Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.     

Gradual inhibition of inducible nitric oxide synthase but not of interleukin-1β production in rat microglial cells of endotoxin-treated mixed glial cell cultures

In cultures of purified microglial cells and astrocytes from newborn rats, the immunocytochemical localization of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) using recently developed antibodies, as well as the release of IL-1β and nitric oxide (NO), was studied following exposure of the cells to endotoxin [lipopolysaccharide (LPS)]. In the absence of LPS, IL-1β- and iNOS-immunoreactive microglial cells and IL-1β or NO release were not observed, whereas in the presence of the endotoxin, the production of NO and IL-1β by microglial cells dramatically exceeded their synthesis and release by astrocytes. Interestingly, microglial cells cultured for 4–8 days in the presence of astrocytes appeared to lose their ability to produce iNOS, whereas the release of IL-1β remained unaltered. Moreover, endotoxin-stimulated microglial cells appeared to regain their ability to synthesize iNOS following their separation from astrocytes. These data show that microglia are primarily responsible for NO and IL-1β production in mixed glial cell cultures upon endotoxin stimulation. Moreover, in the presence of astrocytes the induction of iNOS, but not that of IL-1β in microglial cells is gradually inhibited. © 1996 Wiley-Liss, Inc.     

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.     

Modulation by astrocytes of microglial cell-mediated neuroinflammation: effect on the activation of microglial signaling pathways

The strong inflammatory response observed in neurodegenerative diseases can depend on the impairment of the endogenous control of microglial activation, triggering the release of potentially detrimental factors such as cytokines, nitric oxide (NO) and superoxide anion (O(2)(-)). Our aim was to study the activation of microglial cells and the transduction pathways involved in their modulation by IL-1beta and TNF-alpha. Microglial and mixed glial cell cultures from neonatal rats were exposed to IFN-gamma and/or IL-1beta and TNF-alpha. We analyzed NO secretion and the activation of ERK and STAT1. We found that astrocytes modulated microglial cell activation, decreasing production of NO. IFN-gamma induced an 18- to 25-fold increase in NO, associated to a 3- to 5-fold increase in ERK phosphorylation in microglial cultures. IL-1beta, but not TNF-alpha, inhibited IFN-gamma-induced production of NO in microglia by 87%. It also reduced IFN-gamma-induced phosphoERK (pERK) by 40%, without affecting phosphoSTAT1 (pSTAT1). In contrast, in microglial cultures exposed to media conditioned by astrocytes, IL-1beta did not inhibit pERK, whereas it reduced activation of STAT1. Inducible NO synthase expression induced by IFN-gamma in microglial cultures was reduced when the activation of ERK was prevented. We propose that IL-1beta modulates IFN-gamma-induced production of oxidative molecules through cross talk between STAT1 and MAPK pathways, regulating the amplitude and duration of microglial activation. Modulation of ERK was observed at 30 min, whereas inhibition of pSTAT was observed later (at 4 h), indicating that it was an early and transient phenomenon.     

Astrocytes enhance lipopolysaccharide-induced nitric oxide production by microglial cells

Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells. The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment. However, NO production in endotoxin-treated astrocyte cultures is controversial. We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media. We also identified the NO-producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells. Only microglial cells showed iNOS immunoreactivity. Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures. We then analysed the effect of astrocytes on NO production by microglial cells using microglial-astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells. Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS-treated astrocytes could account, at least in part, for this enhancement.     

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.     

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: A role for transforming growth factor β

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1β (IL-1β) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1β expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1β was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B₄-isolectin and an antibody against GFAP. Production of IL-1β and NO was determined by measurement of IL-1β and nitrite concentrations in cell lysates and the culture medium, respectively. TGFβ, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGFβ. The potential effect of TGFβ was studied by using immunoneutralizing antibodies against TGFβ1 and TGFβ2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGFβ antibodies (3 μg/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1β was not affected. The antibodies against TGFβ1 and TGFβ2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGFβ1 and TGFβ2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGFβ on NO production by microglial cells (possibly) by activation of TGFβ or by increasing the sensitivity of microglial cells for TGFβ. GLIA 19:190–198, 1997. © 1997 Wiley-Liss, Inc.     

Cytokine regulation of CD40 expression in fetal human astrocyte cultures

CD40 can participate in inflammatory processes after binding its cognate ligand (CD40L). We found that fetal human astrocytes constitutively express CD40 mRNA and protein. Upon incubating cultures with proinflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) or with lipopolysaccharide (LPS), CD40 expression was increased. No change in CD40 expression was noted in astrocyte cultures incubated with IL-6, HIV or gp41. Astrocytes also showed increased release of proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 after incubation with CD40L peptide. These observations suggest a role for CD40 in central nervous system (CNS) inflammation and that CD40/CD40L autocrine or paracrine pathways may mediate this role.     

Expression of inducible nitric oxide synthase, interleukin-1 and caspase-1 in HIV-1 encephalitis

Inflammatory cytokines and enzymes such as IL-1 and inducible nitric oxide synthase (iNOS) may play an important role in the pathogenesis of AIDS dementia, a condition associated with infection of the CNS cells by the HIV-1. In this report, we investigated the expression of iNOS, IL-1, and caspase-1 (interleukin-1 converting enzyme) in HIV-1 encephalitis (HIVE) by immunocytochemistry and analyzed their expression with respect to HIV-1 infection and glial activation. In HIVE, all three molecules were expressed at high levels in areas of HIV-1 infection (microglial nodules with HIV-1 p24 immunoreactivity) and in areas of diffuse white matter gliosis. Expression was cell-type specific, with IL-1 and caspase-1 being expressed in macrophages and microglia, and iNOS in activated astrocytes. Multinucleated giant cells, a hallmark of virally infected cells, showed intense staining for both IL-1 and caspase-1, suggesting induction of these molecules by HIV-1. Double immunocytochemistry demonstrated a regional co-localization of astrocyte iNOS and microglial IL-1 and caspase-1. These results support the notion that autocrine and paracrine interactions between HIV-1 infected macrophages and microglia, activated microglia, and astrocytes lead to expression of proinflammatory and neurotoxic molecules. iNOS and caspase-1 may provide additional therapeutic targets for HIVE.     

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.     

Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain

Inducible nitric oxide synthase (iNOS) expression has been shown to be differentially regulated among different cell types and species. In cultures of primary human fetal glial cells, we have shown that astrocytes rather than microglia express iNOS. In the present study, we extended these findings to primary cultures of astrocytes and microglia derived from adult human brains. Mixed cultures of adult brain tissue were stimulated with IL-1β and IFNγ, a combination known to induce iNOS maximally in human fetal cells, and the expression of iNOS was determined by immunocytochemistry. Cell types were determined by morphology as well as immunocytochemistry for GFAP (astrocytes) and CD68 (microglia). The results showed that in cultures of adult human glia, iNOS was expressed following stimulation with cytokines, and the expression was restricted to astrocytes. Astrocyte iNOS immunoreactivity was detected both in the cytosol and in a discrete paranuclear region, a pattern noted in human fetal astrocytes. These results demonstrate that the ability to express iNOS is common to both fetal and adult human astrocytes.