S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.     

Aging and glial responses to lipopolysaccharide in vitro: greater induction of IL-1 and IL-6, but smaller induction of neurotoxicity

Glial activation during aging was analyzed in primary glia cultured from brain regions sampled across the life span. An initial study showed that phenotypes of activated astrocytes and microglia from aging rat cerebral cortex persisted in primary cultures (Neurobiol. Aging 19 (1998), 97). We extend these findings by examining effects of age on the activation of glial cultures from adult rat brain in response to lipopolysaccharide (LPS), an inflammatory stimulus. Mixed glia from F344 male rats, aged 3 and 24 months, cultured from cerebral cortex (Cx), hippocampus (Hc), and striatum (St), were assayed for cytokines implicated in Alzheimer's disease: IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Regional differences across all age groups included consistently lower expression of these cytokines in glia derived from Cx than Hc and St. Aging increased basal IL-6 mRNA and secretion by >or=3-fold in glia from Cx and Hc. Aging also increased LPS-induced IL-1 and IL-6 in Hc more than in Cx, whereas no significant effects of age were seen in St-derived glial cytokines. TNF-alpha secretion did not differ by donor age (basal or LPS-induced). Nitric oxide production by microglia from Cx of aging brains showed a smaller induction in response to LPS, with proportionately less neurotoxicity. Thus, glial activation during aging shows regional selectivity in cytokine expression, with opposite effects of aging on the increased inducibility of IL-1 and IL-6 vs the decreased production of nitric oxide.     

Cytokine regulation of CC and CXC chemokine expression by human astrocytes

Chemokines constitute a large family of secreted proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the central nervous system (CNS), are a source of chemokine production within diseased brain. As such, we have examined the production of chemokines by human astroglioma cell lines and primary human astrocytes treated with a variety of stimuli, including LPS, TNF-alpha, IFN-gamma and IL-1beta. In addition, IL-6 in conjunction with the soluble IL-6 receptor (sIL-6R), and hybrid IL-6 (H-IL-6), a highly active fusion protein of sIL-6R and IL-6, were tested for their ability to induce chemokine expression. The findings presented herein demonstrate that both human astroglioma cell lines and primary human astrocytes express the CXC chemokines IP-10 and IL-8 and the CC chemokines MCP-1 and RANTES in response to TNF-alpha and IL-1beta. IFN-gamma induced the expression of IP-10, but not of IL-8, MCP-1 or RANTES. Surprisingly, IL-6/sIL-6R and H-IL-6 had little or no effect on chemokine expression in these cells. The effect of TGF-beta on chemokine expression in human astroglioma cell lines and astrocytes was also examined. TGF-beta alone had little or no effect on RANTES, MCP-1 and IL-8 expression; however, TGF-beta synergized with TNF-alpha to enhance MCP-1 expression in both astroglioma cells and primary astrocytes. An inhibitory effect of TGF-beta on TNF-alpha and IL-1beta induced RANTES and IL-8 expression was observed in human astroglioma cells. In contrast, TGF-beta enhanced TNF-alpha and IL-1beta induction ofIL-8 production by human astrocytes. These findings document a complex pattern of chemokine regulation by the pleiotropic cytokine TGF-beta with both enhancing and inhibitory effects.     

S100B secretion is stimulated by IL-1β in glial cultures and hippocampal slices of rats: Likely involvement of MAPK pathway

S100B is an astrocyte-derived cytokine implicated in the IL-1β-triggered cytokine cycle in Alzheimer's disease. However, the secretion of S100B following stimulation by IL-1β has not been directly demonstrated. We investigated S100B secretion in cortical primary astrocyte cultures, C6 glioma cells and acute hippocampal slices exposed to IL-1β. S100B secretion was induced by IL-1β in all preparations, involving MAPK pathway and, apparently, NF-кB signaling. Astrocytes and C6 cells exhibited different sensitivities to IL-1β. These results suggest that IL-1β-induced S100B secretion is a component of the neuroinflammatory response, which would support the involvement of S100B in the genesis of neurodegenerative diseases.     

Leptomeningeal cells activate microglia and astrocytes to induce IL-10 production by releasing pro-inflammatory cytokines during systemic inflammation

The leptomeninges covering the surface of the brain parenchyma play the physical role at the cerebrospinal fluid-blood barrier. We report here that leptomeningeal cells may transduce peripheral proinflammatory signals to the central anti-inflammatory response through the activation of glial cells in the brain parenchyma. After adjuvant injection, both microglia and astrocytes in the cerebral cortex localized in the proximity of the leptomeninges were activated. The protein levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in the cortical extracts were significantly increased at different time after adjuvant injection. The TNF-alpha immunoreactivity was most prominent in the leptomeninges covering astrocytes. On the other hand, the IL-10 immunoreactivity was observed in both activated microglia and astrocytes localized along the leptomeninges. Cultured leptomeningeal cells covering the cerebral cortex released TNF-alpha which was significantly increased by lipopolysaccharide (LPS). Upon stimulation with LPS, cultured leptomeningeal cells also secreted interleukin-1beta and interleukin-6 with differential time-courses. When primary cultured rat astrocytes and microglia were treated with the conditioned medium of LPS-activated cultured leptomeningeal cells, the immunoreactivity of IL-10 was markedly increased. These observations strongly suggest that leptomeningeal cells release pro-inflammatory cytokines to activate both microglia and astrocytes during systemic inflammation. The activated astrocytes and microglia may in turn regulate anti-inflammatory response in the brain by providing IL-10.     

High glutamate decreases S100B secretion stimulated by serum deprivation in astrocytes

S100B is a calcium-binding protein expressed and secreted by astrocytes, playing a neurotrophic role in neighboring cells. A protective role of the S100B against glutamate-induced excitotoxicity has recently been proposed. We investigated S100B secretion in rat hippocampal astrocytes exposed to high concentrations of glutamate during serum deprivation (stimulated condition) or not (basal condition), for 30 min. Glutamate at 1 mM had no effect on basal secretion of S100B, but it decreased S100B secretion in serum-deprived astrocytes after 1 h. Secretion was inhibited by Rp-cAMPS or H89. In addition, serum deprivation was accompanied by a transitory increase of intracellular content of cAMP. Our results suggest that high levels of glutamate in a serum-deprived condition could impair S100B secretion from hippocampal astrocytes.     

In vitro S100B secretion is reduced by apomorphine: effects of antipsychotics and antioxidants

Astrocytes express dopamine receptors and respond to dopamine stimulation. However, the role of astrocytes in psychiatric disorders and the effects of antipsychotics on astroglial cells have only been investigated recently. S100B is a glial-derived protein, commonly used as a marker of astroglial activation in psychiatric disorders, particularly schizophrenia. We investigated S100B secretion in three different rat brain preparations (fresh hippocampal slices, C6 glioma cells and primary astrocyte cultures) exposed to apomorphine and antipsychotics (haloperidol and risperidone), aiming to evaluate, ex vivo and in vitro, whether dopamine activation and dopaminergic antagonists modulate astroglial activation, as measured by changes in the extracellular levels of S100B. The serum S100B elevation observed in schizophrenic patients is not reflected by the in vitro decrease of S100B secretion that we observed in hippocampal slices, cortical astrocytes and C6 glioma cells treated with apomorphine, which mimics dopaminergic hyperactivation. This decrease in S100B secretion can be explained by a stimulation of D2 receptors negatively coupled to adenyl cyclase. Antipsychotic medications and antioxidant supplementation were able to prevent the decline in S100B secretion. Findings reinforce the benefits of antioxidant therapy in psychiatric disorders. Based on our results, in hippocampal slices exposed to apomorphine, it may be suggested that antipsychotics could help to normalize S100B secretion by astrocytes.     

Gap junction inhibitors modulate S100B secretion in astrocyte cultures and acute hippocampal slices

Astrocytes sense, integrate, and respond to stimuli generated by neurons or neural injury; this response involves gap junction (GJ) communication. Neuronal vulnerability to injury increased when cocultures of astrocytes and neurons were exposed to GJ inhibitors. However, GJ uncoupling could limit the extension of a lesion. We investigated a possible link between GJ communication and S100B secretion. S100B is a calcium‐binding protein of 21 kDa that is predominantly expressed and secreted by astrocytes, which has trophic paracrine activity on neurite growth, glial proliferation, and neuronal survival. GJ inhibitors were analyzed in isolated astrocytes in primary cultures from hippocampus, acute hippocampal slices, and C6 glioma cells, which were used as a negative control. Our data indicate that GJ blocking stimulates S100B secretion in astrocyte cultures and acute hippocampal slices. Different assays were used to confirm cell integrity during exposure to GJ inhibitors. S100B secretion was observed with different types of GJ inhibitors; the resulting event was dependent on time, the nature of the inhibitor, its putative molecular target of GJ blocking, and/or the cell preparation used. Only carbenoxolone induced a fast and persistent increase in S100B secretion in both preparations. Endothelin‐1 increased S100B secretion in astrocyte cultures at 1 hr, but a decrease was observed at 6 hr or in acute hippocampal slices. Physiologically, a local GJ closure associated with release of S100B in injury conditions favors the idea of a common mechanism available to limit the extension of lesion and increase the chances of cell survival. © 2009 Wiley‐Liss, Inc.     

Peroxisome proliferator-activated receptor-alpha and retinoid X receptor agonists inhibit inflammatory responses of astrocytes

The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) plays a key role in lipid metabolism and inflammation. Recently, we demonstrated that administration of the PPAR-alpha agonists gemfibrozil and fenofibrate, inhibit the clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we investigated the effects of PPAR-alpha agonists on primary mouse astrocytes, a cell type implicated in the pathology of MS and EAE. Our studies demonstrated that the PPAR-alpha agonists fenofibrate, and WY 14643 inhibited NO production by LPS-stimulated astrocytes in a dose-dependent manner. Additionally, PPAR-alpha agonists inhibited the secretion of the pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6 by LPS-stimulated astrocytes. Fenofibrate inhibited NF-kappaB DNA binding activity, suggesting a mechanism by which PPAR-alpha agonists may regulate the expression of genes encoding these pro-inflammatory molecules. Retinoid X receptors (RXRs) physically interact with PPAR-alpha receptors, and the resulting heterodimers regulate the expression of PPAR-responsive genes. Interestingly, a combination of 9-cis RA and the PPAR-alpha agonists fenofibrate or gemfibrozil cooperatively inhibited NO, TNF-alpha, IL-1beta, IL-6, and MCP-1 production by these cells. Collectively, these results raise the possibility that PPAR-alpha and RXR agonists might be effective in the treatment of MS, where activated astrocytes are believed to contribute to disease pathology.     

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-1β on IL-6 and PGE₂ secretion by spinal cord astrocytes without affecting the IL-1-β-evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor-α (TNF-α) on IL-6 and PGE₂ secretion and that of transforming growth factor-β 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. GLIA 21:183–193, 1997. © 1997 Wiley-Liss, Inc.     

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.     

S100B attenuates microglia activation in gliomas: possible role of STAT3 pathway

Despite significant infiltration into tumors, the effector function of macrophages (MPs) and microglia (MG) appears to be suppressed in gliomas. Although STAT3 pathway is thought to play a role in this process, the exact mechanism by which gliomas induce STAT3 activation in MPs and MG is not known. Because activation of receptor for advanced glycation end products (RAGE) can induce STAT3, and because gliomas express high levels of S100B, a RAGE ligand, we hypothesized that MP/MG STAT3 activity may be modulated through S100B-RAGE interaction. Exposure of N9 MG and bone marrow-derived monocytes (BMM) to GL261 glioma condition medium (GCM) and low (nM) levels of S100B increased RAGE expression, induced STAT3 and suppressed MG function in vitro. Furthermore, neutralization of S100B in GCM, partially reversed IL-1β suppression in BMM, suggesting that the inhibitory effect of GCM to be in part due to S100B. Finally, blockage of S100B-RAGE interaction inhibited STAT3 activation in N9 MG and in glioma MG/MP in vivo. These findings suggest that the RAGE pathway may play an important role in STAT3 induction in glioma-associated MG/MPs, and that this process may be mediated through S100B.     

Glycoursodeoxycholic acid and interleukin-10 modulate the reactivity of rat cortical astrocytes to unconjugated bilirubin

The pathogenesis of bilirubin encephalopathy seems to result from accumulation of unconjugated bilirubin (UCB) within the brain. We have recently demonstrated that UCB causes astroglial release of proinflammatory cytokines and glutamate, as well as cell death. The bile acid glycoursodeoxycholic acid (GUDCA) and the anti-inflammatory cytokine interleukin (IL)-10 have been reported to modulate inflammation and cell survival. In this study we investigated the effect of these therapeutic agents on the astroglial response to UCB. Only GUDCA prevented UCB-induced astroglial death. The secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta elicited by UCB in astrocytes was reduced in the presence of GUDCA and IL-10, whereas the suppression of IL-6 was only counteracted by GUDCA. Neither GUDCA nor IL-10 modulated the accumulation of extracellular glutamate. Additionally, IL-10 markedly inhibited UCB-induced nuclear factor-kappaB nuclear translocation and cytokine mRNA expression, whereas GUDCA only prevented TNF-alpha mRNA expression. Moreover, GUDCA inhibited TNF-alpha- and IL-1beta-converting enzymes, preventing the maturation of these cytokines and their consequent release. Collectively, this study shows that IL-10 action is restricted to UCB-induced release of TNF-alpha and IL-1beta from the astrocytes, whereas GUDCA presents a more ubiquitous action on the astroglial reactivity to UCB. Hence, GUDCA may have potential benefits over an IL-10 therapeutic approach in reducing UCB-induced astrocyte immunostimulation and death.     

Interleukin-1 beta, but not IL-1 alpha, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor.

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1 beta (IL-1 beta). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1 alpha and IL-1 beta) and the antagonist (IL-1ra)-members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [(125)I] IL-1 beta to cultured astrocytes was saturable and of high affinity. Mean values for the K(D) and B(max) were calculated to be 60.7+/-7.4 pM and 2.5+/-0.1 fmol mg(-1) protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1 alpha (K(i) of 341.1 pM) and IL-1 beta (K(i) 59.9 pM), as well as the antagonist IL-1ra (K(i) 257.6 pM), displaced specific [(125)I] IL-1 beta binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [(125)I] IL-1 beta binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1 alpha and IL-1 beta revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1 beta (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1 alpha by itself had no stimulatory action on NGF release. The simultaneous application of IL-1 alpha and IL-1 beta elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1 beta. We concluded that IL-1 beta-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1 alpha and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.     

Preferential expression and function of Toll-like receptor 3 in human astrocytes

In contrast to other tissues, the central nervous system (CNS) is essentially devoid of MHC expression and shielded from antibodies by the blood-brain barrier. Therefore, a rapid local innate immune response by resident brain cells is required to effectively fight infectious agents. This study analyzed the expression and function of Toll-like receptors (TLRs) in cultured human astrocytes. Quantitative PCR for TLRs 1 to 10 showed a basal expression of TLR3 that could be enhanced by IFN-gamma, IL-1beta, and IFN-beta. The other TLRs were barely detectable and not inducible by the same cytokines. IFN-gamma-activated astrocytes responded to TLR3 ligand poly (I:C) engagement with IL-6 production, while ligands of other TLRs, like LPS, lipoteichoic acid, peptidoglycan, flagellin, and CpG, had no effect. Poly (I:C) also triggered astrocyte production of TNF-alpha and the chemokines CCL2/MCP-1, CCL5/RANTES, CCL20/MIP-3alpha, and CXCL10/IP-10. The adapter molecules MyD88 (full length and short isoform), TIRAP/Mal, and TICAM-1/TRIF, which are required for TLR signaling, were all expressed by astrocytes. Thus, resting and activated human astrocytes express preferentially TLR3 and, upon TLR3 engagement, produce IL-6 and chemokines active on T cells, B cells, monocytes, and dendritic cells. These data indicate that astrocytes function as sentinels for viral infections in the CNS.