Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains.

There is evidence that the cytokine tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of neurological autoimmune diseases such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). TNF-alpha exerts damaging effects on oligodendrocytes, the myelin-producing cell of the central nervous system (CNS), and myelin itself. We have recently demonstrated TNF-alpha expression from astrocytes induced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), and interleukin 1 beta (IL-1 beta). Astrocytes secrete TNF-alpha in response to LPS alone, and can be primed by IFN-gamma to enhance LPS-induced TNF-alpha production. IFN-gamma and IL-1 beta, cytokines known to be present in the CNS during neurological disease states, do not induce TNF-alpha production alone, but act synergistically to stimulate astrocyte TNF-alpha expression. Inbred Lewis and Brown-Norway (BN) rats differ in genetic susceptibility to EAE, which is controlled in part by major histocompatibility complex (MHC) genes. We examined TNF-alpha gene expression by astrocytes derived from BN rats (resistant to EAE) and Lewis rats (highly susceptible). Astrocytes from BN rats express TNF-alpha mRNA and protein in response to LPS alone, yet IFN-gamma does not significantly enhance LPS-induced TNF-alpha expression, nor do they express appreciable TNF-alpha in response to the combined stimuli of IFN-gamma/IL-1 beta. In contrast, astrocytes from Lewis rats express low levels of TNF-alpha mRNA and protein in response to LPS, and are extremely responsive to the priming effect of IFN-gamma for subsequent TNF-alpha gene expression. Also, Lewis astrocytes produce TNF-alpha in response to IFN-gamma/IL-1 beta. The differential TNF-alpha production by astrocytes from BN and Lewis strains is not due to the suppressive effect of prostaglandins, because the addition of indomethacin does not alter the differential pattern of TNF-alpha expression. Furthermore, Lewis and BN astrocytes produce another cytokine, IL-6, in response to LPS, IFN-gamma, and IL-1 beta in a comparable fashion. Peritoneal macrophages and neonatal microglia from Lewis and BN rats are responsive to both LPS and IFN-gamma priming signals for subsequent TNF-alpha production, suggesting that differential TNF-alpha expression by the astrocyte is cell type specific. Taken together, these results suggest that differential TNF-alpha gene expression in response to LPS and IFN-gamma is strain and cell specific, and reflects both transcriptional and post-transcriptional control mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-6 production by astrocytes: induction by the neurotransmitter norepinephrine.

Astrocytes contribute to the immunocompetence of the central nervous system (CNS) via their expression of class II major histocompatibility complex (MHC) antigens and the production of inflammatory cytokines such as interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Of these cytokines, IL-6 is of particular interest because one of its many immune and inflammatory actions is the promotion of immunoglobulin synthesis, and it is thought that IL-6 expression within the brain exacerbates autoimmune diseases of the CNS, which are marked by local immunoglobulin production. Several stimuli induce astrocyte IL-6 expression, including such inducible endogenous factors as IL-1 beta and TNF-alpha. We have investigated the possibility that a constitutively present endogenous factor, the neurotransmitter norepinephrine (NE), can induce astrocyte IL-6 production. We report that NE induces both IL-6 mRNA and protein in primary neonatal rat astrocytes, with optimal induction at 10 microM. IL-6 protein induction by NE is comparable to that seen with IL-1 beta or TNF-alpha, and NE synergizes with these cytokines for a ten-fold enhanced effect. In contrast to astrocytes, microglia are relatively unresponsive to NE, IL-1 beta and TNF-alpha for IL-6 production. Experiments with the beta-adrenergic receptor agonist isoproterenol, and alpha and beta-adrenergic receptor antagonists (propranolol, phentolamine, atenolol, and yohimbine) indicate that beta 2 and alpha 1-adrenergic receptors are involved in NE induction of astrocyte IL-6 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction and regulation of interleukin-6 gene expression in rat astrocytes.

Cells that produce interleukin-6 (IL-6) require the presence of signaling molecules since this cytokine is not normally constitutively expressed. It is now established that astrocytes produce IL-6; however, the precise inducing molecules and the kinetics of their action have not yet been clearly identified. In the current study, we show that either interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) exert a strong inducing signal for IL-6 in primary rat astrocytes. When the two cytokines are added together the response is synergistic, suggesting that each cytokine may induce IL-6 gene expression by different pathways. Interferon-gamma (IFN-gamma) does not affect IL-6 expression although if it is added in conjunction with IL-1 beta, an augmented induction of IL-6 occurs. In addition to the cytokines, bacterial lipopolysaccharide (LPS) and the calcium ionophore, A23187, induce IL-6 expression. IL-6 expression can be blocked by the glucocorticoid analogue, dexamethasone. IL-6 induction by LPS/Ca2+ ionophore is more sensitive to the suppressive effects of dexamethasone than is IL-6 induction by TNF-alpha/IL-1 beta. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased levels of IL-6 mRNA in both unstimulated and stimulated astrocytes, indicating that ongoing protein synthesis is not required for astrocyte IL-6 gene expression. We propose that astrocyte-produced IL-6 may have a role in augmenting intracerebral immune responses in neurological diseases such as multiple sclerosis (MS), AIDS dementia complex (ADC), and viral infections. These diseases are characterized by infiltration of lymphoid and mononuclear cells into the central nervous system (CNS), and intrathecal production of immunoglobulins. IL-6 may act to promote terminal differentiation of B cells in the CNS, leading to immunoglobulin synthesis.

Signal transduction pathways mediating astrocyte IL-6 induction by IL-1 beta and tumor necrosis factor-alpha.

One immune function of astrocytes is IL-6 production. Synthesis of IL-6 within the central nervous system (CNS) can produce several different responses, acting on glia, neurons, and lymphocytes infiltrating brain tissue, and some of these effects are associated with CNS autoimmune disease. IL-6 gene expression in astrocytes is regulated by cytokines, infectious agents, neuropeptides, and neurotransmitters, and most of these stimuli interact synergistically. To examine the integration of these diverse factors in the control of IL-6 production, we have studied the involvement of underlying signal transduction processes using neonatal rat astrocytes. We have focused on signal transduction related to the stimulation of IL-6 gene expression by IL-1 beta and TNF-alpha. Our results indicate that stimuli related to protein kinase C (PKC), such as PMA and calcium ionophore A23187, increase IL-6 expression, whereas pharmacologic inhibitors of PKC inhibit IL-6 induction by IL-1 beta and TNF-alpha. Furthermore, both IL-1 beta and TNF-alpha stimulate PKC activity in astrocytes. Stimulators of the cAMP pathway, such as cholera toxin, forskolin, and dibutyryl cAMP, also induced astrocyte IL-6 gene expression. However, inhibition of the cAMP pathway effector, protein kinase A, did not reduce the induction of astrocyte IL-6 gene expression in response to IL-1 beta or TNF-alpha, and an ELISA for cAMP detected only very small increases in cAMP synthesis in response to these cytokines. These data suggest that although cAMP does activate astrocyte IL-6 gene expression, it is the PKC pathway that plays a primary role in the stimulation of astrocyte IL-6 gene expression by IL-1 beta and TNF-alpha.

Interferon-gamma-induced astrocyte class II major histocompatibility complex gene expression is associated with both protein kinase C activation and Na+ entry.

Astrocytes can be induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. This study was undertaken to elucidate the intracellular signaling pathways involved in IFN-gamma induction of class II MHC. We examined the effects of Na+/H+ antiporter and protein kinase C (PKC) inhibitors on class II expression and Na+ influx in astrocytes. We found that amiloride and ethyl isopropylamiloride, inhibitors of Na+/H+ exchange, blocked IFN-gamma-induced class II gene expression. IFN-gamma stimulated Na+ influx, and this increased influx was inhibited by amiloride. Treatment of astrocytes with the PKC inhibitor H7 also blocked the increase in Na+ uptake induced by IFN-gamma, indicating that IFN-gamma-induced PKC activation is required for subsequent Na+ influx. IFN-gamma treatment produced an increase of total PKC activity, which was associated with a rapid translocation of PKC activity from cytosolic to particulate fraction. H7 and another PKC inhibitor, staurosporine, inhibited IFN-gamma-induced class II gene expression. However, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, a potent PKC activator, did not affect class II expression. Taken together, our data indicate that both IFN-gamma-induced PKC activation and Na+ influx are required for class II MHC expression in astrocytes but that activation of PKC alone is not sufficient for ultimate expression of this gene.