Molecular mechanisms of microglial activation

Microglial cells are brain macrophages which serve specific functions in the defense of the central nervous system (CNS) against microorganisms, the removal of tissue debris in neurodegenerative diseases or during normal development, and in autoimmune inflammatory disorders of the brain. In cultured microglial cells, several soluble inflammatory mediators such as cytokines and bacterial products like lipopolysaccharide (LPS) were demonstrated to induce a wide range of microglial activities, e.g. increased phagocytosis, chemotaxis, secretion of cytokines, activation of the respiratory burst and induction of nitric oxide synthase. Since heightened microglial activation was shown to play a role in the pathogenesis of experimental inflammatory CNS disorders, understanding the molecular mechanisms of microglial activation may lead to new treatment strategies for neurodegenerative disorders, multiple sclerosis and bacterial or viral infections of the nervous system.     

Caveolin-1-deficient mice show defects in innate immunity and inflammatory immune response during Salmonella enterica serovar Typhimurium infection

A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1(-/-) mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1(-/-) mice. However, infection of Cav-1(-/-) macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1(-/-) mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1(-/-) mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1(-/-) mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1(-/-) macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.     

Enhancement of Nitric Oxide Release in Mouse Inflammatory Macrophages Co-cultivated with Tumor Cells of a Different Origin

In the present study we investigated whether synthesis of nitric oxide (NO) by macrophages is affected by contact with tumor cells. Although it is well known that NO generated by macrophages influences different activities related to tumor progression, there is limited information on the modulatory role of tumor cells on NO release by macrophages. The experimental protocol used in our study consisted in the determination of NO secreted by macrophages, either resident or inflammatory, co-cultivated with tumor cells (B16 melanoma and L929 fibrosarcoma cells) at different cell densities and macrophage:tumor cell ratios. This experimental in vitro protocol simulates the different interactions between macrophages and tumor cells that occur during the development of a tumor mass. We found that the co-cultivation with tumor cells induced an increased secretion of NO in macrophages provided that they express an inflammatory phenotype, and they were challenged with LPS or IFNγ/LPS. Two more variables were found to be critical in the increase of NO generation in inflammatory macrophages cultivated with tumor cells: a high cell density and a prevalence of tumor cells over macrophages. The enhancement of NO secreted in inflammatory macrophages stimulated by tumor cells was not observed in normal murine fibroblasts co-cultivated with tumor cells.     

Macrophage-stimulating protein and RON receptor tyrosine kinase: potential regulators of macrophage inflammatory activities

Macrophage-stimulating protein (MSP) is a serum protein belonging to the plasminogen-related growth factor family. The specific receptor for MSP is the RON (recepteur d'origine nantais) receptor tyrosine kinase - a member of the MET proto-oncogene family. Activation of RON by MSP exerts dual functions on macrophages. The stimulatory activities include the induction of macrophage spreading, migration and phagocytosis. However, MSP also inhibits lipopolysaccharide (LPS)-induced production of inflammatory mediators, including inducible nitric oxide and prostaglandins. These suppressive effects are mediated by RON-transduced signals that block LPS-induced enzymatic cascades that activate nuclear factor kappa-B (NFkappaB) pathways. Recent in vivo studies demonstrated that inactivation of the RON gene results in increased inflammatory responses and susceptibility to LPS-induced septic death in mice, suggesting that RON expression is required for attenuating the extent of inflammatory responses in vivo. Thus, MSP and RON are potential regulators that control macrophage activities during bacterial infection in vivo.     

The role of nitric oxide in multiple sclerosis

 During the past decade nitric oxide has emerged as an important mediator of physiological and pathophysiological processes. Elevated nitric oxide biosynthesis has been associated with nonspecific immune-mediated cellular cytotoxicity and the pathogenesis of chronic, inflammatory autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, and mutiple sclerosis. Recent evidence suggests, however, that nitric oxide is also immunoregulatory and suppresses the function of activated proinflammatory macrophages and T lymphocytes involved in these diseases. This article reviews the role of nitric oxide in the biology of central nervous system glial cells (astrocytes and microglia) as it pertains to the pathogenesis of multiple sclerosis in humans and experimental allergic encephalitis, the animal model of this disease. Although nitric oxide has been clearly implicated as a potential mediator of microglia-dependent primary demyelination, a hallmark of multiple sclerosis, studies with nitric oxide synthase inhibitors in the encephalitis model have been equivocal. These data are critically reviewed in the context of what is know from clinical research on the nitric oxide pathway in multiple sclerosis. Specific recommendations for future preclinical animal model research and clinical research on the nitric oxide pathway in patients are suggested. These studies are necessary to further define the role of nitric oxide in the pathology of multiple sclerosis and to fully explore the potential for nitric oxide synthase inhibitors as novel therapeutics for this disease. Received: 6 June 1996 / Accepted: 24 September 1996     

Effect of CD14 blockade on endotoxin-induced acute lung injury in mice

CD14 functions as a cell surface receptor for endotoxin (lipopolysaccharide [LPS]) and is thought to have an essential role in innate immune responses to infection. Previous studies have revealed attenuation of the systemic response after sepsis by blocking CD14. In this study, we tested the hypothesis that CD14 blockade protects against inflammatory responses associated with LPS pneumonia. We examined the effect of an anti-murine CD14 monoclonal antibody (4C1) on the development of acute lung injury induced by intratracheal LPS in mice. We also measured the production of cytokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2) and nitric oxide by murine peritoneal macrophages exposed to LPS in vitro. Nuclear factor (NF)-kappa B translocation was evaluated in nuclear extracts from lung homogenates. 4C1 significantly attenuated pulmonary edema and neutrophil emigration after LPS administration. The production of cytokines and nitric oxide by LPS-stimulated macrophages was significantly decreased by 4C1 treatment. NF-kappa B translocation induced by LPS instillation was also suppressed by 4C1. These results suggest that blockade of CD14 might attenuate acute lung injury after intratracheal instillation of LPS through the suppression of NF-kappa B translocation. The inhibitory effect of CD14 blockade on cytokine production and nitric oxide release of macrophages might contribute to the attenuation of lung injury.     

Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection.

African trypanosomes induce a generalized state of immunosuppression in their mammalian hosts. One characteristic of this is a suppression of lymphocyte responses to mitogen, which is mediated by suppressor macrophages. We investigated the involvement of nitric oxide in this phenomenon. Both peritoneal and splenic cell cultures from infected mice released nitrite and this was inhibitable by NG-monomethyl L-arginine (L-NMMA). The release of nitrite correlated with suppressed splenic T cell proliferative responses to concanavalin A. It was shown that adherent spleen cells from infected mice mediate suppression, which could be abrogated by L-NMMA. These results suggest that in T. brucei infection, the activation of macrophages to produce nitric oxide leads to impaired lymphocyte responses and immunosuppression.     

Infectious bursal disease virus infection induces macrophage activation via p38 MAPK and NF-kappaB pathways

In the present study, we show that infection with infectious bursal disease virus (IBDV) causes activation of macrophages, the key cells involved in inflammatory and immune-regulatory functions. Exposure of cultured spleen macrophages (SM) from SPF chickens to IBDV resulted in the production of nitric oxide (NO). In addition, there was upregulation of mRNA expression of inducible nitric oxide synthase (iNOS), IL-8 and cyclooxygenase-2 (COX-2). The signal transduction pathways involved in macrophage activation were examined. The role of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) was tested by using specific pharmacological inhibitors. Addition of p38 MAPK inhibitor, SB-203580 and NF-kappaB inhibitor Bay 11-7082, suppressed IBDV-induced NO production and mRNA expression of iNOS, IL-8 and COX-2. The results suggest that IBDV uses cellular signal transduction machinery, in particular the p38 MAPK and NF-kappaB pathways, to elicit macrophage activation. The increased production of NO, IL-8 and COX-2 by macrophages may contribute to bursa inflammatory responses commonly seen during the acute IBDV infection.     

Role of inducible nitric oxide synthase expressed by alveolar macrophages in High Mobility Group Box 1- induced acute lung injury

Objective and design The role of inducible nitric oxide synthase (iNOS) expressed by alveolar macrophages in acute lung injury induced by high mobility group box 1 (HMGB1) was explored. Treatments Primary rat alveolar macrophages (PRAMs) were stimulated with HMGB1 to analyze iNOS expression. Alveolar macrophages and iNOS were inhibited by gadolinium chloride and 1400W in rats challenged by HMGB1 intratracheally. Methods Western Blot was applied to assay iNOS expression in PRAMs. Indices for acute lung injury in rats were measured. Immunocytochemistry was used to localize iNOS in□bronchoalveolar lavage (BAL) cells. The enzyme activities of iNOS and constitutive nitric oxide synthase (cNOS) for BAL cells were determined. Results A time- and concentration-dependent response of iNOS expression in PRAMs to HMGB1 induction was observed. Intratracheal instillation of HMGB1 produced persistently exacerbated acute lung inflammation, induction of iNOS in alveolar macrophages and increased lung nitric oxide production in rats. Abrogation of iNOS or macrophages attenuated lung inflammation, nitric oxide in BAL fluid, and iNOS activity of BAL cells, but had no significant effect on cNOS activity of BAL cells in rats challenged by HMGB1. Conclusions Inducible nitric oxide synthase expressed by alveolar macrophages facilitates the development of HMGB1-induced acute lung injury. Received 12 March 2005; returned for revision 11 January 2006; accepted by M. Parnham 24 January 2006     

Essential role of platelet-activating factor in control of Leishmania (Leishmania) amazonensis infection

In the present study we investigated the role of platelet-activating factor (PAF) and prostaglandins in experimental Leishmania (Leishmania) amazonensis infection and the relationship between these mediators and nitric oxide (NO) production. Mouse peritoneal macrophages elicited with thioglicolate were infected with leishmania amastigotes, and the infection index determined 48 h later. The course of infection was monitored for 5 weeks in mice infected in the footpad with promastigotes by measuring the footpad swelling and parasite load in regional lymph nodes and spleen. The addition of PAF to C57BL/6 mouse macrophages significantly inhibited parasite growth and induced NO production. Treatment of macrophages with a selective PAF antagonist, WEB2086, increased the infection, indicating that endogenously produced PAF regulates macrophage ability to control leishmania infection. This effect of PAF was abolished by addition of the inhibitor of NO synthesis, L-NAME, to the cultures. The addition of prostaglandin E(2) significantly increased the infection and NO production. Treatment with cyclo-oxygenase inhibitor, indomethacin, reduced the infection and PAF-induced release of NO. Thus, the increased NO production induced by PAF seems to be mediated by prostaglandins. The more-selective inhibitors of cyclo-oxygenase 2, nimesulide and NS-398, had no significant effect. Thus, antileishmanial activity correlates better with the presence of PAF or absence of prostaglandins than with NO production. In vivo treatment with PAF antagonists significantly increased leishmania lesions, as well as the parasite load, in regional lymph nodes and spleens. These findings indicate that PAF is essential for the control of leishmania infection.     

Increased Antimycobacterial Immunity in Interleukin-10-Deficient Mice

Macrophage effector functions are essential for clearing mycobacterial infections. Interleukin 10 (IL-10) negatively regulates macrophages and could be a factor inhibiting effective antimycobacterial immunity. We previously showed that transgenic mice which produce excess IL-10 from T cells are susceptible to infection, even though these mice continue to produce gamma interferon (IFN-gamma) at levels similar to those in controls. Here, we extend our genetic analysis of the functions of IL-10 in antimycobacterial immunity by testing the hypothesis that IL-10-deficient (IL-10(-/-)) mice should be more resistant to mycobacteria than control mice. Mycobacterium bovis bacillus Calmette-Guérin-infected IL-10(-/-) mice had significantly lower bacterial burdens than control mice early in the infection. Contrary to expectations, however, IL-10(-/-) mice did not have increased levels of IFN-gamma, either from T cells or in the plasma, suggesting that other mechanisms are responsible for the increased resistance. However, macrophages from IL-10(-/-) mice produced increased levels of inflammatory cytokines, including IFN-gamma, as well as nitric oxide and prostaglandins, which could account for increased antimycobacterial immunity. Our genetic analysis revealed that IL-10 is an inhibitor of early mycobacterial clearance. The data also suggest that IL-10 negatively regulates numerous macrophage functions as well as playing a role in down-regulating the general inflammatory response, especially in conditions where an infection must be controlled through macrophage activity.     

Nitric oxide is an important mediator of renal tubular epithelial cell death in vitro and in murine experimental hydronephrosis

Macrophages play a pivotal role in tissue injury and fibrosis during renal inflammation. Although macrophages may induce apoptosis of renal tubular epithelial cells, the mechanisms involved are unclear. We used a microscopically quantifiable co-culture assay to dissect the cytotoxic interaction between murine bone marrow-derived macrophages and Madin-Darby canine kidney cells and primary murine renal tubular epithelial cells. The induction of tubular cell apoptosis by cytokine-activated macrophages was reduced by inhibitors of nitric oxide synthase whereas tubular cell proliferation was unaffected. Furthermore, cytokine-activated macrophages derived from mice targeted for the deletion of inducible nitric oxide synthase were noncytotoxic. We then examined the role of nitric oxide in vivo by inhibiting inducible nitric oxide synthase in the model of murine experimental hydronephrosis. l-N(6)-(1-iminoethyl)-lysine was administered in the drinking water between days 5 and 7 after ureteric obstruction. Macrophage infiltration was comparable between groups, but treatment significantly inhibited tubular cell apoptosis at day 7. Tubular cell proliferation was unaffected. Inducible nitric oxide synthase blockade also reduced interstitial cell apoptosis and increased collagen III deposition. These data indicate that nitric oxide is a key mediator of macrophage-directed tubular cell apoptosis in vitro and in vivo and also modulates tubulointerstitial fibrosis.     

Rat, mouse and human neutrophils stimulated by a variety of activating agents produce much less nitrite than rodent macrophages.

The role of reactive nitrogen intermediates (RNI) in the antimicrobial activities of neutrophils from various mammalian species is unclear. However, it has been reported that rodent neutrophils possess the inducible form of nitric oxide synthase and that inflammatory neutrophils from rats produce potentially antimicrobial levels of RNI. In the present study, neutrophils from humans, rats and mice were evaluated for production of nitrite, a stable end-product of RNI. Human neutrophil preparations (> 95% neutrophils) isolated from peripheral blood were stimulated for 2-24 hr with agents known to trigger the Ca(2+)-dependent constitutive nitric oxide synthase, or to stimulate synthesis of the inducible nitric oxide synthase. Superoxide dismutase was added to some cultures to decrease the levels of superoxide, a compound reported to react with RNI and yield products other than nitrite. Even though the cells were viable and responsive to stimuli, they did not produce nitrite concentrations indicative of antimicrobial potential. Preparations of inflammatory (casein-elicited) mouse neutrophils also failed to produce high concentrations of nitrite. Inflammatory rat neutrophils (2.5 x 10(6)/ml) produced nitrite concentrations of approximately 40 microM in 24-hr cultures, but plots of nitrite production versus cell number for neutrophil and macrophage preparations indicated that contaminating macrophages could account for all the nitrite production in the neutrophil preparations. Thus, neutrophils from rats, mice and humans seem comparable in their inability to produce high levels of nitrite in response to a variety of stimuli. This suggests that in most circumstances the constitutive nitric oxide synthase known to be present in these cells is limited to the production of low levels of nitric oxide for intercellular signalling. In addition, this raises questions about the presence or functional status of inducible nitric oxide synthase in rodent neutrophils.     

Role of extracellular and intracellular nitric oxide in the regulation of macrophage responses

We studied the role of nitric oxide in the stress response and apoptosis. Intracellular nitric oxide potentiated the stress response. However, intracellular nitric oxide suppressed the stress response in macrophages of proinflammatory and antiinflammatory phenotypes. Intracellular nitric oxide promoted apoptosis in macrophages of the proinflammatory phenotype, but inhibited this process in cells of the antiinflammatory phenotype. Exogenous nitric oxide synthesized by macrophages protected them from lipopolysaccharide-induced apoptosis. Our results indicate that nitric oxide produces various effects on the stress response and apoptosis in macrophages, which depends on modus operandi. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 4, pp. 386–388, April, 2006     

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha induce nitric oxide release and enhance parasite killing in Leishmania infantum-infected human macrophages

Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1α significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-γ. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L-N ^Gmonomethylarginine (L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1α mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection. Received: 9 November 2001 / Accepted: 16 September 2002 Correspondence to O. Brandoniso     

Superoxide dismutase-overexpressing mice are resistant to ozone-induced tissue injury and increases in nitric oxide and tumor necrosis factor-alpha

Reactive oxygen intermediates have been implicated in lung injury induced by inhaled irritants. The present studies used mice overexpressing Cu/Zn-superoxide dismutase (SOD+/+) to analyze their role in ozone-induced lung inflammation and cytotoxicity. Treatment of wild-type mice with ozone (0.8 ppm, 3 h) resulted in increased bronchoalveolar lavage fluid protein, which was maximal after 24-48 h. Significant increases in lung macrophages and 4-hydroxyalkenals were also observed. In contrast, bronchoalveolar lavage fluid protein and macrophage content and 4-hydroxyalkenals were at control levels in ozone-treated SOD+/+ mice. There was also no evidence of peroxynitrite-mediated lung damage, demonstrating that SOD+/+ mice are resistant to ozone toxicity. Whereas alveolar macrophages from wild-type mice produced increased amounts of nitric oxide and expressed more inducible nitric oxide synthase, phospholipase A(2), and tumor necrosis factor-alpha after ozone inhalation, this was not evident in cells from SOD+/+ mice. Ozone-induced decreases in interleukin-10 were also not observed. In wild-type mice, ozone inhalation resulted in activation of nuclear factor-kappaB, which regulates proinflammatory gene activity. This response was significantly reduced in SOD+/+ mice. These data demonstrate that antioxidant enzymes play a critical role in ozone-induced tissue injury and in inflammatory mediator production.     

Necessity and sufficiency of beta interferon for nitric oxide production in mouse peritoneal macrophages.

Bacterial lipopolysaccharide and some cytokines can activate macrophages to secrete nitric oxide. Macrophage-derived nitric oxide is a key cytotoxic factor for microbicidal and tumoricidal processes. We report here that a monoclonal antibody specific for beta interferon inhibited lipopolysaccharide-induced nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages and macrophage-like cell line RAW 264.7. In addition, exogenous added beta interferon enabled lipopolysaccharide-hyporesponsive thioglycolate-elicited C3H/HeJ peritoneal macrophages to produce nitric oxide in response to lipopolysaccharide. These data support the concept that beta interferon provides an essential signal(s) for lipopolysaccharide-triggered nitric oxide production by mouse macrophages. Heat-killed Staphylococcus aureus, a gram-positive bacterium which was unable to initiate nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages in vitro, promoted nitric oxide formation in the presence of beta interferon, suggesting that beta interferon may be a general cofactor necessary for bacterium-derived stimulus-induced nitric oxide production in these macrophages. However, neither beta interferon nor tumor necrosis factor alpha, alone or in combination, triggered nitric oxide production in thioglycolate-elicited mouse peritoneal macrophages, demonstrating that these macrophage-derived cytokines, while necessary, were not sufficient by themselves for the induction of nitric oxide production in these cells. On the other hand, gamma interferon and tumor necrosis factor alpha acted together to induce nitric oxide production in vitro in the absence of lipopolysaccharide in thioglycolate-elicited mouse peritoneal macrophages, indicating that these two types of interferons provided different signals during the activation of these macrophages.     

Differential effects of osteopontin on the cytotoxic activity of macrophages from young and old mice.

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.