The role of microglia and astrocytes in CNS immune surveillance and immunopathology.
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Animals↗
Biomedical subjects
F Aloisi
Publications and source records attributed to F Aloisi.
HIV-1 Nef alters the expression of betaII and epsilon isoforms of protein kinase C and the activation of the long terminal repeat promoter in human astrocytoma cells.
In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.
AIDS Dementia Complex↗
Human myelin basic protein specific T cell lines display differential cytotoxicity against astrocytes, but are consistently cytotoxic against monocytes.
In experimental allergic encephalomyelitis (EAE) myelin basic protein (MBP) specific T cells differ in their encephalitogenic potential. To investigate the functional diversity of human MBP specific T cell lines, we analysed their cytotoxic activity against human astrocytes and monocytes. Five out of 14 MBP specific T cell lines killed astrocytes in the presence of MBP. Nevertheless, all lines lysed blood derived monocytes. T cell lines that lysed astrocytes efficiently in the presence of MBP, recognized peptide aa 80-99/86-105 in context with HLA-DRB5 * 0101, peptide aa 50-69/61-83 in context with HLA-DRB1 * 1501 and peptides aa 139-153, and aa 148-162 in context with HLA-DRB1 * 0101. There was no correlation of MBP-mediated lysis of astrocytes with TCR-Vbeta usage, HLA-restriction and the production of tumor-necrosis-factor-alpha (TNF-alpha), lymphotoxin (LT) and interferon-gamma (IFN-gamma). Different lysis of astrocytes, however, revealed a functional heterogeneity of MBP specific T cells, which was not observed by using monocytes as targets.
Astrocytes↗
Microglia are more efficient than astrocytes in antigen processing and in Th1 but not Th2 cell activation.
Microglia and astrocytes, two glial cell populations of the central nervous system, present Ag and stimulate T cell proliferation, but it is unclear whether they preferentially activate Th1 or Th2 responses. We have investigated the efficiency of microglia and astrocytes in the presentation of OVA peptide 323-339 or native OVA to Th1 and Th2 cell lines from DO11.10 TCR transgenic mice. Upon stimulation with IFN-gamma, microglia express MHC class II molecules, CD40, and ICAM-1 and efficiently present OVA 323-339, leading to T cell proliferation and production of IL-2 and IFN-gamma by Th1 and of IL-4 by Th2 cells. IFN-gamma-treated astrocytes, which express MHC class II and ICAM-1, present OVA 323-339 less efficiently to Th1 cells but are as efficient as microglia in inducing IL-4 secretion by Th2 cells. However, astrocytes are much less potent than microglia in presenting naturally processed OVA peptide to either T cell subset, indicating inefficient Ag processing. The capacity of astrocytes and microglia to stimulate Th1 and Th2 cells depends on their MHC class II expression and does not involve ICAM-1, B7-1, or B7-2 molecules. However, CD40-CD40L interactions contribute to Th1 activation by microglia. These data suggest that microglia may play a role in the activation of Th1 and Th2 cells, whereas astrocytes would restimulate mainly Th2 responses in the presence of appropriate peptides. This differential capacity of brain APC to restimulate Th1 and Th2 responses may contribute to the reactivation and regulation of local inflammatory processes during infectious and autoimmune diseases.
Animals↗
Interferon gamma gene expression in rat central nervous system glial cells.
The expression of interferon gamma (TFN-gamma) mRNA in purified cultures of glial cells and leptomeningeal fibroblasts from the neonatal rat brain was analysed using semi-quantitative reverse transcriptase-polymerase chain reaction. IFN-gamma specific transcripts were clearly detected in rat astrocytes and were upregulated after treatment with IFN-gamma itself or cycloheximide. In microglial cells, IFN-gamma transcripts were barely detectable in basal conditions and were upregulated by lipopolysaccharide and, to a lesser extent, by IFN-gamma or cycloheximide. IFN-gamma mRNA was neither detected not inducible in oligodendrocytes and leptomeningeal cells. The demonstration that IFN-gamma is expressed in astrocytes and microglia widens the putative role of these cells in local immunoregulation and antiviral responses.
Animals↗
Virological and molecular parameters of HIV-1 infection of human embryonic astrocytes.
Two different strains of HIV-1, the lymphotropic HIV-IIIB and the monocytotropic HIV-Ba-L, were able to infect tertiary cultures of astrocytes established from the human embryonic brain. The infection did not require contact with infected cells, as astrocytes were exposed to infectious cell-free supernatants. Except for an early transient peak of p24 consistently observed after infection with HIV-Ba-L, the infection of astrocytes appeared to be nonproductive. However, viral production was always observed when infected astrocytes were cocultured with permissive cells (CEM-SS or monocytes). To exclude the possibility that undetectable levels of virus are chronically produced by astrocytes, we exposed permissive cells to p24 negative supernatants taken from infected cultures. In such conditions permissive cells were never infected. Infection of astrocytes by HIV-1 was further supported by the finding that provirus persisted in these cells. Indeed, by a nested PCR, we detected HIV-1 DNA even one month after infection. Moreover, at the transcriptional level we observed expression of the multiply spliced RNA (tat and nef primers). Noteworthy, this pattern of HIV-1 expression did not change appreciably when astrocytes were pretreated and cultivated in the presence of IL-1 beta. Altogether, our data support the concept that astrocytes may play a role in the spread of HIV-1 infection within the brain and in the pathogenesis of neuro-AIDS.
Astrocytes↗
Regulation of prostanoid synthesis in microglial cells and effects of prostaglandin E2 on microglial functions.
Brain prostanoid levels are normally low but can increase after ischemia and during inflammatory and infectious diseases. High prostanoid levels can affect brain function in several ways. In particular, prostaglandin E2 (PGE2) might exert both immunodepressive and proinflammatory actions. The present short review focuses on the regulation of prostanoid synthesis in microglial cultures and on the possible role of PGE2 in the down-regulation of microglial activation induced by lipopolysaccharide (LPS). Our studies were carried out using purified mouse or rat microglial cultures. LPS induced a dose-dependent expression of the inducible isoform of cyclooxygenase (COX-2), both in neonatal and adult microglial cultures. In the latter, the inducibility of COX-2 increased with time in culture, paralleling the acquisition of a more 'activated' microglial phenotype, and appeared to account for the time-dependent increase in the PGE2/TXB2 production ratio. The LPS-induced COX-2 expression and prostanoid production were down-regulated by potentially neurotoxic agents, such as nitric oxide (NO), the proinflammatory cytokine IFN-gamma (which acted both directly and indirectly, through its NO-inducing activity) and the HIV regulatory protein tat. On the other hand, COX-2 expression was up-regulated by the macrophage-deactivating cytokine TGF-beta 1, by exogenous PGE2 itself, which acted through EP2 receptors linked to cyclic AMP generation, and by non steroidal anti-inflammatory drugs. Interestingly, PGE2 utilized the same EP2 receptor-mediated signal transduction mechanism to down-regulate the expression of the inducible NO synthase and the production of NO. Largely, but not exclusively, through its effect on cyclic AMP, PGE2 can also: i) depress the expression of major histocompatibility complex class II antigens and of the costimulatory molecule B7-2; ii) down-regulate TNF and up-regulate IL-10 microglial production; iii) inhibit microglial IL-12 secretion. These observations, together with literature data on in vivo models of central nervous system (CNS) diseases, suggest a neuroprotective role of PGE2 in pathological conditions.
Animals↗
Synergistic stimulation of MHC class I and IRF-1 gene expression by IFN-gamma and TNF-alpha in oligodendrocytes.
In order to understand the molecular basis of the synergistic action of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) on rat oligodendrocyte development, we studied some aspects of the signalling pathways involved in the regulation of the major histocompatibility complex (MHC) class I and the interferon regulatory factor 1 (IRF-1) gene expression. Two well-defined inducible enhancers of the MHC class I gene promoter, the MHC class I regulatory element (MHC-CRE) and the interferon consensus sequence (ICS), were analysed. Neither IFN-gamma nor TNF-alpha was capable of inducing MHC-CRE binding activity when administrated alone. Following the exposure of oligodendrocytes to IFN-gamma, TNF-R1 expression was transcriptionally induced by the binding of signal transducer and activator of transcription (STAT-1) homodimers to the IFN-gamma activated site (GAS) present in the gene promoter. The upregulation of TNF-R1 allowed TNF-alpha to induce the binding of nuclear factor-kappaB (NF-kappaB) to the MHC-CRE site. With respect to ICS element, IFN-gamma induced IRF-1 binding, that was further enhanced upon co-treatment with TNF-alpha. The existence of a synergism between IFN-gamma and TNF-alpha in stimulating IRF-1 expression at the transcriptional level was supported by IRF-1 promoter analysis: IFN-gamma directly induced the binding of STAT-1 homodimers to the GAS element, while NF-kappaB binding to the kappaB sequence was activated by TNF-alpha only after IFN-gamma treatment. This transcriptional regulation of IRF-1 gene by IFN-gamma and TNF-alpha was confirmed at the mRNA level. The synergism demonstrated in the present study highlights the importance of cytokine interactions in magnifying their biological effects during brain injury and inflammation.
Animals↗
IL-12 production by central nervous system microglia is inhibited by astrocytes.
IL-12, a 75-kDa heterodimeric cytokine composed of two chains (p35 and p40), is a central regulator of immune responses and may be implicated in the pathogenesis of certain inflammatory diseases of the central nervous system (CNS). We have examined the capacity of two CNS APC, microglia and astrocytes, to produce IL-12 upon stimulation with cytokines, LPS, or a neurotropic virus. In purified microglial cultures from neonatal mouse brains, expression of IL-12 p35 and p40 mRNA is induced by LPS and is stimulated maximally by combined IFN-gamma/LPS treatment, as detected by semiquantitative reverse-transcriptase PCR. LPS induces secretion of IL-12 p40, but not of IL-12 p75, as detected by specific ELISA. Combined stimulation with IFN-gamma/LPS enhances IL-12 p40 secretion and induces IL-12 p75 secretion by microglia. Conversely, mouse astrocytes do not express IL-12 p35 mRNA and do not secrete IL-12 p75 under any condition tested. IL-12 production by activated microglia is inhibited by IL-10, PGE2, and cAMP-elevating agents. Coculture of microglia with astrocytes or exposure of microglia to astrocyte-conditioned medium also results in marked reduction of IL-12 p75 and p40 secretion by IFN-gamma/LPS-stimulated microglia, indicating a regulatory role of astrocytes on IL-12 production. This novel mechanism of IL-12 regulation may play an important role in the control of immune responses during infection or in Th1 cell-mediated autoimmune diseases of the CNS.
Animals↗
Functional characterization of substance P receptors on cultured human spinal cord astrocytes: synergism of substance P with cytokines in inducing interleukin-6 and prostaglandin E2 production.
Following brain injury, astrocytes express receptors for cytokines and neuropeptides and secrete several regulatory mediators that have a well established role in inflammation, immunity, and tissue development or repair. To elucidate the role of substance P (SP), a neurotransmitter peptide of the tachykinin family, in inducing astrocyte secretory activities, we have examined the expression of SP receptors and the functional consequences of their activation in cultured astrocytes from the human embryonic brain or spinal cord. Radioligand binding studies revealed that only one type of SP receptors, the high affinity NK-1 receptor, was present on human astrocytes and that spinal cord astrocytes expressed about 6 times as many SP binding sites as brain astrocytes. Following SP treatment, a substantial inositol phosphate formation was observed in spinal cord astrocytes only. Stimulation of spinal cord astrocytes with SP alone did not induce secretion of cytokines [interleukin-6 (IL-6), granulocyte-macrophage-CSF, macrophage chemoattractant protein-1 or leukemia inhibitory factor] or prostaglandin E2 (PGE2). Interestingly, however, SP selectively potentiated the inducing effect of IL-1beta on IL-6 and PGE2 secretion by spinal cord astrocytes without affecting the IL-1-beta-evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor-alpha (TNF-alpha) on IL-6 and PGE2 secretion and that of transforming growth factor-beta on PGE2 secretion. These results suggest that SP can enhance immunoregulatory and neurotrophic astroglial functions mediated by IL-6 and PGE2 by acting in concert with a set of cytokines whose cerebral expression has been reported during development and in a variety of diseases.
Astrocytes↗
Analysis of B7-1 and B7-2 costimulatory ligands in cultured mouse microglia: upregulation by interferon-gamma and lipopolysaccharide and downregulation by interleukin-10, prostaglandin E2 and cyclic AMP-elevating agents.
Recent evidence indicates that membrane-bound costimulatory molecules of the B7 family are important for T-cell activation and are upregulated in IFN gamma-stimulated human microglia and in multiple sclerosis active lesions. In this study we have performed a detailed analysis of B7-1 and B7-2 expression and regulation in cultured mouse glial cells using immunocytochemical and semi-quantitative reverse transcriptase-polymerase chain reaction techniques. In an immortalized mouse microglial cell line (BV-2), expression of B7-1 and B7-2 was enhanced by interferon-gamma (IFN gamma). IFN gamma was a weak inducer of B7-2 mRNA and immunoreactivity in microglia primary cultures obtained from the neonatal mouse brain, whereas lipopolysaccharide, tumour necrosis factor-alpha, colony-stimulating factors and interleukin-1 beta did not affect microglial B7-2 expression. Combined IFN gamma and lipopolysaccharide treatment very effectively upregulated the B7-2 gene expression and immunoreactivity in microglia, but not in astrocytes. In both glial cell types, expression of B7-1 was not induced by any of the above agents. Among known microglia/macrophage deactivators, interleukin-10, prostaglandin E2 and cAMP-elevating agents, but not transforming growth factor-beta 1 and interleukin-4, inhibited B7-2 transcripts and immunoreactivity in IFN gamma/LPS-stimulated microglia, thus suggesting possible paracrine and autocrine mechanisms for regulating the expression of this important T-cell costimulatory signal in the brain.
Animals↗
Targeting IL-12, the key cytokine driving Th1-mediated autoimmune diseases.
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Animals↗
Cytokine-regulated expression of platelet-derived growth factor gene and protein in cultured human astrocytes.
To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF, AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-beta 1 (TGF-beta 1) or tumor necrosis factor-alpha (TNF-alpha); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1beta (IL-1beta) by itself had little or no effect but synergized with TGF-beta 1 in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B- chains mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-beta 1, TNF-alpha, TNF-alpha/TGF-beta, or IL-1-beta/TGF-beta 1, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-beta 1, TNF-alpha, and IL-1 beta are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.
Astrocytes↗
The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions.
B7 is a costimulatory molecule which is expressed on antigen-presenting cells and which plays a pivotal role in T cell activation and proliferation. To elucidate mechanisms regulating intracerebral immune responses, expression of B7 was examined in cultured microglial cells and in brain tissue from control and multiple sclerosis patients. Using immunocytochemical and polymerase chain reaction techniques, we show that B7 was expressed in cultured microglial cells from the human embryonic brain. Microglia also bound the soluble form of the B7 receptor CTLA-4 (CTLA-4-Ig). B7 gene expression and binding of anti-B7 antibodies and CTLA-4-Ig increased after treatment with interferon-gamma. B7 was not inducible in human astrocytes. Human microglia expressed other costimulatory molecules, such as intercellular adhesion molecule-1, LFA-1 and LFA-3. In sections of multiple sclerosis brains, B7 immunoreactivity was detected on activated microglia and infiltrating macrophages within active lesions. In chronic lesions, only perivascular cells were stained. B7 immunoreactivity was undetectable in sections from Alzheimer's disease or normal brain tissue. These data suggest that B7 may be involved in T cell activation and lesion development in multiple sclerosis and that the regulated expression of B7 on microglia may contribute to the local stimulation of T cell proliferation and effector functions.
Acute Disease↗
Regulation of leukemia inhibitory factor synthesis in cultured human astrocytes.
We have examined the ability of human astrocytes to synthesize and secrete leukemia inhibitory factor (LIF), which is a multifunctional cytokine that controls cell proliferation and differentiation in many tissues, including the nervous system. Astrocyte-enriched cultures, prepared from 8- to 9-wk-old embryonic brains, expressed LIF mRNA and secreted LIF protein. LIF synthesis was significantly increased by the cytokines IL-1 beta, TNF-alpha, and TGF-beta 1, but not by IFN-gamma, IL-6, or LPS. No major differences in basal and cytokine-inducible LIF production were detected among astrocyte populations obtained from different brain areas. LIF synthesis was lower in serum-free than in serum-containing astrocyte cultures. A role for protein kinase C in the regulation of astrocyte LIF production was suggested by the findings that phorbol esters induced both LIF mRNA and protein and that the cytokine-induced LIF increase was partially antagonized by relatively selective inhibitors of protein kinase C such as H7 and staurosporine. Human leptomeningeal fibroblasts also expressed LIF gene and protein. Astrocytes produced LIF and responded to cytokines with increased LIF synthesis only after being subcultured, and not when grown in primary cultures in close contact with neurons. Our findings suggest that in vivo induction of astrocyte LIF secretion might occur in pathologic conditions as a consequence of both alterations of neuronal-glial interactions and a local increase in the level of inflammatory cytokines.
Astrocytes↗
Human astrocytes are only partially competent antigen presenting cells. Possible implications for lesion development in multiple sclerosis.
Highly purified astrocyte cultures from human embryonic brain were examined for their capacity to present antigen to human leukocyte antigen (HLA) class II compatible, cytolytic CD4+ T lymphocytes. Most astrocytes constitutively expressed HLA class I products and LFA-3 (CD58). Constitutive expression of HLA class II, LFA-1 alpha (CD11a) and ICAM-1 (CD54) was lower and varied among different cultures, while LFA-2 (CD2) was absent. IFN-gamma alone or in combination with TNF-alpha strongly enhanced expression of HLA class I, HLA-DR, -DP, -DQ, LFA-1 alpha and ICAM-1, but did not affect expression of LFA-2 (CD2) and LFA-3 (CD58). TNF-alpha alone induced only HLA class I and ICAM-1, but not HLA class II or LFA-1 alpha. Cytokine treated, but not untreated astrocytes were able to present protein (auto-)antigens to specific T lymphocyte lines. Astrocytes expressing appropriate major histocompatibility complex class II products were lysed by CD4+ T cells specific for myelin basic protein or tetanus toxoid. The lytic response was antigen dose dependent and HLA-DR restricted. It could be blocked by antibodies against HLA-DR determinants and against the adhesion molecules LFA-1 alpha and ICAM-1. In remarkable contrast to their susceptibility to T cell lysis, antigen presenting astrocytes were not only completely unable to induce T cell proliferation but even inhibited proliferation. The results indicate that, although human astrocytes have the potential to present protein antigens to CD4+ T cells, they do not induce the co-stimulatory factors required to trigger the complete T cell activation programme.
Antigen-Presenting Cells↗
Multiple sclerosis. Immunomodulatory effects of human astrocytes on T cells.
Using a human culture system, we have previously shown that interferon-gamma-and tumour necrosis factor-alpha-stimulated astrocytes are capable of presenting antigens to T lymphocytes, but do not support antigen-dependent T cell proliferation. To gain further insight into the mechanisms involved in the local regulation of intracerebral T cell responses, we have investigated the effects of astrocytes on T cell proliferation induced by peripheral blood-derived mononuclear cells (PBMC). We found that astrocytes derived from human embryonic brain were able to suppress PBMC-dependent proliferation of antigen-specific, CD4+ T cell lines. Interferon-gamma production by PBMC-stimulated T cells was also suppressed by astrocytes, and this inhibition was seen as early as 6 h after initiation of co-culture. The inhibitory effect was observed in the presence of both HLA matched and mismatched astrocytes and was mediated by astrocyte-derived soluble factor(s) rather than by direct cellular contact. Inhibition of T cell proliferation was incompletely reverted by indomethacin, suggesting that prostaglandins were partially involved in the suppressive effect. The cytotoxic mediator nitric oxide was not involved in astrocyte-mediated inhibition. These observations led us to further investigate the contribution of other mediators known to down-regulate inflammatory processes. Our astrocyte cultures did not synthesize interleukin (IL)-4 or IL-10, whereas they secreted both the latent and active forms of transforming growth factor-beta 2. Transforming growth factor-beta was, however, found not to participate in astrocyte-induced inhibition in vitro. The inhibitory properties of human astrocytes may contribute to confinement of inflammatory lesions in multiple sclerosis and other inflammatory diseases of the central nervous system.
Astrocytes↗
Norepinephrine and vasoactive intestinal peptide induce IL-6 secretion by astrocytes: synergism with IL-1 beta and TNF alpha.
Resident glial cells and invading inflammatory cells are responsible for cytokine production within the brain. Astrocytes are known to secrete a variety of cytokines upon stimulation with cytokines themselves, protein kinase C activators, bacterial or viral constituents. Astrocytes also have surface receptors for a wide number of neurotransmitters and neuropeptides and some of these substances affect astrocyte immune functions, such as major histocompatibility complex (MHC) class II antigen expression. To elucidate the activity of neuromediators on cytokine secretion by glial cells, we studied the secretion of interleukin-6 (IL-6) by cultured rat astrocytes after incubation with various neurotransmitters and neuropeptides. Norepinephrine (NE) and the beta-adrenergic agonist isoproterenol (IPT) induced IL-6 secretion in a dose-dependent fashion. NE effect was predominantly mediated by beta 2-adrenergic receptors with a minor contribution of alpha 1-adrenergic receptors. The induction of IL-6 release by dibutyryl-cAMP indicated that IL-6 secretion secondary to beta 2-adrenergic receptor activation probably occurs through cAMP signalling pathways. Vasoactive intestinal peptide (VIP) was the sole neuropeptide able to induce IL-6 secretion. NE and VIP promoted IL-6 mRNA synthesis and both substances synergized with interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) in inducing IL-6 release. Our findings provide further evidence that neurons modulate astrocyte cytokine production and thereby regulate central nervous system immune functions.
Animals↗