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.     

Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures

We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells.     

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.     

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.     

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.     

Role of astrocyte-derived tissue-type plasminogen activator in the regulation of endotoxin-stimulated nitric oxide production by microglial cells

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces nitric oxide (NO) production in microglial cells. Earlier we demonstrated that endotoxin induced NO production by microglial cells is inhibited in the presence of astroglial cells by transforming growth factor β (TGFβ). Both microglial and astroglial cells produce TGFβ in a biologically inactive form, which can be activated by plasmin generated by plasminogen activators (PA). In the present paper we describe studies on the mechanism by which glial cells may activate inactive TGFβ and its potential inhibitory effect on NO production by microglial cells. Inhibition of plasmin increased NO production in endotoxin-treated mixed glial cell cultures. Subsequently, antibodies against tissue-type plasminogen activator (tPA) increased NO production in endotoxin-treated mixed glial cell cultures while amiloride, an inhibitor for urokinase (uPA), had no effect. We hereby concluded that tPA is the crucial PA involved in plasmin production resulting in inhibition of NO production in mixed glial cell cultures. Zymography and Northern blot analysis of purified astroglial, microglial, and mixed glial cell cultures demonstrated that astroglial cells produce tPA and a plasminogen activator inhibitor (PAI-1) and are thereby responsible for the production of plasmin which may activate the inactive TGFβ in these cultures. In conclusion, astroglial-derived tPA plays a major role in the inhibition of NO production by endotoxin-treated microglial cells through enhanced plasmin production and possible subsequent TGFβ activation. GLIA 22:130–137, 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.     

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-γ (IFNγ). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFNγ. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFNγ/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.     

Cloning and expression of inducible nitric oxide synthase from rat astrocytes

In primary cultures of rat astroglial cells exposure to bacterial endotoxin lipopolysaccharide (LPS) causes induction of a Ca²⁺-independent form of the nitric oxide synthase (iNOS) enzyme. We have now cloned the mRNA encoding astroglial iNOS using a combination of cDNA library screening and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides directed against conserved regions of all NOS enzymes. The sequence of astroglial iNOS cDNA is highly similar to the mouse macrophage sequence, having an overall homology of 92% at the DNA level and 93% at the protein level. As in other NOSs, canonical binding sites for redox cofactors are present. The 3′-untranslated region displays 4 consensus AU-pentamers, 2 polyadenylation sites, and terminates in a stretch of 17 adenosine residues. In situ hybridization studies with LPS-treated astrocyte cultures demonstrated the presence of iNOS mRNA in the majority of astroglial cells, identified by antibody staining to the glial fibrillary acidic protein (GFAP). PCR analysis showed that LPS stimulated synthesis of astrocyte iNOS mRNA, which was detected as early as 2 hr after exposure to LPS, peaked at 4 hr, and slowly declined over the next 20 hr. These results confirm that astrocytes can express iNOS and provide tools for the subsequent analysis of iNOS gene expression in rodent brain © 1994 Wiley-Liss, Inc.     

Microglial reactivity to beta-amyloid is modulated by astrocytes and proinflammatory factors

The brains of Alzheimer's disease (AD) patients present activated glial cells, amyloid plaques and dystrophic neurites. The core of amyloid plaques is composed of aggregated amyloid peptide (Abeta), a peptide known to activate glial cells and to have neurotoxic effects. We evaluated the capability of glial cells to mediate Abeta(1-42) cytotoxicity in hippocampal cultures. Conditioned media obtained from microglial cultures exposed to Abeta induced apoptosis of hippocampal cells. This pro-apoptotic effect was not observed in hippocampal cultures exposed to conditioned media obtained from mixed glial (astrocytes and microglia) cultures that had been exposed to Abeta. Microglia exposed to Abeta responded with reactive morphological changes, induction of iNOS, elevated nitric oxide production and decreased reductive metabolism. All these responses were attenuated by the presence of astrocytes. This astrocyte modulation was however, not observed when glial cells were exposed to proinflammatory factors (LPS+Interferon-gamma) alone or in combination with Abeta. Our results suggest that astrocytes and proinflammatory molecules are determining factors in the response of microglia to Abeta.     

Microglial regulation of immunological and neuroprotective functions of astroglia

Microglia and astroglia play critical roles in the development, function, and survival of neurons in the CNS. However, under inflammatory conditions the role of astrogliosis in the inflammatory process and its effects on neurons remains unclear. Here, we used several types of cell cultures treated with the bacterial inflammogen LPS to address these questions. We found that the presence of astroglia reduced inflammation‐driven neurotoxicity, suggesting that astrogliosis is principally neuroprotective. Neutralization of supernatant glial cell line‐derived neurotrophic factor (GDNF) released from astroglia significantly reduced this neuroprotective effect during inflammation. To determine the immunological role of astroglia, we optimized a highly‐enriched astroglial culture protocol and demonstrated that LPS failed to induce the synthesis and release of TNF‐α and iNOS/NO. Instead we found significant enhancement of TNF‐α and iNOS expression in highly‐enriched astroglial cultures required the presence of 0.5–1% microglia, respectively. Thus suggesting that microglial‐astroglial interactions are required for LPS to induce the expression of pro‐inflammatory factors and GDNF from astroglia. Specifically, we found that microglia‐derived TNF‐α plays a pivotal role as a paracrine signal to regulate the neuroprotective functions of astrogliosis. Taken together, these findings suggest that astroglia may not possess the ability to directly recognize the innate immune stimuli LPS, but rather depend on crosstalk with microglia to elicit release of neurotrophic factors as a counterbalance to support neuronal survival from the collateral damage generated by activated microglia during neuroinflammation. GLIA 2015;63:118–131     

Pro- and anti-inflammatory cytokines regulate the ERK pathway: Implication of the timing for the activation of microglial cells

Pro-inflammatory molecules induce glial activation and the release of potentially detrimental factors capable of generating oxidative damage, such as nitric oxide (NO) and superoxide anion (O2.-). Activated glial cells (astrocytes and microglia) are associated to the inflammatory process in neurodegenerative diseases. A strong inflammatory response could escape endogenous control becoming toxic to neurons and contributing to the course of the disease. We evaluated in a hippocampal cells-microglia co-culture model, if the pro-inflammatory condition induced by lipopolysaccharide + interferon-gamma (LPS+IFN-gamma) promoted damage directly or if damage was secondary to glial activation. In addition, we explored the effect of the anti-inflammatory cytokine transforming growth factor-beta1 (TGF-beta1), and pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on the regulation of the inflammatory response of microglia. We found that LPS+IFN-gamma-induced damage on hippocampal cultures was dependent on the presence of microglial cells. In hippocampal cultures exposed to LPS+IFN-gamma, TGF-beta1 was induced whereas in microglial cell cultures LPS+IFN-gamma induced the secretion of IL-1beta. TGF-beta1 and IL-1beta but not TNF-alpha decreased the NO production by 70-90%. PD98059, an inhibitor of MAP kinase (MEK), reduced the IFN-gamma-induced NO production by 40%. TGF-beta and IL-1beta reduced the IFN-gamma induced phosphorylation of ERK1,2 by 60% and 40%, respectively. However, the effect of IL-1beta was observed at 30 min and that of TGF-beta1 only after 24 h of exposure. We propose that acting with different timing, TGF-beta1 and IL-1beta can modulate the extracellular signal-regulated kinase ERK1,2, as a common element for different transduction pathways, regulating the amplitude and duration of glial activation in response to LPS+IFN-gamma. Cross-talk among brain cells may be key for the understanding of inflammatory mechanisms involved in pathogenesis of neurodegenerative diseases.     

Characterization of a novel brain-derived microglial cell line isolated from neonatal rat brain.

We observed highly aggressively proliferating immortalized (HAPI) cells growing in cultures that had been enriched for microglia. The cells were initially obtained from mixed glial cultures prepared from 3-day-old rat brains. HAPI cells are typically round with few or no processes when cultured in 10% serum containing medium. As the percentage of serum in the medium is decreased, the HAPI cells have more processes. HAPI cells stain for the isolectin B4, OX-42, and GLUT5, which are markers for microglial cells, but the cells do not immunolabel with A2B5, a marker of cells in the oligodendroglial cell lineage, or with the astrocyte-specific marker, glial fibrillary aciidic protein (GFAP). In addition, HAPI cells are capable of phagocytosis. We conclude that HAPI cells are of microglia/macrophage lineage. Exposing HAPI cells to lipopolysaccharide (LPS) induces the mRNAs for tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). LPS exposure also induces secretion of TNF-alpha and production of nitric oxide (NO) in HAPI cells. Because activation of microglia is associated with an increase in iron accumulation and ferritin expression, we tested the hypothesis that iron status affects the production of TNF-alpha and NO. Our studies demonstrate that both iron chelation and iron loading diminished the LPS-induced effect of TNF-alpha and NO. The results of this study indicate that HAPI cells possess the characteristics of microglia/brain macrophages, providing an alternative cell culture model for the study of microglia. In addition, we demonstrate that the activation of microglial cells could be modified by iron.     

Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia

Oligodendrocytes are the primary cells injured in periventricular leukomalacia (PVL), a predominant form of brain white matter lesion in preterm infants. To explore the possible linkage between white matter injury and maternal infection, purified rat O-2A progenitor (Oligodendrocyte-type 2 astrocyte progenitor) cell cultures were used as a model in studying the effects of lipopolysaccharide (LPS), an endotoxin, on survival and differentiation of oligodendrocytes and the involvement of other glial cells in the effects of LPS. O-2A progenitor cells were cultured from optic nerves of 7-day-old rat pups in a chemically defined medium (CDM). Astrocyte and microglia cell cultures were prepared from the cortex of 1-day-old rat brains in the CDM. Direct treatment of LPS (1 microg/ml) to O-2A cells had no effect on viability or differentiation of these cells. When O-2A progenitor cells were cultured in the conditioned medium obtained from either astrocyte or microglial cell cultures for 48 hr, survival rate and differentiation of O-2A cells into mature oligodendrocytes were greatly enhanced as measured by the MTT assay and immunocytochemistry. The conditioned medium obtained from astrocytes or microglia treated with LPS for 48 hr, however, failed to show such a promotional effect on viability and differentiation of O-2A cells. When 5 microg/ml LPS was used to stimulate astrocytes or microglia, the conditioned medium from these glial cell cultures caused O-2A cell injury. The overall results indicate that astrocytes and microglia may promote viability and differentiation of O-2A progenitor cells under physiological conditions, but they may also mediate cytotoxic effects of LPS on oligodendrocytes under an infectious disease biochemical environment.     

GFAP-independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells

The directed generation of pure astrocyte cultures from pluripotent stem cells has proven difficult. Generation of defined pluripotent-stem-cell derived astrocytes would allow new approaches to the investigation of plasticity and heterogeneity of astrocytes. We here describe a two-step differentiation scheme resulting in the generation of murine embryonic stem cell (mESC) derived astrocytes (MEDA), as characterized by the upregulation of 19 astrocyte-associated mRNAs, and positive staining of most cells for GFAP (glial fibrillary acidic protein), aquaporin-4 or glutamine synthetase. The MEDA cultures could be cryopreserved, and they neither contained neuronal, nor microglial cells. They also did not react to the microglial stimulus lipopolysaccharide, while inflammatory activation by a complete cytokine mix (CCM) or its individual components (TNF-α, IL1-β, IFN-γ) was readily observed. MEDA, stimulated by CCM, became susceptible to CD95 ligand-induced apoptosis and produced NO and IL-6. This was preceded by NF-kB activation, and up-regulation of relevant mRNAs. Also GFAP-negative astrocytes were fully inflammation-competent. Neurotrophic support by MEDA was found to be independent of GFAP expression. In summary, we described here the generation and functional characterization of microglia-free murine astrocytes, displaying phenotypic heterogeneity as is commonly observed in brain astrocytes.