Strongly compromised inflammatory response to brain injury in interleukin-6-deficient mice

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin-6 (IL-6) during the inflammatory response in the brain following disruption of the blood-brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto-parietal cortex in interleukin-6-deficient (IL-6-/-) and normal (IL-6+/+) mice. In IL-6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and metallothionein-I+II (MT-I+II) were increased in these cells, while the brain-specific MT-III was only moderately upregulated. In IL-6-/- mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage-induced GM-CSF and MT-I+II expression were also markedly depressed compared to IL-6+/+ mice. In contrast, MT-III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT-I+II but not MT-III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL-6+/+ and IL-6-/- mice. The present results demonstrate that IL-6 is crucial for the recruitment of myelo-monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL-6 is important for neuroprotection and the induction of GM-CSF and MT expression. The opposing effect of IL-6 on MT-I+II and MT-III levels in the damaged brain suggests MT isoform-specific functions.     

Control of astrocytosis by interleukin-1 and transforming growth factor-β1 in human brain

Astrocytosis is a common neurocellular manifestation of brain pathology in individuals with a variety of diseases. It is comprised of astrocytic hyperplasia (an increase in number of astrocytes) and astrocytic hypertrophy (an increase in size of astrocytes). The precise cause (s) of astrocytosis remains unknown. We morphometrically measured the relative extent of astrocytosis in brains of 22 individuals who died with seven different diseases. The relative amounts of interleukin-1 (IL-1) and transforming growth factor-beta 1 (TGF-β1) immunoreactive products (IRPs) were next assessed in sections serial to those in which astrocytosis was measured because these cytokines were shown in animal and in vitro experiments to be associated with astrocytosis. The data demonstrate that astrocytosis and these cytokines were co-localized in all examined human tissues. Relative increase in density of astrocytes was correlated with the increase in total IL-1 but not TGF-β1. In contrast, the increase in size of astrocytes was correlated with TGF-β1 associated only with astrocytes but not with total IL-1. Both IL-1 and TGF-β1 IRPs were present in GFAP IRP-containing and other cells, as assessed by double label immunocytochemistry. These observations suggest that IL-1 acts on astrocytes by both, paracrine and autocrine mechanisms whereas, TGF-β1 only acts by an autocrine mechanism. Because these correlations were statistically significant and also because a change in number and size of astrocytes constitutes the most frequent response of astrocytes to several diseases or injury, we conclude that these cytokines may mediate the most common pathological change in human 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.     

Dual effect of glia maturation factor on astrocytes : Differentiation and release of interleukin-1 like factors

C6 glioma cells, and primary cultures of mouse astrocytes, stimulated with lipopolysaccharide (LPS) release an interleukin-1 like factor (IL-1) which enhances lectin-induced T-lymphocyte proliferation and promotes the release of interleukin-2 (IL-2) by ConA-stimulated thymocytes. In the present study, the glia maturation factor (GMF) was found not only to induce differentiation of glioblasts, but also to elicit the secretion of IL-1 like factors by cultured mouse astrocytes and their precursor cells. GMF was also effective in triggering IL-1 release by macrophages. Contamination of the 23 000 MW GMF preparation with LPS was excluded by the Limulus lysate assay and by using C3H/HeJ LPS-nonresponder mice whose glia and macrophages responded to GMF but not to LPS, by IL-1 release. Through its ability to induce glial differentiation and IL-1 release, GMF may represent an important endogenous signal, triggering both reactive gliosis and the development of an immune response within the central nervous system.     

Interleukin-1 and Tumor Necrosis Factor-α Synergistically Mediate Neurotoxicity: Involvement of Nitric Oxide and of N-Methyl-D-aspartate Receptors

The cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α, produced by glial cells within the brain, appear to contribute to the neuropathogenesis of several inflammatory neurodegenerative diseases; however, little is known about the mechanism underlying cytokine-induced neurotoxicity. Using human fetal brain cell cultures composed of neurons and glial cells, we investigated the injurious effects of IL-1 β and TNF-α, cytokines which are known to induce nitric oxide (NO) production by astrocytes. Although neither cytokine alone was toxic, IL-1 β and TNF-α in combination caused marked neuronal injury. Brain cell cultures treated with IL-1 β plus TNF-α generated substantial amounts of NO. Blockade of NO production with a NO synthase inhibitor was accompanied by a marked reduction (about 45%) of neuronal injury, suggesting that NO production by astrocytes plays a role in cytokine-induced neurotoxicity. Addition of N-methly-D-aspartate (NMDA) receptor antagonists to brain cell cultures also blocked IL-1 β plus TNF-α-induced neurotoxicity (by 55%), implicating the involvement of NIMDA receptors in cytokine-induced neurotoxicity. Treatment of brain cell cultures with IL-1 β plus TNF-α was found to inhibit [³H]-glutamate uptake and astrocyte glutamine synthetase activity, two major pathways involved in NMDA receptor-related neurotoxicity. These in vitro findings suggest that agents which suppress NO production or inhibit NMDA receptors may protect against neuronal damage in cytokine-induced neurodegenerative diseases.     

Robust expression of TNF-alpha, IL-1beta, RANTES, and IP-10 by human microglial cells during nonproductive infection with herpes simplex virus

Cytokine (TNF-alpha/beta, IL-1beta, IL-6, IL-18, IL-10, and IFN-alpha/beta/gamma) and chemokine (IL-8, IP-10, MCP-1, MIP-1alpha/beta, and RANTES) production during herpes simplex virus (HSV) 1 infection of human brain cells was examined. Primary astrocytes as well as neurons were found to support HSV replication, but neither of these fully permissive cell types produced cytokines or chemokines in response to HSV. In contrast, microglia did not support extensive viral replication; however, ICP4 was detected by immunochemical staining, demonstrating these cells were infected. Late viral protein (nucleocapsid antigen) was detected in <10% of infected microglial cells. Microglia responded to nonpermissive viral infection by producing considerable amounts of TNF-alpha, IL-1beta, IP-10, and RANTES, together with smaller amounts of IL-6, IL-8, and MIP-1alpha as detected by RPA and ELISA. Surprisingly, no interferons (alpha, beta, or gamma) were detected in response to viral infection. Pretreatment of fully permissive astrocytes with TNF-alpha prior to infection with HSV was found to dramatically inhibit replication, resulting in a 14-fold reduction of viral titer. In contrast, pretreatment of astrocytes with IL-1beta had little effect on viral replication. When added to neuronal cultures, exogenous TNF-alpha or IL-1beta did not suppress subsequent HSV replication. Exogenously added IP-10 inhibited HSV replication in neurons (with a 32-fold reduction in viral titer), however, similar IP-10 treatment did not affect viral replication in astrocytes. These results suggest that IP-10 possesses direct antiviral activity in neurons and support a role for microglia in both antiviral defense of the brain as well as amplification of immune responses during neuroinflammation.     

Rat Schwann cells produce interleukin-1

There is increasing evidence that Schwann cells of peripheral nerves may be able to function as accessory cells, interacting with the immune system in T cell-mediated immune responses, by expression of the major histocompatibility complex (MHC) class II molecules. In addition to MHC class II-associated presentation of antigen to T lymphocytes, the release of a co-stimulatory factor, interleukin-1 (IL-1), is an essential function of accessory cells for T cell activation. In this study, we investigated if Schwann cells were able to produce IL-1. Purified cultures of neonatal and adult rat Schwann cells were incubated with various stimulatory agents. Supernatants and cell lysates were collected from these cultures and IL-1 activity was assayed. Both neonatal and adult rat Schwann cells produced IL-1 activity in response to bacterial antigens and the IL-1 activity was often higher in the cell lysate than in the supernatant. When stimulated neonatal or adult rat Schwann cells were examined with antibody against IL-1, strong immunolabelling was seen intracellularly, but no IL-1 was detected on the cell surface. Since IL-1 plays an important role in the initiation of immune responses, these observations support the view that Schwann cells may function as antigen-presenting cells, thereby taking part in neuroimmunological responses within peripheral nerves.     

Rat microglial interleukin-3

Interleukin-3 (IL-3, multi-CSF) is a growth factor for a variety of hematopoietic progenitor cells. Recently, microglial cells, the resident macrophages of the central nervous system (CNS) have been shown to proliferate in the presence of IL-3 both in vivo and in culture. Data obtained from cultured astrocytes gave rise to the hypothesis that astrocytes synthesize the microglial growth factor. This is the first report identifying rat microglial cells themselves as a source of IL-3. Culture media conditioned by isolated microglia enhanced microglial proliferation above fresh media controls. IL-3 polypeptide was detected in both conditioned media (CM) and in microglial cells by Western blotting and immunoprecipitation. Furthermore, anti-IL-3 antibodies were able to inhibit microglial proliferation induced by conditioned media. mRNA^IL-3 was present in single microglial cells as revealed by in situ hybridization. Total RNA prepared from purified microglia yielded a single PCR amplification product. Identity of the PCR product was confirmed by Southern blot hybridization using a cDNA^IL-3 probe and by DNA sequencing. Expression of mRNA^IL-3 was observed in both absence and presence of lipopolysaccharide, a bacterial endotoxin, that commonly induces expression of inflammatory cytokines and inhibits microglial proliferation. It is concluded that IL-3 expression in microglial cells is an early marker of inflammatory events in the brain preceding the expression of other cytokines and most likely ensuring the recruitment of enhanced numbers of immunocompetent cells at sites of lesion. In the light of weak immune reactions in the brain, it is hypothesized that the expression of a characteristic T cell feature in monocyte-derived microglia may be a partial compensation of T cell functions in brain lesions.     

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.     

Cultures of astrocytes and microglia express interleukin 18

Interleukin 18 (IL-18 or interferon-gamma inducing factor) is a recently discovered pro-inflammatory cytokine and powerful stimulator of the cell-mediated immune response. IL-18 is produced by several sources including monocytes/macrophages, keratinocytes and the zona reticularis and zona fasciculata of the adrenal cortex. IL-18 occurs in brain but its cellular source in the CNS has never been investigated. The presence of IL-18 and its response to stimulation in the brain was tested with primary cultures of microglia, astrocytes and hippocampal neurons. IL-18 mRNA was present in astrocytes and microglia, but not in neurons. The endotoxin lipopolysaccharide (LPS) did not affect IL-18 in astrocytes, but LPS robustly increased IL-18 mRNA in microglia. IL-18 protein was constitutively expressed in astrocytes and induced in microglia by LPS. The levels of interleukin-1beta converting enzyme (ICE), an activating enzyme, and caspase 3 (CPP32), an inactivating enzyme, were assessed to investigate the presence of the appropriate processing enzymes in the cultured cells. ICE was present at constitutive levels in microglia and astrocytes suggesting that these cell types may produce and secrete matured IL-18. Active forms of CPP32 were not detectable in either cell type indicating the absence of a degradative pathway of IL-18. The present results demonstrate that microglia and astrocytes are sources of brain IL-18 and add a new member to the family of cytokines produced in the brain.     

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.     

Rat microglial cells secrete predominantly the precursor of interleukin-1beta in response to lipopolysaccharide

Little is known on the forms of interleukin-1beta (IL-1beta) that are produced by microglial cells in the nervous system. Mixed glial cell cultures of rats produced IL-1beta in response to lipopolysaccharide (LPS). Using Western blot, pro-IL-1beta was found to be localized both intracellularly and in the supernatant, whereas mature IL-1beta was found only in the supernatant but in lower quantities than pro-IL-1beta. Immunocytochemistry confirmed that microglial cells are the exclusive source of IL-1beta. Blockade of the IL-1beta-converting enzyme (ICE) by Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO) decreased the levels of mature IL-1beta but had no effect on pro-IL-1beta. Release of pro-IL-1beta was not associated with cell death nor with the extracellular release of ICE. Using gelatin zymography, glial cells were found to express constitutive matrix metalloproteinases (MMP) in the form of MMP-2. Exposure to LPS induced MMP-9 expression in a time-dependent manner similar to the pro-IL-1beta expression profile. MMP activation and inhibition experiments indicated a possible role of MMPs in the cleavage of pro-IL-1beta but not in the generation of mature IL-1beta. Microglial cells share with macrophages the ability to release large amounts of pro-IL-1beta of which the extracellular role remains to be determined.     

Interferon-gamma induced IA antigen expression on cultured neuroglial cells and brain macrophages from rat spinal cord and cerebrum.

The inducibility of major histocompatibility complex class II (Ia) antigens on glial cells of the brain suggests that neuroglia have immunoregulatory functions within the central nervous system (CNS), i.e., recognition and presentation of antigens. The aim of the present study was to investigate rat recombinant-interferon-gamma (r-IFN-gamma) induced Ia antigen expression in rat cerebral cultures containing type-1 astrocytes and macrophages, and in rat spinal cord cultures enriched in type-2 astrocytes or oligodendrocytes. We compared induction of Ia antigen expression in glial cell cultures derived from Lewis rats, which are very susceptible to experimental allergic encephalomyelitis (EAE), with those from Wistar rats, which are but modestly EAE susceptible. After 5 days in culture we found in Wistar rat type-1 astrocyte-enriched cultures that Ia antigens were expressed by 19% of the astrocytes, whereas we found that in Lewis rat type-1 astrocyte cultures a considerably higher number of astrocytes expressed Ia antigens (53%). However, no significant difference were found in Ia antigen expression between type-2 astrocytes derived from Wistar rat spinal cord (49%) and Lewis rat type-2 astrocytes (56%). In contrast, in oligodendrocyte-enriched cell cultures derived from either Lewis or Wistar rats no Ia antigen expression was found. Interestingly, we found in type-1 astrocyte-enriched cerebral cultures a large number (approx. 46% of the cells) of brain macrophages (amoeboid microglia), all expressing Ia antigens after treatment with r-IFN-gamma.     

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.     

Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures

The effects of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N-methyl-D-aspartate (NMDA) and treated with IL-1beta (6 ng/mL) or IL-1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL-1beta did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA-lesioned OHSCs with IL-1beta exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA-lesioned cultures with IL-1ra significantly attenuated NMDA-induced neuronal damage and reduced the number of microglial cells, whereas application of IL-1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL-1beta directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL-1beta induces microglial activation but is not neurotoxic per se; (iii) IL-1beta enhances excitotoxic neuronal damage and microglial activation and (iv) IL-1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.     

Neuroprotective actions of endogenous interleukin-1 receptor antagonist (IL-1ra) are mediated by glia

The pro-inflammatory cytokine interleukin-1 (IL-1), contributes to neuronal inflammation and cell death induced by ischemia, excitotoxicity, or trauma, while administration of IL-1 receptor antagonist (IL-1ra) reduces neuronal injury. The aim of the present study was to test the hypothesis that endogenous IL-1ra is neuroprotective in vivo and in vitro, and to identify its mechanism of actions. Mice lacking IL-1ra (IL-1ra knock-out (KO]) exhibited a dramatic increase in neuronal injury (3.6-fold increase in infarct size) induced by transient cerebral ischemia compared to wild-type (WT) animals. Basal cell death of cultured cortical neurons from WT and IL-1ra KO was identical, and treatment with NMDA or AMPA (20 microM) increased cell death to the same extent in WT and IL-1ra KO neurons. However, basal and NMDA- or AMPA-induced cells death was significantly higher in glial-neuronal co-cultures from IL-1ra KO than from WT mice. We further showed that pure microglial cultures, but not pure astrocytes cultures, released IL-1ra in response to treatment with conditioned medium from NMDA- or AMPA-treated primary neurons. These results demonstrate that endogenous IL-1ra produced by microglia is neuroprotective in cerebral ischemia or excitotoxicity.     

The effect of CXCL1 on human fetal oligodendrocyte progenitor cells

Chemokine CXCL1 is abundantly present in proliferative zones during brain development and in regions of remyelination, suggesting that it influences development of oligodendrocyte progenitors (OPC) in these regions. We studied in vitro the effects and possible mechanisms by which CXCL1 acts on human fetal OPC. In organotypic slice cultures of human fetal cortical ventricular/subventricular (VZ/SVZ) zones, blocking of CXCL1 signaling reduced significantly the proliferation of OPC. Moreover, exogenously added CXCL1 induced increase of OPC proliferation. Treatments of purified OPC cultures and cell depletion experiments demonstrated that this effect of CXCL1 was mainly indirect, mediated through astrocytes. We identified that CXCL1 acted through the extracellular signal regulated kinase (ERK1/2) pathway, activated primarily in astrocytes. In vitro, astrocytes stimulated with CXCL1 released several cytokines, but only the release of interleukin-6 (IL-6) was completely blocked by inhibition of ERK1/2 pathway. When released IL-6 was neutralized in slices, a decrease in OPC proliferation was demonstrated, while addition of IL-6 was able to return OPC proliferation in astrocyte-depleted slices to the control level. These results suggest that in the human fetal brain CXCL1 promotes proliferation of early OPC, acting in part through an ERK1/2-dependent pathway and release of IL-6 from astrocytes.