Discoidin domain receptor 1 mediates collagen-induced inflammatory activation of microglia in culture

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor tyrosine kinase with an extracellular domain homologous to discoidin 1 of a soil-living amoeba Dictyostelium discoideum. We have previously demonstrated that DDR1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Because collagen is one of the main components of extracellular matrix in the central nervous system, we hypothesized that collagen also induces inflammatory activation of brain microglia, and DDR1 may mediate collagen-induced microglial activation. Using BV-2 mouse microglial cells and mouse primary microglial cultures, we have demonstrated that (1) collagen induces inflammatory activation of microglia as evidenced by production of nitric oxide, expression of inducible nitric oxide synthase, COX-2, CD40, and matrix metalloproteinase-9; (2) DDR1 is expressed in microglia and is phosphorylated by collagen treatment; and (3) collagen-induced microglial activation is abrogated by DDR1 blockade but not by integrin neutralization. We have further shown that p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-kappa B are involved in the collagen-DDR1-induced microglial activation. Our results suggest that collagen can induce inflammatory activation of brain microglia and that DDR1 mediates this effect of collagen in an integrin-independent manner.     

Brain macrophages synthesize interleukin-1 and interleukin-1 mRNAs in vitro

Amoeboid microglial cells (brain macrophages) were purified from early post-natal mouse brain cultures. The percentage of cells stained with an anti-Mac-1 antibody was greater than 95%. Stimulation of these brain macrophages by lipopolysaccharides induced the synthesis of interleukin-1 (IL-1), which, in part, remained associated with the cell surface and, in part, was released into the culture medium. In contrast, pure primary astrocyte cultures and cell lines of transformed or immortalised astrocytes did not synthesise significant amounts of IL-1, demonstrating that amoeboid microglia and not astrocytes synthesise IL-1 in vitro. These physiological data were confirmed by RNA hybridisation studies showing that, on LPS treatment, brain macrophages synthesise significant amounts of IL-1 alpha and IL-1 beta mRNAs.     

Gp-41-mediated astrocyte inducible nitric oxide synthase mRNA expression: involvement of interleukin-1beta production by microglia.

Mechanisms underlying human immunodeficiency virus-1 encephalopathy are not completely known; however, recent studies suggest that the viral protein gp41 may be neurotoxic via activation of inducible nitric oxide synthase (iNOS) in glial cells. In the present study, we investigated the NO-generating activity of primary human fetal astrocytes in response to gp41 and the relationship to microglial cell production of interleukin-1 (IL-1). Gp41 failed to trigger iNOS mRNA expression in highly enriched (>99%) astrocyte or microglial cell cultures. However, gp41-treated microglia released a factor(s) that triggered iNOS mRNA expression and NO production in astrocytes. Because IL-1 receptor antagonist protein blocked gp41-induced NO production, a pivotal role was suggested for microglial cell IL-1 production in astrocyte iNOS expression. Also, gp41 induced IL-1beta mRNA expression and IL-1 production in microglial cell but not astrocyte cultures. Using specific inhibitors, we found that gp41-induced IL-1beta production in microglia was mediated via a signaling pathway involving protein-tyrosine kinase. These data support the hypothesis that gp41 induces astrocyte NO production indirectly by triggering upregulation of microglial cell IL-1 expression.     

Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response

The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.     

Cytomegalovirus induces cytokine and chemokine production differentially in microglia and astrocytes: antiviral implications

Glial cells function as sensors for infection within the brain and produce cytokines to limit viral replication and spread. We examined both cytokine (TNF-alpha, IL-1beta, and IL-6) and chemokine (MCP-1, MIP-1alpha, RANTES, and IL-8) production by primary human glial cells in response to cytomegalovirus (CMV). Although CMV-infected astrocytes did not produce antiviral cytokines, they generated significant quantities of the chemokines MCP-1 and IL-8 in response to viral infection. On the other hand, supernatants from CMV-stimulated purified microglial cell cultures showed a marked increase in the production of TNF-alpha and IL-6, as well as chemokines. Supernatants from CMV-infected astrocyte cultures induced the migration of microglia towards chemotactic signals generated from infected astrocytes. Antibodies to MCP-1, but not to MIP-1alpha, RANTES, or IL-8, inhibited this migratory activity. These findings suggest that infected astrocytes may use MCP-1 to recruit antiviral cytokine-producing microglial cells to foci of infection. To test this hypothesis, cocultures of astrocytes and microglial cells were infected with CMV. Viral gene expression in these cocultures was 60% lower than in CMV infected purified astrocyte cultures lacking microglia. These results support the hypothesis that microglia play an important antiviral role in defense of the brain against CMV. The host defense function of microglial cells may be directed in part by chemokines, such as MCP-1, produced by infected astrocytes.     

Alpha-smooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factor-beta 1 (TGF-beta1)

Aims: Rapid and extensive activation of astrocytes occurs subsequent to many forms of central nervous system (CNS) injury. Recent studies have revealed that the expression profile of reactive astrocytes comprises antigens present during astrocyte development. Elevated levels of the injury‐related cytokine transforming growth factor‐beta 1 (TGF‐β1) secreted by microglial cells and invading macrophages have been correlated with the reactive astrocyte phenotype and glial scar formation. Methods: In the present study, the expression profile of alpha‐smooth muscle actin (α‐SMA) and nestin, two cytoskeletal proteins expressed during astrocyte development, was studied in multiple sclerosis (MS) lesions. In addition, α‐SMA and nestin organization and expression were analysed in rat primary astrocyte cultures in response to TGF‐β1. Results: In active lesions and in the hypercellular margin of chronic active MS lesions, immunostaining for α‐SMA revealed a subpopulation of reactive astrocytes, whereas the majority of reactive astrocytes expressed nestin. α‐SMA and nestin expressing reactive astrocytes were in close relationship with TGF‐β1 expressing macrophages or microglia. In addition, TGF‐β1 expression within α‐SMA or nestin expressing astrocytes was also detected. Our in vitro experiments showed that TGF‐β1 regulated the organization and expression of α‐SMA and nestin in astrocytes. Conclusions: Reactive astrocytes in active MS lesions re‐express α‐SMA and nestin. We suggest that the in vivo re‐expression might be under regulation of TGF‐β1. These results further clarify the regulation of astrocyte activity after CNS injury, which is important for the astroglial adaptation to pathological situations.     

Induction of human immunodeficiency virus type 1 replication in human glial cells after proinflammatory cytokines stimulation: Effect of IFNγ, IL1β, and TNFα on differentiation and chemokine production in glial cells

Although evidence for human immunodeficiency virus 1 (HIV-1) presence in the central nervous system (CNS) of infected patients is well established, the intensity of viral replication within the brain is not usually known. In vitro, human embryonic microglial cells internalized HIV-1 through a CD4-dependent pathway but were not permissive to viral replication. We observed that HIV replication was induced when CNS cell cultures were stimulated for 14 days by a combination of proinflammatory cytokines including IFNγ, IL1β, and TNFα. After long-term cytokine stimulation, morphologically differentiated glial cells appeared, in which HIV-1 tat antigen was detected after infection. Thus, variations in the stage of maturation/activation of CNS cells under inflammatory conditions probably play a major role in facilitating massive production of HIV-1. We then studied the effect of prolonged cytokine stimulation on the secretion of inflammatory mediators by glial cells. An early increased secretion of prostaglandin F2α and chemokines (RANTES>>MIP-1α>>MIP-1β) was observed, due to both microglia and astrocytes. In contrast to persistent PGF2α production, an extinction of RANTES and MIP-1β but not of MIP-1α secretion occurred during the 14 days of stimulation and was inversely correlated with the ability of glial cells to replicate HIV-1. The study of the secretory factors produced in response to a persistent inflammation could provide a better understanding of the modulation of HIV replication in glial cells. GLIA 23:304–315, 1998. © 1998 Wiley-Liss, Inc.     

GM-CSF and M-CSF modulate beta-chemokine and HIV-1 expression in microglia

Significant numbers of patients with acquired immunodeficiency syndrome (AIDS) develop CNS infection primarily in macrophages and microglial cells. Therefore, the regulation of human immunodeficiency virus type 1 (HIV-1) infection and activation of the brain mononuclear phagocytes subsequent to infection are important areas of investigation. In the current report, we studied the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) in the expression of antiviral beta-chemokines and HIV-1 p24 in cultures of primary human fetal microglia. We found that stimulation with GM-CSF or M-CSF induced macrophage inflammatory proteins (MIP-1alpha and MIP-1beta) and augmented RANTES expression, after HIV-1 infection of microglia. This was not due to the effect of GM-CSF on viral expression because GM-CSF was neither necessary nor stimulatory for viral infection, nor did GM-CSF enhance the expression of env-pseudotyped reporter viruses. Blocking GM-CSF-induced microglial proliferation by nocodazole had no effect on beta-chemokine or p24 expression. The functional significance of the GM-CSF-induced beta-chemokines was suggested by the finding that, in the presence of GM-CSF, exogenous beta-chemokines lost their anti-HIV-1 effects. We further show that although HIV-1-infected microglia produced M-CSF, they failed to produce GM-CSF. In vivo, GM-CSF expression was localized to activated astrocytes and some inflammatory cells in HIV-1 encephalitis, suggesting paracrine activation of microglia through GM-CSF. Our results demonstrate a complex interplay between CSFs, chemokines, and virus in microglial cells and may have bearing on the interpretation of data derived in vivo and in vitro.     

Hypoxic induction of caspase-11/caspase-1/interleukin-1beta in brain microglia

Caspase-11 is an inducible protease that plays an important role in both inflammation and apoptosis. Inflammatory stimuli induce and activate caspase-11, which is required for the activation of caspase-1 or interleukin-1beta (IL-1beta) converting enzyme (ICE). Caspase-1 in turn mediates the maturation of proinflammatory cytokines such as IL-1beta, which is one of the crucial mediators of neurodegeneration in the central nervous system. Here, we report that hypoxic exposure of cultured brain microglia (BV-2 mouse microglia cells and rat primary microglial cultures) induces expression and activation of caspase-11, which is accompanied by activation of caspase-1 and secretion of mature IL-1beta and IL-18. Hypoxic induction of caspase-11 was observed in both mRNA and protein levels, and was mediated through p38 mitogen-activated protein kinase pathway. Transient global ischemia in rats also induced caspase-11 expression and IL-1beta production in hippocampus supporting our in vitro findings. Caspase-11-expressing cells in hippocampus were morphologically identified as microglia. Taken together, our results indicate that hypoxia induces a sequential event-caspase-11 induction, caspase-1 activation, and IL-1beta release-in brain microglia, and point out the importance of initial caspase-11 induction in hypoxia-induced inflammatory activation of microglia.     

An efficient method to limit microglia-dependent effects in astroglial cultures

Microglia, the CNS resident macrophages, and astrocytes, the most abundant glial cell population, are both implicated in brain pathologies and can exhibit a pro-inflammatory phenotype. Microglial cells are known to rapidly and strongly react to brain insults. They will promote astrocyte activation and may lead to a vicious, self-perpetuating cycle of chronic inflammation. To obtain a better understanding of the individual role of both cell types, primary cells are frequently used in in vitro studies, but the purity of specific cell cultures remains rarely investigated. The aim of this study is to determine the effect of specific removal of microglial cells on the inflammatory properties of different glial cultures. Here, the removal of microglial contamination from mixed glial cultures to obtain astrocyte-enriched cultures was achieved using a magnetic cell sorting approach. Compared to mixed cultures, we clearly showed that these enriched cultures are only weakly activated by pro-inflammatory agents (lipopolysaccharide, interferon-γ or beta-amyloid peptide). This finding was confirmed using twice-sorted astrocyte-enriched cultures and microglia-free cultures composed of neurosphere-derived astrocytes. Thus, we present evidence that the magnitude of the pro-inflammatory response is linked to the percentage of microglia in cultures. Due to their high reactivity to various insults or pro-inflammatory stimuli, microglia-derived effects could be credited to astrocytes in mixed glial cultures. Therefore, we highlight the importance of monitoring the presence of microglia in glial cultures since they can affect the interpretation of the results, especially when inflammatory processes are studied.     

Expression of beta-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: potential role of chemokines in the developing CNS.

Chemokines play specific roles in directing the recruitment of leukocyte subsets into inflammatory foci within the central nervous system (CNS). The involvement of these cytokines as mediators of inflammation is widely accepted. Recently, it has become evident that cells of the CNS (astrocytes, microglia, and neurons) not only synthesize, but also respond functionally or chemotactically to chemokines. We previously reported developmental events associated with colonization of the human fetal CNS by mononuclear phagocytes (microglial precursors), which essentially takes place within the first two trimesters of life. As part of the array of signals driving colonization, we noted specific anatomical distribution of chemokines and chemokine receptors expressed during this period. In order to further characterize expression of these molecules, we have isolated and cultured material from human fetal CNS. We demonstrate that unstimulated subconfluent human fetal glial cultures express high levels of CCR2 and CXCR4 receptors in cytoplasmic vesicles. Type I astrocytes, and associated ameboid microglia in particular, express high levels of surface and cytoplasmic CXCR4. Of the chemokines tested (MIP-1alpha, MIP-1beta, MCP-1, MCP-3, RANTES, SDF-1, IL-8, IP-10), only MIP-1alpha, detected specifically on microglia, was expressed both constitutively and consistently. Low variable levels of MCP-1, MIP-1alpha, and RANTES were also noted in unstimulated glial cultures. Recombinant human chemokines rhMCP-1 and rhMIP-1alpha also displayed proliferative effects on glial cultures at [10 ng/ml], but displayed variable effects on CCR2 levels on these cells. rhMCP-1 specifically upregulated CCR2 expression on cultured glia at [50 ng/ml]. It is gradually becoming evident that chemokines are important in embryonic development. The observation that human fetal glial cells and their progenitors express specific receptors for chemokines and can be stimulated to produce MCP-1, as well as proliferate in response to chemokines, supports a role for these cytokines as regulatory factors during development.     

GM-CSF promotes proliferation of human fetal and adult microglia in primary cultures

Proliferation of microglia/macrophages is a common finding in many central nervous system diseases. To identify mitogenic signals for human microglia, we examined primary cultures of human fetal and adult microglia after stimulation with cytokines, colony stimulating factors (CSFs), or LPS, using proliferating cell nuclear antigen (PCNA) expression as an index of cell proliferation. The results showed that both M-CSF and GM-CSF induced microglial proliferation in fetal and adult human cultures, but that GM-CSF provided a much stronger stimulus. At 96 h post-stimulation, the mean PCNA labeling index was 2.4 for M-CSF and 13.3 for GM-CSF in fetal microglia; in adult microglia, the PCNA labeling index was 4.7 for M-CSF and 9.0 for GM-CSF. The effect of GM-CSF on fetal microglia was dose dependent and synergistic with M-CSF. LPS abolished the basal level of PCNA labeling in adult microglia, but in fetal microglia, caused a slight increase in PCNA labeling (1.9) at 96 h and consistently enhanced microglial cell survival and differentiation into highly branched cells. The production of GM-CSF in purified human fetal astrocyte and microglial cultures was examined after stimulation with LPS, TNF-α, or IL-1β. Unlike M-CSF, neither cell type produced GM-CSF in unstimulated cultures; however, when stimulated with IL-1β, astrocytes expressed GM-CSF mRNA and protein, which accumulated in the culture through 72 h. In microglia, LPS was the only effective inducing agent. An immunocytochemical study performed to identify in vivo sources of GM-CSF revealed selective labeling of reactive astrocytes in active lesions of multiple sclerosis and senile plaques of Alzheimer's disease. Our data demonstrate that both fetal and adult human microglia are capable of proliferation in response to CSFs, GM-CSF being the more effective stimulus.     

S100B expression in and effects on microglia

We evaluated the intracellular and extracellular biological role of S100B protein with respect to microglia. S100B, which belongs to the multigenic family of Ca2+-binding proteins, is abundant in astrocytes where it is found diffusely in the cytoplasm and is associated with membranes and cytoskeleton constituents. S100B protein is also secreted by astrocytes and acts on these cells to stimulate nitric oxide secretion in an autocrine manner. However, little is known about the relationship between S100B and microglia. To address this issue, we used primary microglia from newborn rat cortex and the BV-2 microglial cell line, a well-established cell model for the study of microglial properties. S100B expression was assessed by immunofluorescence in primary microglia and by RT-PCR, Western blotting, and immunofluorescence in BV-2 cells. S100B was found in microglia in the form of a filamentous network as well as diffusely in the cytoplasm and associated with intracellular membranes. S100B relocated around phagosomes during BV-2 phagocytosis of opsonized Cryptococcus neoformans. Furthermore, interferon-gamma (IFN-gamma) treatment caused cell shape changes and redistribution of S100B, and downregulation of S100B mRNA expression in BV-2 cells. Treatment of BV-2 cells with nanomolar to micromolar amounts of S100B resulted in increased IFN-gamma-induced expression of inducible nitric oxide synthase mRNA as well as nitric oxide secretion. Taken together, these data suggest a possible role for S100B in the accomplishment/regulation of microglial cell functions.     

TGFβ produced by IL‐10 redirected astrocytes attenuates microglial activation

While there clearly is an intimate relationship between astrocytes and microglia, few studies have examined these potentially dynamic interactions. In this study, cytokine‐mediated communication between microglia and astrocytes under inflammatory conditions was investigated. We have previously shown that activated microglia produce Interleukin (IL)‐10, a regulatory cytokine that plays an important role in resolving neuroinflammation. Nonetheless, the mechanism by which IL‐10 attenuates pro‐inflammatory cytokine expression in the brain is unclear. Here, we show that IL‐10 redirected astrocytes regulate the activation of microglia in a transforming growth factor (TGF)‐β dependent manner. In support of this concept, astrocytes in the brain maintained higher IL‐10 receptor (IL‐10R1) expression and primary astrocytes in culture were markedly more sensitive to the anti‐inflammatory effects of IL‐10 compared with microglia. Moreover, studies using primary cultures and an astrocyte‐microglia coculture system revealed that astrocytes mediated the anti‐inflammatory effects of IL‐10 on microglia through the production of TGFβ. For instance, only when astrocytes were present did IL‐10 stimulation reduce the expression of IL‐1β and increase expression of anti‐inflammatory mediators fractalkine receptor (CX₃CR1) and interleukin 4 receptor‐α (IL‐4Rα) in microglia. Importantly, these IL‐10‐astrocyte dependent effects on microglia were blocked by a TGFβ inhibitor. Furthermore, inhibition of TGFβ signaling in the brain resulted in prolonged sickness behavior and amplified pro‐inflammatory cytokine expression in mice challenged with lipopolysaccharide. Taken together, IL‐10 stimulated the production of TGFβ by astrocytes, which in turn, attenuated microglial activation. Overall, these findings provide novel insight into the mechanisms by which astrocytes modulate microglia under inflammatory conditions. GLIA 2014;62:881–895     

Microglia and macrophages are major sources of locally produced transforming growth factor-β1 after transient middle cerebral artery occlusion in rats

The potentially neurotrophic cytokine transforming growth factor-β₁ (TGF-β₁) is locally expressed following human stroke and experimental ischemic lesions, but the cellular source(s) and profile of induction have so far not been established in experimental focal cerebral ischemia. This study presents the time course and a cellular localization of TGF-β₁ mRNA, visualized by in situ hybridization combined with immunohistochemical staining for microglia, macrophages, or astrocytes, on brain sections from adult spontaneously hypertensive rats subjected to transient proximal occlusion of their middle cerebral artery. Six hours after ischemia, an early and transient neuronal and microglial expression of TGF-β₁ mRNA was observed in the extraischemic cingulate and frontal cortices. Both early and protracted expression of TGF-β₁ mRNA in the caudate-putamen and neocortical infarcts and in the caudate-putamen penumbra colocalized with OX42/ ED1-immunoreactive microglia and macrophages, whereas TGF-β₁ mRNA in the neocortical penumbra colocalized with OX42/ ED1-immunoreactive cells of a microglial morphology. No astrocytes were double-labeled. The number of TGF-β₁ mRNA-expressing microglia and macrophages increased strongly during the first week. Thereafter, TGF-β₁ mRNA became increasingly restricted to the neocortical penumbra (3 weeks), and after 3 months it was confined to activated microglia in the anterior commisure. Our data establish activated microglia and macrophages as the major source of TGF-β₁ mRNA following experimental focal cerebral ischemia. Consequently, TGF-β₁-mediated functions may be exerted by microglia both in the early degenerative phase, and later in combination with blood-borne macrophages, in the remodeling and healing phase after focal cerebral ischemia. GLIA 24:437–448, 1998. © 1998 Wiley-Liss, Inc.     

Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells

Primary rat astrocyte cultures were used to isolate a macrophage population that does not adhere to the confluent glial cells. The cells multiplied vigorously in coculture with astrocytes during the 14 d culture period, provided that functionally active lipopolysaccharide (LPS) was either absent or present in very low concentrations. Based on morphological, immunocytochemical, and pharmacological data, it was concluded that the isolated cells were microglia, the resident macrophages of the brain. The findings characterized them as a distinct cell population that shares features both of peritoneal macrophages and of astroglial cells. Like peritoneal macrophages, the isolated cells were able to phagocytize as shown by their ingestion of latex beads and uptake of L-leucyl methylester. Furthermore, they were immunocytochemically stainable by a specific monoclonal antibody (ED 1) against a macrophage-specific antigen (Dijkstra et al., 1985). They also synthesized prostaglandin E2 (PGE2) and secreted interleukin 1 (IL-1) upon stimulation with LPS. Upon stimulation with the ionophore A23187, PGD2, the predominant prostaglandin of the brain, was the major PG metabolite released by these cells. In contrast to peritoneal macrophages, microglial cells were able to multiply. Proliferation of microglial cells in coculture with astrocytes was suppressed when 2 ng LPS/ml or higher concentrations were added to astroglial culture media. These astrocyte cultures, which contained approximately 1% microglia, were used to investigate the influence of LPS on prostaglandin and IL-1 secretion in order to compare astroglial and microglial features. Increasing LPS concentrations induced increased PGE2 secretion, whereas PGD2 secretion was essentially unaffected by LPS. The critical influence of LPS contaminations in most of the commercially available animal sera used for astrocyte cultures on cellular composition in general and on metabolism of hormones and growth factors in particular is discussed.