.

    2015-01-01

    Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs. PMID:26280020

  10. The ligand-bound thyroid hormone receptor in macrophages ameliorates kidney injury via inhibition of nuclear factor-κB activities.

    PubMed

    Furuya, Fumihiko; Ishii, Toshihisa; Tamura, Shogo; Takahashi, Kazuya; Kobayashi, Hidetoshi; Ichijo, Masashi; Takizawa, Soichi; Kaneshige, Masahiro; Suzuki-Inoue, Katsue; Kitamura, Kenichiro

    2017-03-08

    In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor α (TRα) in macrophages and examined the role of ligand-bound TRα in macrophage polarization-mediated anti-inflammatory effects. TRα-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRα displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRα-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1β levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRα on macrophages plays a protective role in kidney inflammation through the inhibition of NF-κB pathways, possibly by affecting the pro- and anti-inflammatory balance that controls the development of CKD.

  11. The ligand-bound thyroid hormone receptor in macrophages ameliorates kidney injury via inhibition of nuclear factor-κB activities

    PubMed Central

    Furuya, Fumihiko; Ishii, Toshihisa; Tamura, Shogo; Takahashi, Kazuya; Kobayashi, Hidetoshi; Ichijo, Masashi; Takizawa, Soichi; Kaneshige, Masahiro; Suzuki-Inoue, Katsue; Kitamura, Kenichiro

    2017-01-01

    In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor α (TRα) in macrophages and examined the role of ligand-bound TRα in macrophage polarization-mediated anti-inflammatory effects. TRα-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRα displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRα-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1β levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRα on macrophages plays a protective role in kidney inflammation through the inhibition of NF-κB pathways, possibly by affecting the pro- and anti-inflammatory balance that controls the development of CKD. PMID:28272516

  12. Activation of pulmonary macrophages for fungicidal activity by gamma-interferon or lymphokines.

    PubMed Central

    Brummer, E; Stevens, D A

    1987-01-01

    The ability of murine recombinant gamma interferon (IFN) or lymphokines to enhance the fungicidal activity of murine pulmonary macrophages (PuM) was studied in in vitro. PuM monolayers were incubated overnight with IFN, lymph node cells (LNC) plus concanavalin A, supernatants from Con A stimulated LNC or spleen cell cultures (Con A Sup), or tissue culture medium (TCM) +/- Con A (5 micrograms/ml) or +/- lipopolysaccharide (LPS, 10 ng to 10 micrograms/ml). After treatment, culture fluids were removed and PuM were challenged for 4 h with the yeast-form Blastomyces dermatitidis or 2 h with Candida albicans. Inoculum colony forming units (CFU) of B. dermatitidis were significantly reduced by PuM treated with 1000 U/ml of IFN (25 +/- 3%), Con A Sup (25 +/- 3%) or LNC plus Con A (37-44%), but not by TCM, ConA or LPS. Candida albicans was killed by PuM treated with Con A Sup (33 +/- 8%) or LNC plus Con A (30-43%), but not by TCM, Con A, or LPS, and the activity of Con A Sup was neutralized by anti-IFN antibody. Candida albicans was not significantly killed by PuM treated with IFN doses ranging from 1 to 10(5) U/ml; nor did addition of LPS to IFN, or prolonged (3 day) treatment with IFN, result in significant killing of C. albicans by PuM. However, IFN (100 U/ml) could activate resident peritoneal macrophages for significant candidacidal activity (63%). These data indicate that PuM can be activated for fungicidal activity, and that PuM differ from resident peritoneal macrophages with regard to induction of candidacidal activity by recombinant gamma-IFN. PMID:3124995

  13. The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro.

    PubMed

    Khabbazi, Samira; Xie, Nan; Pu, Wenjun; Goumon, Yannick; Parat, Marie-Odile

    2016-01-01

    Macrophages are abundant in the tumor microenvironment where they adopt a pro-tumor phenotype following alternative polarization induced by paracrine factors from cancer and stromal cells. In contrast, classically activated macrophages have tumoricidal activities, such that the polarization of tumor-associated macrophages has become a novel therapeutic target. Toll-like receptor 4 engagement promotes classical activation of macrophages, and recent literature suggests TLR4 agonism to prevent metastasis and promote survival in experimental metastasis models. A growing number of studies indicate that TLR4 can respond to opioids, including the opioid receptor-inactive morphine metabolite morphine-3-glucuronide (M3G). We measured the activation of TLR4 in a reporter cell line exogenously expressing TLR4 and TLR4 co-receptors, and confirmed that M3G weakly but significantly activates TLR4. We hypothesized that M3G would promote the expression of classical activation signature genes in macrophages in vitro. We exposed mouse and human macrophage cell lines to M3G or the TLR4 activator lipopolysaccharide (LPS), alone or in combination with interferon gamma (IFN-γ). The classical macrophage activation markers tested were iNOS, CD86, IL-6, or TNF-α in RAW 264.7 cells and IL-6, IL-12, IL-23, TNF-α, CXCL10, and CXCL11 in THP1 cells. Our results show that despite exhibiting TLR4-activation ability, M3G does not elicit the expression of classical activation markers in LPS-responsive macrophages.

  14. The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro

    PubMed Central

    Khabbazi, Samira; Xie, Nan; Pu, Wenjun; Goumon, Yannick; Parat, Marie-Odile

    2016-01-01

    Macrophages are abundant in the tumor microenvironment where they adopt a pro-tumor phenotype following alternative polarization induced by paracrine factors from cancer and stromal cells. In contrast, classically activated macrophages have tumoricidal activities, such that the polarization of tumor-associated macrophages has become a novel therapeutic target. Toll-like receptor 4 engagement promotes classical activation of macrophages, and recent literature suggests TLR4 agonism to prevent metastasis and promote survival in experimental metastasis models. A growing number of studies indicate that TLR4 can respond to opioids, including the opioid receptor-inactive morphine metabolite morphine-3-glucuronide (M3G). We measured the activation of TLR4 in a reporter cell line exogenously expressing TLR4 and TLR4 co-receptors, and confirmed that M3G weakly but significantly activates TLR4. We hypothesized that M3G would promote the expression of classical activation signature genes in macrophages in vitro. We exposed mouse and human macrophage cell lines to M3G or the TLR4 activator lipopolysaccharide (LPS), alone or in combination with interferon gamma (IFN-γ). The classical macrophage activation markers tested were iNOS, CD86, IL-6, or TNF-α in RAW 264.7 cells and IL-6, IL-12, IL-23, TNF-α, CXCL10, and CXCL11 in THP1 cells. Our results show that despite exhibiting TLR4-activation ability, M3G does not elicit the expression of classical activation markers in LPS-responsive macrophages. PMID:27909407

  15. Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis.

    PubMed

    Siegmund, Daniela; Kums, Juliane; Ehrenschwender, Martin; Wajant, Harald

    2016-09-22

    Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes.

  16. Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages.

    PubMed

    Myers, Jesse T; Tsang, Albert W; Swanson, Joel A

    2003-11-15

    Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-gamma, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91(phox-/-) and NO synthase 2(-/-) mice showed that vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2',4,5,6,7,7'-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space

  17. Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis

    PubMed Central

    Siegmund, Daniela; Kums, Juliane; Ehrenschwender, Martin; Wajant, Harald

    2016-01-01

    Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes. PMID:27899821

  18. Methamphetamine Inhibits Toll-Like Receptor 9-Mediated Anti-HIV Activity in Macrophages

    PubMed Central

    Cen, Ping; Ye, Li; Su, Qi-Jian; Wang, Xu; Li, Jie-Liang; Lin, Xin-Qin

    2013-01-01

    Abstract Toll-like receptor 9 (TLR9) is one of the key sensors that recognize viral infection/replication in the host cells. Studies have demonstrated that methamphetamine (METH) dysregulated host cell innate immunity and facilitated HIV infection of macrophages. In this study, we present new evidence that METH suppressed TLR9-mediated anti-HIV activity in macrophages. Activation of TLR9 by its agonist CpG-ODN 2216 inhibits HIV replication, which was demonstrated by increased expression of TLR9, interferon (IFN)-α, IFN regulatory factor-7 (IRF-7), myeloid differentiation factor 88 (MyD88), and myxovirus resistance gene A (MxA) in macrophages. However, METH treatment of macrophages greatly compromised the TLR9 signaling-mediated anti-HIV effect and inhibited the expression of TLR9 downstream signaling factors. Dopamine D1 receptor (D1R) antagonists (SCH23390) could block METH-mediated inhibition of anti-HIV activity of TLR9 signaling. Investigation of the underlying mechanisms of the METH action showed that METH treatment selectively down-regulated the expression of TLR9 on macrophages, whereas it had little effect on the expression of other TLRs. Collectively, our results provide further evidence that METH suppresses host cell innate immunity against HIV infection by down-regulating TLR9 expression and its signaling-mediated antiviral effect in macrophages. PMID:23751096

  19. Attenuated expression of interferon-β and interferon-λ1 by human alternatively activated macrophages.

    PubMed

    El Fiky, Ashraf; Perreault, Roger; McGinnis, Gwendolyn J; Rabin, Ronald L

    2013-12-01

    Macrophages can be polarized into classically (CAM) or alternatively (AAM) activated macrophages with IFN-γ or IL-4, respectively. CAM are associated with type 1 immune responses and are implicated in autoimmunity; AAM are associated with type 2 responses and are implicated in allergic diseases. An impediment in investigating macrophage biology using primary human monocyte derived macrophages is the wide inter-donor heterogeneity and the limited quantity of cells that survive in vitro polarization. To overcome this impediment, we established a protocol to generate CAM and AAM cultures derived from the THP-1 human promonocytic cell line. In this report, we demonstrate that THP-CAM and -AAM express gene and protein markers that define their primary human monocyte derived counterparts, such as IL-1β, CXCL10, and CXCL11 for CAM, and MRC1, IL-4 and CCL22 for AAM. In addition, we demonstrate that STAT6 is selectively activated in THP-AAM which, upon LPS stimulation, have an attenuated or delayed expression of IFN-β, IFN-λ1, and IFN α/β pathway genes compared to their CAM counterparts. Taken together, these findings may help further investigate human diseases associated with the alternatively activated macrophage phenotype using this reproducible in vitro macrophage model. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  20. Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages.

    PubMed

    Soehnlein, O; Kenne, E; Rotzius, P; Eriksson, E E; Lindbom, L

    2008-01-01

    Macrophages represent a multi-functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil-derived granule proteins in the anti-microbial activity of macrophages. Neutrophil secretion obtained by antibody cross-linking of the integrin subunit CD18 (X-link secretion) or by treatment with N-Formyl-Met-Leu-Phe (fMLP secretion) induced a several-fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca(2+). Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X-link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti-microbial therapy.

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

    PubMed Central

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

    1987-01-01

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

  2. Pneumocystis carinii cell wall beta-glucans initiate macrophage inflammatory responses through NF-kappaB activation.

    PubMed

    Lebron, Frances; Vassallo, Robert; Puri, Vishwajeet; Limper, Andrew H

    2003-07-04

    beta-Glucans are major structural components of fungi. We have recently reported that the pathogenic fungus Pneumocystis carinii assembles a beta-glucan-rich cell wall that potently activates alveolar macrophages to release pro-inflammatory cytokines and chemokines. Purified P. carinii beta-glucans predictably induce both cytokine generation and associated neutrophilic lung inflammation. Herein, we demonstrate that P. carinii beta-glucan-induced macrophage stimulation results from activation of NF-kappaB. Although analogous to macrophage activation induced by bacterial lipopolysaccharide (LPS), P. carinii beta-glucan-induced macrophage NF-kappaB activation exhibits distinctly different kinetics, with slower induction and longer duration compared with LPS stimulation. Macrophage activation in response to P. carinii beta-glucan was also substantially inhibited with the NF-kappaB antagonist pyrrolidine dithiocarbamate. In addition to different kinetics of NF-kappaB activation, P. carinii beta-glucan and LPS also utilize different receptor systems to induce macrophage activation. Macrophages from Toll-like receptor 4-deficient and wild type mice produced equivalent amounts of tumor necrosis factor alpha when stimulated with P. carinii beta-glucan. However, Toll-like receptor 4-deficient macrophages were refractory to stimulation with LPS. In contrast, MyD88-deficient macrophages exhibited a significant (though partial) blunted response to P. carinii beta-glucan. These data demonstrate that P. carinii beta-glucan acts as potent inducer of macrophage activation through NF-kappaB utilizing cellular receptors and signaling pathways distinct from LPS.

  3. Development of ostrich thrombocytes and monocyte-derived macrophages in culture and the control of Toxoplasma gondii reproduction after macrophage activation.

    PubMed

    Miranda, Farlen J B; Damasceno-Sá, João Cláudio; DaMatta, Renato A

    2016-01-01

    Raising ostriches became an important economic activity after their products became commodities. The health of farm animals is of paramount importance, so assessing basic immunological responses is necessary to better understand health problems. We developed a method to obtain ostrich thrombocytes and macrophages. The thrombocytes died by apoptosis after 48 h in culture, and the macrophages expanded in size and increased the number of acidic compartments. Macrophages were activated by chicken interferon-γ, producing high levels of nitric oxide. Toxoplasma gondii was able to infect these macrophages, and activation controlled parasitic reproduction. T. gondii, however, persisted in these cells, and infection reduced the production of nitric oxide. These results are important for the future assessment of the basic cellular and immunobiology of ostriches and demonstrate T. gondii suppression of nitric oxide production.

  4. Macrophage activation and leishmanicidal activity by galactomannan and its oxovanadium (IV/V) complex in vitro.

    PubMed

    Adriazola, Izabela Ono; Evangelista do Amaral, Alex; Amorim, Juliana Carolina; Correia, Beatriz Lourenço; Petkowicz, Carmen Lúcia Oliveira; Mercê, Ana Lucia Ramalho; Noleto, Guilhermina Rodrigues

    2014-03-01

    Compounds that activate macrophage antimicrobial activity are potential targets for treatment of leishmaniasis. The present study investigated the in vitro immunomodulatory effects of a galactomannan (GALMAN-A) isolated from seeds of Mimosa scabrella and its oxovanadium (IV/V) complex (GALMAN-A:VO(2+)/VO(3+)) on macrophage activity. GALMAN-A increased nitric oxide levels by ~33% at a concentration of 250μg/ml, while GALMAN-A:VO(2+)/VO(3+) decreased nitric oxide levels by ~33% at a concentration of 50μg/ml. Furthermore, GALMAN-A increased interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) levels by 5.5 and 2.3 times, respectively, at a concentration of 25μg/ml; at the same concentration, GALMAN-A:VO(2+)/VO(3+) promoted an increase in IL-1β and IL-6 production by 8 and 5.5 times, respectively. However, neither GALMAN-A nor GALMAN-A:VO(2+)/VO(3+) affected tumor necrosis factor alpha (TNF-α) or interleukin-10 (IL-10) levels. Importantly, both GALMAN-A and GALMAN-A:VO(2+)/VO(3+) exhibited leishmanicidal activity on amastigotes of Leishmania (L.) amazonensis, reaching ~60% activity at concentrations of 100 and 25μg/ml, respectively. These results indicate that GALMAN-A is three times more potent and its oxovanadium complex is twelve times more potent than Glucantime (300μg/ml), which is the drug of choice in leishmaniasis treatment. The IC50 value for GALMAN-A:VO(2+)/VO(3+) was 74.4μg/ml (0.58μg/ml of vanadium). Thus, the significant activation of macrophages and the noted leishmanicidal effect demonstrate the need for further studies to clarify the mechanisms of action of these compounds.

  5. Kaurane diterpenes protect against apoptosis and inhibition of phagocytosis in activated macrophages

    PubMed Central

    de las Heras, B; Hortelano, S; Girón, N; Bermejo, P; Rodríguez, B; Boscá, L

    2007-01-01

    Background and purpose: The kaurane diterpenes foliol and linearol are inhibitors of the activation of nuclear factor κB, a transcription factor involved in the inflammatory response. Effects of these diterpenes on apoptosis and phagocytosis have been analysed in cultured peritoneal macrophages and in the mouse macrophage cell line, RAW 264.7. Experimental approach: Macrophages were maintained in culture and activated with pro-inflammatory stimuli in the absence or presence of diterpenes. Apoptosis and the phagocytosis in these cells under these conditions were determined. Key results: Incubation of macrophages with a mixture of bacterial lipopolysaccharide (LPS)/interferon-γ (IFN-γ) induced apoptosis through a NO-dependent pathway, an effect significantly inhibited by foliol and linearol in the low μM range, without cytotoxic effects. Apoptosis in macrophages induced by NO donors was also inhibited. The diterpenes prevented apoptosis through a mechanism compatible with the inhibition of caspase-3 activation, release of cytochrome c to the cytosol and p53 overexpression, as well as an alteration in the levels of proteins of the Bcl-2 family, in particular, the levels of Bax. Cleavage of poly(ADP-ribose) polymerase, a well-established caspase substrate, was reduced by these diterpenes. Treatment of cells with foliol and linearol decreased phagocytosis of zymosan bioparticles by RAW 264.7 cells and to a greater extent by peritoneal macrophages. Conclusions and implications: Both diterpenes protected macrophages from apoptosis and inhibited phagocytosis, resulting in a paradoxical control of macrophage function, as viability was prolonged but inflammatory and phagocytic functions were impaired. PMID:17618303

  6. In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed.

    PubMed

    Vainchtein, I D; Vinet, J; Brouwer, N; Brendecke, S; Biagini, G; Biber, K; Boddeke, H W G M; Eggen, B J L

    2014-10-01

    Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) characterized by loss of myelin accompanied by infiltration of T-lymphocytes and monocytes. Although it has been shown that these infiltrates are important for the progression of MS, the role of microglia, the resident macrophages of the CNS, remains ambiguous. Therefore, we have compared the phenotypes of microglia and macrophages in a mouse model for MS, experimental autoimmune encephalomyelitis (EAE). In order to properly discriminate between these two cell types, microglia were defined as CD11b(pos) CD45(int) Ly-6C(neg) , and infiltrated macrophages as CD11b(pos) CD45(high) Ly-6C(pos) . During clinical EAE, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecules (CD80, CD86, and CD40) and proinflammatory genes [interleukin-1β (IL-1β) and tumour necrosis factor- α (TNF-α)]. In contrast, CD11b(pos) CD45(high) Ly-6C(pos) infiltrated macrophages were strongly activated and could be divided into two populations Ly-6C(int) and Ly-6C(high) , respectively. Ly-6C(high) macrophages contained less myelin than Ly-6C(int) macrophages and expression levels of the proinflammatory cytokines IL-1β and TNF-α were higher in Ly-6C(int) macrophages. Together, our data show that during clinical EAE, microglia are only weakly activated whereas infiltrated macrophages are highly immune reactive.

  7. Guanylate-binding protein 5 is a marker of interferon-γ-induced classically activated macrophages

    PubMed Central

    Fujiwara, Yukio; Hizukuri, Yoshiyuki; Yamashiro, Kyoko; Makita, Naoyuki; Ohnishi, Koji; Takeya, Motohiro; Komohara, Yoshihiro; Hayashi, Yasuhiro

    2016-01-01

    Macrophage activation is the main immunological process occurring during the development of several diseases, and the heterogeneity of macrophage activation or differentiation has been suggested to be involved in disease progression. In the present study, we attempted to identify molecules specifically expressed on human classically activated macrophages (M1) to investigate the significance of the M1-like phenotype in human diseases. Human monocyte-derived macrophages were differentiated into M1, M2a, M2b and M2c phenotypes, and also M1(−) (the M1 phenotype differentiated with interferon-γ) to eliminate the strong effects of lipopolysaccharides (LPS) on the gene expression profile. The gene expression profiles of those macrophage phenotypes were analyzed by a cDNA microarray analysis and were used for a bioinformatics examination to identify the markers of the M1 phenotype that are expressed in both M1 and M1(−). The gene expression profiles of murine macrophages were also evaluated. We identified guanylate-binding protein 5 (GBP5), which is associated nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3)-mediated inflammasome assembly in the M1 macrophages of both humans and mice. Notably, the expression of GBP5 protein was detected in cultured M1(−) as well as in M1 macrophages by western blotting, which means that GBP5 is a more generalized marker of the M1 phenotype compared with the M1 markers that can be induced by LPS stimulation. GBP5 is a useful candidate marker of the M1 phenotype. PMID:27990286

  8. Guanylate-binding protein 5 is a marker of interferon-γ-induced classically activated macrophages.

    PubMed

    Fujiwara, Yukio; Hizukuri, Yoshiyuki; Yamashiro, Kyoko; Makita, Naoyuki; Ohnishi, Koji; Takeya, Motohiro; Komohara, Yoshihiro; Hayashi, Yasuhiro

    2016-11-01

    Macrophage activation is the main immunological process occurring during the development of several diseases, and the heterogeneity of macrophage activation or differentiation has been suggested to be involved in disease progression. In the present study, we attempted to identify molecules specifically expressed on human classically activated macrophages (M1) to investigate the significance of the M1-like phenotype in human diseases. Human monocyte-derived macrophages were differentiated into M1, M2a, M2b and M2c phenotypes, and also M1(-) (the M1 phenotype differentiated with interferon-γ) to eliminate the strong effects of lipopolysaccharides (LPS) on the gene expression profile. The gene expression profiles of those macrophage phenotypes were analyzed by a cDNA microarray analysis and were used for a bioinformatics examination to identify the markers of the M1 phenotype that are expressed in both M1 and M1(-). The gene expression profiles of murine macrophages were also evaluated. We identified guanylate-binding protein 5 (GBP5), which is associated nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3)-mediated inflammasome assembly in the M1 macrophages of both humans and mice. Notably, the expression of GBP5 protein was detected in cultured M1(-) as well as in M1 macrophages by western blotting, which means that GBP5 is a more generalized marker of the M1 phenotype compared with the M1 markers that can be induced by LPS stimulation. GBP5 is a useful candidate marker of the M1 phenotype.

  9. Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in lens epithelial cells.

    PubMed

    Pintwala, Robert; Postnikoff, Cameron; Molladavoodi, Sara; Gorbet, Maud

    2015-03-01

    Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and production of reactive oxygen species via the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate were examined in macrophages. α-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation (p < 0.03) versus control (no intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive oxygen species production in macrophages after one day (p < 0.03) and increased expression of α-smooth muscle actin in HLE B-3 after six days, although only poly(2-hydroxyethyl methacrylate) induced a significant difference versus control (p < 0.01). Our results imply that-contrary to prior uveal biocompatibility understanding-macrophage adherence is not necessary for a strong inflammatory response to an intraocular lens, with hydrophilic surfaces inducing higher activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal

  10. Autophagy in pulmonary macrophages mediates lung inflammatory injury via NLRP3 inflammasome activation during mechanical ventilation.

    PubMed

    Zhang, Yang; Liu, Gongjian; Dull, Randal O; Schwartz, David E; Hu, Guochang

    2014-07-15

    The inflammatory response is a primary mechanism in the pathogenesis of ventilator-induced lung injury. Autophagy is an essential, homeostatic process by which cells break down their own components. We explored the role of autophagy in the mechanisms of mechanical ventilation-induced lung inflammatory injury. Mice were subjected to low (7 ml/kg) or high (28 ml/kg) tidal volume ventilation for 2 h. Bone marrow-derived macrophages transfected with a scrambled or autophagy-related protein 5 small interfering RNA were administered to alveolar macrophage-depleted mice via a jugular venous cannula 30 min before the start of the ventilation protocol. In some experiments, mice were ventilated in the absence and presence of autophagy inhibitors 3-methyladenine (15 mg/kg ip) or trichostatin A (1 mg/kg ip). Mechanical ventilation with a high tidal volume caused rapid (within minutes) activation of autophagy in the lung. Conventional transmission electron microscopic examination of lung sections showed that mechanical ventilation-induced autophagy activation mainly occurred in lung macrophages. Autophagy activation in the lungs during mechanical ventilation was dramatically attenuated in alveolar macrophage-depleted mice. Selective silencing of autophagy-related protein 5 in lung macrophages abolished mechanical ventilation-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and lung inflammatory injury. Pharmacological inhibition of autophagy also significantly attenuated the inflammatory responses caused by lung hyperinflation. The activation of autophagy in macrophages mediates early lung inflammation during mechanical ventilation via NLRP3 inflammasome signaling. Inhibition of autophagy activation in lung macrophages may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.

  11. Interleukin-6 signaling promotes alternative macrophage activation to limit obesity-associated insulin resistance and endotoxemia

    PubMed Central

    Mauer, Jan; Chaurasia, Bhagirath; Goldau, Julia; Vogt, Merly C.; Ruud, Johan; Nguyen, Khoa D.; Theurich, Sebastian; Hausen, A. Christine; Schmitz, Joel; Brönneke, Hella S.; Estevez, Emma; Allen, Tamara L.; Mesaros, Andrea; Partridge, Linda; Febbraio, Mark A.; Chawla, Ajay; Wunderlich, F. Thomas; Brüning, Jens C.

    2014-01-01

    Obesity and insulin resistance are closely associated with the development of low-grade inflammation. Interleukin 6 (IL-6) is linked to obesity-associated inflammation, however its role in this context remains controversial. Here, we show that mice with inactivated Il6ra gene in myeloid cells (Il6raΔmyel) displayed exaggerated deterioration of glucose homeostasis upon diet-induced obesity due to enhanced insulin resistance. Insulin target tissues showed increased inflammation and a shift in macrophage polarization. IL-6 induced IL-4-receptor expression and augmented the response to IL-4 in macrophages in a cell-autonomous manner. Il6raΔmyel mice were resistant to IL-4-mediated alternative macrophage polarization and exhibited increased susceptibility to LPS-induced endotoxemia. These results reveal IL-6 signaling as an important determinant for alternative macrophage-activation and assign IL-6 an unexpected homeostatic role to limit inflammation. PMID:24681566

  12. Differential effects of osteopontin on the cytotoxic activity of macrophages from young and old mice.

    PubMed

    Rollo, E E; Denhardt, D T

    1996-08-01

    Osteopontin (OPN) is a secreted phosphoprotein found in body fluids (e.g. plasma, urine, milk) and in mineralized tissues. Its expression is increased in many transformed cells and in normal cells exposed to various cytokines. When stimulated with the inflammatory mediators lipopolysaccharide and interferon-gamma, mouse macrophages secrete nitric oxide (NO) as a cytotoxic agent effective against microbial invaders and tumour cells. This report documents (1) that thioglycollate-elicited peritoneal macrophages, activated with the inflammatory mediators, produced less NO and exhibited reduced cytotoxicity towards target cells when they were obtained from old animals than when they were obtained from young animals; and (2) that OPN was able to inhibit both the induced NO synthesis and cytotoxicity, but more effectively in macrophages from the young animals than those from the old animals. This may be due to the observed higher level of OPN expression in macrophages from old animals.

  13. Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity.

    PubMed

    Ennist, D L; Jones, K H

    1983-07-01

    A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.

  14. Guinea Pig Neutrophils Infected with Mycobacterium tuberculosis Produce Cytokines Which Activate Alveolar Macrophages in Noncontact Cultures▿

    PubMed Central

    Sawant, Kirti V.; McMurray, David N.

    2007-01-01

    The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, we investigated the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce interleukin-8 (IL-8; CXCL8) and tumor necrosis factor alpha (TNF-α) and to activate alveolar macrophages. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12, and 24 h. IL-8 protein production in cocultures, as measured by using an enzyme-linked immunosorbent assay, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected cocultures than in uninfected cocultures and in cultures of the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time reverse transcription-PCR, were elevated at 24 h in infected cocultures and infected cells cultured alone. In order to elucidate the contributions of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner by using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper wells, and alveolar macrophages were cultured in the lower wells. The release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to that from unexposed alveolar macrophages. Significant up-regulation of IL-1β and TNF-α mRNA levels in alveolar macrophages was observed at 24 and 30 h, respectively, compared to those in cells not exposed to soluble neutrophil products. Treatment with anti-guinea pig TNF-α polyclonal antibody completely abolished the response of alveolar macrophages to neutrophil products. This finding suggests that TNF-α produced by infected neutrophils may be involved in the activation of

  15. Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

    PubMed

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

    2015-06-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Slamf8 is a negative regulator of Nox2 activity in macrophages

    PubMed Central

    Wang, Guoxing; Abadía-Molina, Ana C; Berger, Scott B; Romero, Xavier; O'Keeffe, Michael; Rojas-Barros, Domingo I.; Aleman, Marta; Liao, Gongxian; Maganto-García, Elena; Fresno, Manuel; Wang, Ninghai; Detre, Cynthia; Terhorst, Cox

    2012-01-01

    Slamf8 (CD353) is a cell surface receptor that is expressed upon activation of macrophages by interferon-gamma or bacteria. Here we report that a very high Nox2 activity enzyme was found in Slamf8−/− macrophages in response to E.coli or S.aureus, but also to phorbol myristate acetate. The elevated Nox2 activity in Slamf8−/− macrophages was also demonstrated in E.coli or S.aureus phagosomes by using a pH indicator system, and was further confirmed by a reduction of the enzyme activity after transfection of the receptor into Slamf8-deficient primary macrophages or RAW 264.7 cells. Upon exposure to bacteria and/or phorbol myristate acetate, PKC activity in Slamf8−/− macrophages is increased. This results in an enhanced phosphorylation of p40phox, one key component of the Nox2 enzyme complex, which in turn leads to greater Nox2 activity. Taken together, the data show that upon response to inflammation-associated stimuli the inducible receptor Slamf8 negatively regulates inflammatory responses. PMID:22593622

  17. TLR4-mediated activation of mouse macrophages by Korean mistletoe lectin-C (KML-C).

    PubMed

    Park, Hong-Jai; Hong, Ju-ho; Kwon, Hyung-Joon; Kim, Youngchan; Lee, Kwan-Hee; Kim, Jong-Bae; Song, Seong K

    2010-06-04

    Korean mistletoe lectin (KML-C) is an adjuvant that activates systemic and mucosal immune cells to release cytokines including TNF-alpha, which induces immunity against viruses and cancer cells. Although the immunomodulatory activity of KML-C has been well established, the underlying mechanism of action of KML-C has yet to be explored. When mouse peritoneal macrophages were treated with KML-C, both transcription and translation of TLR4 were upregulated. KML-C-induced TLR4 downstream events were similar to those activated by LPS: the upregulation of interleukin-1 receptor-associated kinase-1 (IRAK1); resulting in macrophage activation and TNF-alpha production. When TLR4 was blocked using a TLR4-specific neutralizing antibody, TNF-alpha production from the macrophages was significantly inhibited. Moreover, TLR4-deficient mouse macrophages treated with KML-C also secreted greatly reduced level of TNF-alpha secretion. Finally, TLR4 molecules were co-precipitated with KML-C, to which agarose beads were conjugated, indicating that those molecules are associated. These data indicate that KML-C activates mouse macrophages to secrete TNF-alpha by interacting with the TLR4 molecule and activating its signaling pathways.

  18. Vitamin D binding protein-macrophage activating factor inhibits HCC in SCID mice.

    PubMed

    Nonaka, Koichi; Onizuka, Shinya; Ishibashi, Hiromi; Uto, Yoshihiro; Hori, Hitoshi; Nakayama, Toshiyuki; Matsuura, Nariaki; Kanematsu, Takashi; Fujioka, Hikaru

    2012-01-01

    A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC. The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d. DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group. DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. ATP Release from Dying Autophagic Cells and Their Phagocytosis Are Crucial for Inflammasome Activation in Macrophages

    PubMed Central

    Ayna, Gizem; Krysko, Dmitri V.; Kaczmarek, Agnieszka; Petrovski, Goran; Vandenabeele, Peter; Fésüs, László

    2012-01-01

    Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P2X7 purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses. PMID:22768222

  20. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    PubMed Central

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  1. Antihistoplasma effect of activated mouse splenic macrophages involves production of reactive nitrogen intermediates.

    PubMed Central

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

    1994-01-01

    The mechanism by which recombinant murine gamma interferon (rMuIFN-gamma) and bacterial lipopolysaccharide (LPS) activate mouse resident splenic macrophages to inhibit the intracellular growth of the fungus Histoplasma capsulatum was examined. Growth inhibition depended on L-arginine metabolism. The growth inhibitory state normally induced by rMuIFN-gamma and LPS in resident splenic macrophages did not occur when the macrophages were cultured in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of L-arginine metabolism. Resident splenic macrophages treated with rMuIFN-gamma and LPS produced nitrite (NO2-), an end product of L-arginine metabolism. When macrophages were cultured in the presence of NG-monomethyl-L-arginine together with rMuIFN-gamma and LPS, only baseline levels of NO2- were detected. Spleen cells from H. capsulatum-infected mice produced high levels of NO2- in culture. The production of NO2- correlated with in vitro inhibition of the intracellular growth of H. capsulatum. Anti-tumor necrosis factor alpha antibody did not block NO2- production by the immigrant splenic macrophages and did not abolish the antihistoplasma activity. PMID:8168960

  2. Distinct inflammatory properties of late-activated macrophages in inflammatory myopathies

    PubMed Central

    Rostasy, KM; Schmidt, J; Bahn, E; Pfander, T; Piepkorn, M; Wilichowski, E; Schulz-Schaeffer, J

    2008-01-01

    Summary Distinct mechanisms such as humeral immunity in dermatomyositis (DM) and T-cell-mediated cytotoxicity in polymyositis (PM) contribute to the pathology of inflammatory myopathies. In addition, different subsets of macrophages are present in both diseases. Herein, the characteristics of 25F9-positive macrophages in skeletal muscle inflammation are outlined. Muscle biopsies of subjects with DM and PM were studied by immunohistochemical multi-labelling using the late-activation marker 25F9, together with markers characterizing macrophage function including IFN-γ, iNOS, and TGF-β. In PM, a robust expression of IFN-γ, iNOS, and TGF-β was observed in inflammatory cells. Double- and serial-labelling revealed that a subset of 25F9-positive macrophages in the vicinity of injured muscle fibres expressed iNOS and TGF-β, but not IFN-γ. In DM, IFN-γ, iNOS and TGF-β were also expressed in inflammatory cells in the endomysium. Double- and serial-labelling studies in DM indicated that 25F9-positive macrophages expressed TGF-β and to a lesser degree iNOS, but not IFN-γ. In conclusion, our data suggest that late-activated macrophages contribute to the pathology of inflammatory myopathies. PMID:19364061

  3. Distinct inflammatory properties of late-activated macrophages in inflammatory myopathies.

    PubMed

    Rostasy, K M; Schmidt, J; Bahn, E; Pfander, T; Piepkorn, M; Wilichowski, E; Schulz-Schaeffer, J

    2008-10-01

    Distinct mechanisms such as humeral immunity in dermatomyositis (DM) and T-cell-mediated cytotoxicity in polymyositis (PM) contribute to the pathology of inflammatory myopathies. In addition, different subsets of macrophages are present in both diseases. Herein, the characteristics of 25F9-positive macrophages in skeletal muscle inflammation are outlined. Muscle biopsies of subjects with DM and PM were studied by immunohistochemical multi-labelling using the late-activation marker 25F9, together with markers characterizing macrophage function including IFN-gamma, iNOS, and TGF-beta. In PM, a robust expression of IFN-gamma, iNOS, and TGF-beta was observed in inflammatory cells. Double- and serial-labelling revealed that a subset of 25F9-positive macrophages in the vicinity of injured muscle fibres expressed iNOS and TGF-beta, but not IFN-gamma. In DM, IFN-gamma, iNOS and TGF-beta were also expressed in inflammatory cells in the endomysium. Double- and serial-labelling studies in DM indicated that 25F9-positive macrophages expressed TGF-beta and to a lesser degree iNOS, but not IFN-gamma. In conclusion, our data suggest that late-activated macrophages contribute to the pathology of inflammatory myopathies.

  4. Mycobacterium tuberculosis Activates Human Macrophage Peroxisome Proliferator-Activated Receptor γ Linking Mannose Receptor Recognition to Regulation of Immune Responses

    PubMed Central

    Rajaram, Murugesan V. S.; Brooks, Michelle N.; Morris, Jessica D.; Torrelles, Jordi B.; Azad, Abul K.; Schlesinger, Larry S.

    2010-01-01

    Mycobacterium tuberculosis enhances its survival in macrophages by suppressing immune responses in part through its complex cell wall structures. Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor superfamily member, is a transcriptional factor that regulates inflammation and has high expression in alternatively activated alveolar macrophages and macrophage-derived foam cells, both cell types relevant to tuberculosis pathogenesis. In this study, we show that virulent M. tuberculosis and its cell wall mannose-capped lipoarabinomannan induce PPARγ expression through a macrophage mannose receptor-dependent pathway. When activated, PPARγ promotes IL-8 and cyclooxygenase 2 expression, a process modulated by a PPARγ agonist or antagonist. Upstream, MAPK-p38 mediates cytosolic phospholipase A2 activation, which is required for PPARγ ligand production. The induced IL-8 response mediated by mannose-capped lipoarabinomannan and the mannose receptor is independent of TLR2 and NF-κB activation. In contrast, the attenuated Mycobacterium bovis bacillus Calmette-Guérin induces less PPARγ and preferentially uses the NF-κB–mediated pathway to induce IL-8 production. Finally, PPARγ knockdown in human macrophages enhances TNF production and controls the intracellular growth of M. tuberculosis. These data identify a new molecular pathway that links engagement of the mannose receptor, an important pattern recognition receptor for M. tuberculosis, with PPARγ activation, which regulates the macrophage inflammatory response, thereby playing a role in tuberculosis pathogenesis. PMID:20554962

  5. Effect of age on proteasomal activity of T cells and macrophages

    USDA-ARS?s Scientific Manuscript database

    T cell function is impaired with aging. Proteasome activity in T cells is important for T cell activation and its activity in macrophages is required for processing antigens in order to be presented via class I major histocompatibility complex to CD8+ T cells. Since studies have demonstrated that pr...

  6. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages

    PubMed Central

    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Alencar, Severino M.; Rosalen, Pedro L.; Mayer, Marcia P. A.

    2015-01-01

    Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases. PMID:26660901

  7. Alternatively activated macrophages determine repair of the infarcted adult murine heart

    PubMed Central

    Shiraishi, Manabu; Shintani, Yasunori; Shintani, Yusuke; Ishida, Hidekazu; Saba, Rie; Yamaguchi, Atsushi; Adachi, Hideo; Yashiro, Kenta

    2016-01-01

    Alternatively activated (also known as M2) macrophages are involved in the repair of various types of organs. However, the contribution of M2 macrophages to cardiac repair after myocardial infarction (MI) remains to be fully characterized. Here, we identified CD206+F4/80+CD11b+ M2-like macrophages in the murine heart and demonstrated that this cell population predominantly increases in the infarct area and exhibits strengthened reparative abilities after MI. We evaluated mice lacking the kinase TRIB1 (Trib1–/–), which exhibit a selective depletion of M2 macrophages after MI. Compared with control animals, Trib1–/– mice had a catastrophic prognosis, with frequent cardiac rupture, as the result of markedly reduced collagen fibril formation in the infarct area due to impaired fibroblast activation. The decreased tissue repair observed in Trib1–/– mice was entirely rescued by an external supply of M2-like macrophages. Furthermore, IL-1α and osteopontin were suggested to be mediators of M2-like macrophage–induced fibroblast activation. In addition, IL-4 administration achieved a targeted increase in the number of M2-like macrophages and enhanced the post-MI prognosis of WT mice, corresponding with amplified fibroblast activation and formation of more supportive fibrous tissues in the infarcts. Together, these data demonstrate that M2-like macrophages critically determine the repair of infarcted adult murine heart by regulating fibroblast activation and suggest that IL-4 is a potential biological drug for treating MI. PMID:27140396

  8. Brazilian Red Propolis Attenuates Inflammatory Signaling Cascade in LPS-Activated Macrophages.

    PubMed

    Bueno-Silva, Bruno; Kawamoto, Dione; Ando-Suguimoto, Ellen S; Alencar, Severino M; Rosalen, Pedro L; Mayer, Marcia P A

    2015-01-01

    Although previous studies suggested an anti-inflammatory property of Brazilian red propolis (BRP), the mechanisms involved in the anti-inflammatory effects of BRP and its activity on macrophages were still not elucidated. This study aimed to evaluate whether BRP attenuates the inflammatory effect of LPS on macrophages and to investigate its underlying mechanisms. BRP was added to RAW 264.7 murine macrophages after activation with LPS. NO production, cell viability, cytokines profile were evaluated. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. BRP at 50 μg/ml inhibited NO production by 78% without affecting cell viability. Cd80 and Cd86 were upregulated whereas mrc1 was down regulated by BRP indicating macrophage polarization at M1. BRP attenuated the production of pro-inflammatory mediators IL-12, GM-CSF, IFN-Ɣ, IL-1β in cell supernatants although levels of TNF- α and IL-6 were slightly increased after BRP treatment. Levels of IL-4, IL-10 and TGF-β were also reduced by BRP. BRP significantly reduced the up-regulation promoted by LPS of transcription of genes in inflammatory signaling (Pdk1, Pak1, Nfkb1, Mtcp1, Gsk3b, Fos and Elk1) and of Il1β and Il1f9 (fold-change rate > 5), which were further confirmed by the inhibition of NF-κB and MAPK signaling pathways. Furthermore, the upstream adaptor MyD88 adaptor-like (Mal), also known as TIRAP, involved in TLR2 and TLR4 signaling, was down- regulated in BRP treated LPS-activated macrophages. Given that BRP inhibited multiple signaling pathways in macrophages involved in the inflammatory process activated by LPS, our data indicated that BRP is a noteworthy food-source for the discovery of new bioactive compounds and a potential candidate to attenuate exhacerbated inflammatory diseases.

  9. IFN-γ prevents adenosine receptor (A2bR) upregulation to sustain the macrophage activation response

    PubMed Central

    Cohen, Heather B.; Ward, Amanda; Hamidzadeh, Kajal; Ravid, Katya; Mosser, David M.

    2015-01-01

    The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. Here, we demonstrate that following TLR stimulation, macrophages up regulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This up-regulation of A2bR leads to the induction of a macrophage with an immunoregulatory phenotype and the down regulation of inflammation. IFN-γ priming of macrophages, selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNFα and IL-12 in response to TLR ligation. The pharmacological inhibition or the genetic deletion of the A2bR results in a hyper-inflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense, by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the anti-microbial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated. PMID:26355158

  10. Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2

    SciTech Connect

    Li, Bin; Abdalrahman, Akram; Lai, Yimu; Janicki, Joseph S