The Pro-inflammatory Cytokine TNF-α Regulates the Activity and Expression of the Serotonin Transporter (SERT) in Astrocytes

Pro-inflammatory cytokines have been implicated in the precipitation of depression and related disorders, and the antidepressant sensitive serotonin transporter (SERT) may be a major target for immune regulation in these disorders. Here, we focus on astrocytes, a major class of immune competent cells in the brain, to examine the effects of pro-longed treatment with tumor necrosis factor-alpha (TNF-α) on SERT activity. We first established that high-affinity serotonin uptake into C6 glioma cells occurs through a SERT-dependent mechanism. Functional SERT expression is also confirmed for primary astrocytes. In both cell types, exposure to TNF-α resulted in a dose- and time-dependent increase in SERT-mediated 5-HT uptake, which was sustained for at least 48 h post-stimulation. Further analysis in primary astrocytes revealed that TNF-α enhanced the transport capacity (V_max) of SERT-specific 5-HT uptake, suggesting enhanced transporter expression, consistent with our observation of an increase in SERT mRNA levels. We confirmed that in both, primary astrocytes and C6 glioma cells, treatment with TNF-α activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment with the p38 MAPK inhibitor SB203580 attenuated the TNF-α mediated stimulation of 5-HT transport in both, C6 glioma and primary astrocytes. In summary, we show that SERT gene expression and activity in astrocytes is subject to regulation by TNF-α, an effect that is at least in part dependent on p38 MAPK activation.     

Interleukin 1 and Tumor Necrosis Factor-α Stimulate the Production of Colony-Stimulating Factor 1 by Murine Astrocytes

Astrocytes have the ability to secrete colony-stimulating factor 1 (CSF-1), a growth factor known to stimulate the proliferation of brain macrophages. We have studied the effect of cytokines such as interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and interleukin 6 (IL-6) on the production of CSF-1 by cultured primary astrocytes and an astrocytic cell line derived from embryonic mouse brain. We observed that both TNF alpha and IL-1 increased CSF-1 mRNA and protein levels in the astrocytic cultures. In contrast, IL-6 was ineffective. The CSF-1 mRNA levels were strongly reduced by incubating immortalized astrocytic cells with staurosporine, a protein kinase C inhibitor, both in the absence and in the presence of cytokines. Conversely, 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, increased CSF-1 mRNA levels. These results suggest a mechanism whereby mononuclear phagocytes could favor their own recruitment in the CNS by producing cytokines.     

The inflammatory response of keratinocytes and its modulation by vitamin D: the role of MAPK signaling pathways

The hormonal form of vitamin D, calcitriol, and its analogs are known for their beneficial effect in the treatment of inflammatory skin disorders. Keratinocytes play a role in epidermal inflammatory responses invoked by breeching of the epidermal barrier, by infectious agents and by infiltrating immune cells. We studied the role of calcitriol in the initiation of keratinocyte inflammatory response by the viral and injury mimic polyinosinic-polycytidylic acid (poly(I:C)) and in its maintenance by tumor-necrosis-factor α (TNFα) and investigated the role of the mitogen-activated protein kinase cascades in these processes and their regulation by calcitriol. The inflammatory response of human HaCaT keratinocytes to poly(I:C) or TNFα was assessed by measuring mRNA levels of 13 inflammation-related molecules by real-time PCR microarray and by in-depth investigation of the regulation of interleukin 8, intercellular-adhesion-molecule 1, and TNFα expression. We found that while calcitriol had only a minor effect on the keratinocyte response to poly(I:C) and a modest effect on the early response (2 h) to TNFα, it markedly attenuated the later response (16-24 h) to TNFα. The expression of CYP27B1, the enzyme responsible for calcitriol production, was marginally increased by poly(I:C) and markedly by TNFα treatment. This pattern suggests that while allowing the initial keratinocyte inflammatory response to proceed, calcitriol contributes to its timely resolution. Using pharmacological inhibitors we found that while the p38 MAPK and the extracellular signal-regulated kinase have only a minor role, c-Jun N-terminal kinase plays a pivotal role in the induction of the pro-inflammatory genes and its modulation by calcitriol.     

Mechanism of isoproterenol-induced RGS2 up-regulation in astrocytes

Regulators of G protein signaling (RGSs) are inducibly expressed in response to various stimuli and the up-regulation of RGSs leads to significant decreases in GPCR responsiveness. Isoproterenol, an adrenergic receptor agonist, stimulated RGS2 mRNA in C6 rat astrocytoma cells. The up-regulation of RGS2 mRNA was abrogated by genistein, a protein tyrosine kinase inhibitor (PTK), and by broad-spectrum protein kinase C (PKC) inhibitors (staurosporine and GF109203X). alpha-Adrenergic antagonist (prazocin), beta-adrenergic antagonist (prazocin), and pertussis toxin only partially blocked the RGS2 up-regulation, suggesting that the RGS2 up-regulation is concomitantly mediated by Galphai, Galphas, and Galphaq. It is interesting to note that SB203580, a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, completely inhibited the isoproterenol-mediated RGS2 expression. In addition, isoproterenol also markedly stimulated RGS2 mRNA in rat primary astrocytes, which were sensitive to SB203580 and staurosporine. Therefore, our data suggest that adrenergic receptor-mediated signaling (induced by isoproterenol) may be involved in the regulation of RGS2 expression in astrocytes via activating PTK, PKC, and p38 MAPK.     

Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus.

Synthesis of complement proteins and their regulation in resident cells of the central nervous system are important pathophysiologic factors that can affect the outcome of inflammatory central nervous system diseases. Primary cultures of rat astrocytes constitutively express C3 mRNA and produce C3 protein; both of them were enhanced by LPS or by a live as well as inactivated Newcastle disease virus, a neurotropic paramixovirus. TNF, IL-1 beta, and IL-8 also increased the levels of C3 mRNA and protein whereas IL-1 alpha and IL-6 had no effect, although all of these cytokines are inducible by LPS. LPS stimulation in the presence of cycloheximide decreased the LPS-mediated C3 mRNA induction by 60%. These data suggest that LPS effect on C3 regulation is mediated directly by LPS as well as by LPS-induced cytokines. Interestingly, C3 mRNA induced by Newcastle disease virus or inactivated Newcastle disease virus was inhibited by protein kinase inhibitors, H-7 and staurosporine, whereas these inhibitors had no effect on C3 induction mediated by LPS or cytokines, indicating the existence of different signal transduction pathways.     

Endothelins Stimulate c-fos and Nerve Growth Factor Expression in Astrocytes and Astrocytoma

Abstract: Endothelin receptors have been identified on astrocytes and astrocytoma, but their physiological significance has remained elusive. It is shown here that endothelins induce c- fos in primary cultures of mouse embryo astrocytes, as well as in two subclones of rat astrocytoma C6 cells, although with different kinetics. In addition, nerve growth factor expression is stimulated, as seen by mRNA accumulation and protein secretion, in primary astrocytes and one of the two C6 subclones, with an apparent correlation with the transience of c- fos induction. The activation of protein kinase C appears as an obligatory step during these processes, because (a) inhibition of protein kinase C by staurosporine blocks the induction by endothelin or phorbol esters of both c- fos and nerve growth factor, and (b) phorbol esterevoked down-regulation of protein kinase C completely abolishes the c- fos induction by endothelin, but not that by the β-adrenergic agonist isoproterenol, a known activator of the cyclic AMP-dependent pathway. Our results support the hypothesis that c- fos product might be implicated in nerve growth factor expression by astrocytes, and also suggest that endothelins may participate in vivo in the modulation of the glial neurotrophic activity during brain development or wound healing.     

Induction of BAALC and down regulation of RAMP3 in astrocytes treated with differentiation inducers

Genes controlling proliferation and differentiation of astrocytes are likely to play an important role in the pathogenesis of astrocytic gliomas and could serve as therapeutic targets. mRNA differential display analysis identified two genes, viz. BAALC and RAMP3, whose expression is altered in primary astrocytes treated with differentiation inducers. BAALC, which has been reported to be expressed in neural progenitor cells, was found to be up regulated in both normal and malignant astrocytes on inhibition of proliferation. RAMP3 functions as a modulator of the receptor for adrenomedullin (AM). AM has been suggested to act as autocrine/paracrine growth factor for gliomas. Our studies show RAMP3 down regulation on staurosporine treatment of astrocytes, suggesting protein kinase C inhibition as a possible strategy for inhibiting AM activity and thereby growth of glioma cells.     

Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology.

Interleukin-10 (IL-10) is a key inhibitory signal of inflammatory responses that regulates the production of potentially pathogenic cytokines like tumor necrosis factor (TNF). We show here that the development of chronic intestinal inflammation in IL-10-deficient mice requires the function of TNF, indicating that the IL-10/TNF axis regulates mucosal immunity. We further show that IL-10 targets the 3' AU-rich elements (ARE) of TNF mRNA to inhibit its translation. Moreover, IL-10 does not alter TNF mRNA stability, and its action does not require the presence of the stability-regulating ARE binding factor tristetraprolin, indicating a differential assembly of stability and translation determinants on the TNF ARE. Inhibition of TNF translation by IL-10 is exerted mainly by inhibition of the activating p38/MAPK-activated protein kinase-2 pathway. These results demonstrate a physiologically significant cross-talk between the IL-10 receptor and the stress-activated protein kinase modules targeting TNF mRNA translation. This cross-talk is necessary for optimal TNF production and for the maintenance of immune homeostasis in the gut.     

Inflammatory factors stimulate expression of group II phospholipase A2 in rat cultured astrocytes. Two distinct pathways of the gene expression

Inflammatory factors such as tumor necrosis factor (TNF), interleukin 1 (IL-1), and lipopolysaccharide (LPS) greatly enhance the expression of group II phospholipase A2 (PLA2-II) mRNA, leading to increased secretion of PLA2-II enzyme from rat-cultured astrocytes. The potent antiinflammatory agent dexamethasone suppressed the PLA2-II expression induced by LPS. In vivo studies also demonstrated that the level of PLA2-II mRNA in the brain increased with intravenous injection of LPS. These results suggest that PLA2-II in the brain plays important roles in the inflammatory response. Agents which increase intracellular cAMP concentration did not stimulate PLA2-II expression by themselves but selectively enhanced TNF-induced PLA2-II expression about 5-fold. Phorbol ester, a well known protein kinase C activator, increased the PLA2-II expression. H-7, a protein kinase C inhibitor, inhibited the LPS-induced PLA2-II expression, but did not inhibit the TNF-induced one. Therefore, we conclude that the TNF-activated pathway differs from the LPS-activated one: the former is enhanced by cAMP and the latter involves protein kinase C.     

Antiinflammatory effect of rosiglitazone via modulation of mRNA stability of interleukin 10 and cyclooxygenase 2 in astrocytes

Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E₂, PGE₂) and antiinflammatory marker (interleukin 10, IL-10) levels. The protein level of cyclooxygenase 2 (COX-2) was also increased. Rosiglitazone strengthened LPS-induced mRNA expression of COX-2 and IL-10 but not TNFα. Rosiglitazone is an agonist of nuclear receptor PPARγ, but its impact on IL-10 expression was not influenced by a PPARγ antagonist, GW9662, suggesting PPARγ-independent effect of rosiglitazone. The degradation of mRNA is one of the steps of inflammation regulation and might be affected by small molecules. In experiments with actinomycin D, we found that mRNA half-lives of IL-10, COX-2, and TNFα in naive astrocytes were 70, 44, and 19 min, respectively. LPS stimulation caused 2-fold increase in IL-10 and COX-2 mRNA decay rates, whereas addition of rosiglitazone restored them to the initial level. TNFα decay rate was not changed by these stimulations. This suggests that mRNA decay rate could be regulated by small molecules. Moreover, rosiglitazone could be used as a substance stimulating the resolution of inflammation without influence on proinflammatory signals. These results open new perspectives in the search for inflammation resolution modulators.     

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.     

AKAP12 in astrocytes induces barrier functions in human endothelial cells through protein kinase Czeta

Interactions between astrocytes and blood vessels are essential for the formation and maintenance of the blood-neural barrier (BNB). Astrocyte-derived A-kinase anchor protein 12 (AKAP12) influences BNB formation, but the mechanism of regulation of BNB functions by AKAP12 is not fully understood. We have defined a new pathway of barriergenesis in human retina microvascular endothelial cells (HRMECs) involving astrocytic AKAP12. Treatment of HRMECs with conditioned media from AKAP12-overexpressing astrocytes reduced phosphorylation of protein kinase Czeta (PKCzeta), decreased the levels of vascular endothelial growth factor (VEGF) mRNA and protein, and increased thrombospondin-1 (TSP-1) levels, which led to antiangiogenesis and barriergenesis. Transfection of a small interference RNA targeting PKCzeta decreased VEGF levels and increased TSP-1 levels in HRMECs. Rho is a putative downstream signal of PKCzeta, and inhibition of Rho kinase with a specific inhibitor, Y27632, decreased VEGF levels and increased TSP-1 levels. We therefore suggest that AKAP12 in astrocytes differentially regulates the expression of VEGF and TSP-1 via the inhibition of PKCzeta phosphorylation and Rho kinase activity in HRMECs.     

CXCL10 production from cytomegalovirus-stimulated microglia is regulated by both human and viral interleukin-10

Glial cells orchestrate immunocyte recruitment to focal areas of viral infection within the brain and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since recruitment of T lymphocytes plays a critical role in resolving cytomegalovirus (CMV) infection, we investigated the production of a T-cell chemoattractant, CXCL10 (gamma interferon-inducible protein 10) in response to viral infection of human glial cells. Infection with CMV was found to elicit the production of CXCL10 from primary microglial cells but not from astrocytes. This CXCL10 expression was not dependent on secondary protein synthesis but did require the phosphorylation of p38 mitogen-activated protein (MAP) kinase. In addition, migration of activated lymphocytes toward supernatants from CMV-stimulated microglial cells was partially suppressed by anti-CXCL10 antibodies. Since regulation of central nervous system inflammation is essential to allow viral clearance without immunopathology, microglial cells were then treated with anti-inflammatory cytokines. CMV-induced CXCL10 production from microglial cells was suppressed following treatment with interleukin-10 (IL-10) and IL-4 but not following treatment with transforming growth factor beta. The IL-10-mediated inhibition of CXCL10 production was associated with decreased CMV-induced NF-kappa B activation but not decreased p38 MAP kinase phosphorylation. Finally, CMV infection of fully permissive astrocytes resulted in mRNA expression for the viral homologue to human IL-10 (i.e., cmvIL-10 [UL111a]) in its spliced form and conditioned medium from CMV-infected astrocytes inhibited virus-induced CXCL10 production from microglial cells through the IL-10 receptor. These findings present yet another mechanism through which CMV may subvert host immune responses.     

Regulation of cyclooxygenase 2 expression by agonists of PPAR nuclear receptors in the model of endotoxin tolerance in astrocytes

Endotoxin tolerance (ET) represents a state of an altered immune response induced by multiple stimulations of a cell, a tissue, or an organism with lipopolysaccharide. Characteristics of ET include downregulation of induction of proinflammatory genes (TNFα, IL6, and others) and enhancement of induction of antiinflammatory genes (IL10, TGFβ). ET generally has protective functions; nevertheless, it might result in a state of innate immune deficiency and cause negative outcomes. A current issue is the search for the mechanisms controlling the level of inflammation in the course of endotoxin tolerance. In this work, we investigated the change in cyclooxygenase 2 (Cox2) expression in the model of endotoxin tolerance in astrocytes and analyzed the possibility of regulating this process applying nuclear receptor PPAR agonists. Our results indicate that: 1) endotoxin tolerance can be induced in astrocytes and results in TNFα and Cox2 mRNA induction decrease upon secondary stimulation; 2) tolerance is revealed on the level of TNFα release and Cox2 protein expression; 3) PPAR agonists GW7647, L-165041, and rosiglitazone control Cox2 mRNA expression levels under conditions of endotoxin tolerance. In particular, rosiglitazone (a PPARγ agonist) induces Cox2 mRNA expression, while GW7647 (a PPARα agonist) and L-165041 (a PPARβ agonist) suppress the expression. Our results demonstrate that Cox2 can be upand downregulated during endotoxin tolerance in astrocytes, and PPAR agonists might be effective for controlling this target under conditions of multiple proinflammatory stimulations of brain tissues with endotoxin.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Induction of cyclooxygenase-2 expression by manganese in cultured astrocytes

Inflammatory and oxidative events are present in neurodegenerative disorders and appear to contribute to initiation and/or progression of the disease. Within the brain, redox-active metals, such as manganese, play an important role as components of proteins essential for neural function. However, increasing evidence implies its participation in neurodegenerative diseases involving immune modulation. Prostaglandins (PGs) are lipid mediators that participate in the regulation of physiological and pathophysiological processes, particularly during brain inflammation. In this study, we investigated whether the immune modulating action of manganese involved regulation of PGE₂ production in cortical astrocytes. Within non-toxic concentrations, manganese caused an elevation in the expression of cyclooxygenase-2 (COX-2) mRNA and protein and increased PGE₂ release. Manganese potentiated COX-2 expression and PGE₂ generation by lipopolysaccharide/interferon-γ-activated astrocytes. The inductive action of manganese was accompanied by generation of oxidative stress, activation of mitogen-activated protein kinases (MAPKs), AKT, and protein kinase C- (PKC-), and increased NF-κB and AP-1 DNA binding activities. The generation of reactive oxygen species (ROS) was critical to manganese-induced changes in astrocytes, including MAPKs, PKC-, NF-κB, AP-1, and COX-2 expression but not AKT. Collectively, these data indicate that manganese might cause changes in neural activity through the modulation of oxidative and inflammatory events in astrocytes.