Interleukin (IL)-6 is a pleiotropic cytokine whose production by astrocytes in the CNS of transgenic mice (termed GF-IL6) causes neuroinflammation and neurodegeneration. The binding of IL-6 to its receptor (IL6R) triggers gp130-mediated activation of STAT1 and STAT3 as well as SHP2 phosphatase and ERK1/2. We determined the relative contribution of STAT1 to IL-6 signaling and actions in vivo in the brain of GF-IL6 mice. GF-IL6 mice that were null for STAT1 (termed GF-IL6STAT1 KO) were viable, bred normally and physically indistinguishable from GF-IL6 controls. The level of phosphotyrosine (p-Y) STAT1 was increased significantly in GF-IL6 mice but not detectable in GF-IL6STAT1 KO animals. Phospho-STAT3 and phospho-ERK1/2 were increased markedly in GF-IL6 mice and were not altered by the absence of STAT1. Both the density and distribution of phospho-STAT3-positive cells (mainly astrocytes, microglia and endothelial cells) was similar in GF-IL6 and GF-IL6STAT1 KO mice. Despite a minor decrease in IL-1 and TNF mRNA, the overall inflammatory phenotype of GF-IL6 mice was not altered significantly by the absence of STAT1. IFN-regulated genes activated by STAT1 homodimers via the GAS element (e.g. CXCL9) showed a small increase in GF-IL6 but not GF-IL6STAT1 KO animals. When compared with transgenic mice with astrocyte-targeted production of the type I IFN, IFN-alpha, the increased levels of p-Y-STAT1 and IFN-regulated gene expression were considerably lower in GF-IL6 mice. In conclusion, although IL-6 can activate STAT1 this plays minimal, if any, role in IL-6 signaling and actions in the CNS.
"Because our studies used whole hippocampus and cerebellum, we were unable to identify the cell types or subcellular compartments that showed the increased STAT3 and pSTAT3 levels in these CNS regions. In previous studies of the IL-6 tg cerebellum, pSTAT3 was detected primarily in the nucleus of glial cells (Sanz et al., 2008), suggesting that glial cells are the primary cell type responding to the chronic expression of IL-6 in the IL-6 tg cerebellum. However, the primary site of neuronal effects may be at non-nuclear sites. "
[Show abstract][Hide abstract] ABSTRACT: Emerging research has identified that neuroimmune factors are produced by cells of the central nervous system (CNS) and play critical roles as regulators of CNS function, directors of neurodevelopment and responders to pathological processes. A wide range of neuroimmune factors are produced by CNS cells, primarily the glial cells, but the role of specific neuroimmune factors and their glial cell sources in CNS biology and pathology have yet to be fully elucidated. We have used transgenic mice that express elevated levels of a specific neuroimmune factor, the cytokine IL-6 or the chemokine CCL2, through genetic modification of astrocyte expression to identify targets of astrocyte produced IL-6 or CCL2 at the protein level. We found that in non-transgenic mice constitutive expression of IL-6 and CCL2 occurs in the two CNS regions studied, the hippocampus and cerebellum, as measured by ELISA. In the CCL2 transgenic mice elevated levels of CCL2 were evident in the hippocampus and cerebellum, whereas in the IL-6 transgenic mice, elevated levels of IL-6 were only evident in the cerebellum. Western blot analysis of the cellular and synaptic proteins in the hippocampus and cerebellum of the transgenic mice showed that the elevated levels of CCL2 or IL-6 resulted in alterations in the levels of specific proteins and that these actions differed for the two neuroimmune factors and for the two brain regions. These results are consistent with cell specific profiles of action for IL-6 and CCL2, actions that may be an important aspect of their respective roles in CNS physiology and pathophysiology.
"Stat1 null mice are viable and only have minor defects in immune responses postnatally . Astrocyte formation in these animals is normal, indicating that STAT1 may be dispensable for gliogenesis . On the other hand, genetic elimination of Stat3 results in severe astrogliosis defects, which suggest that STAT1 may not be as potent as STAT3 –. "
[Show abstract][Hide abstract] ABSTRACT: The JAK-STAT signaling pathway has been implicated in astrocyte differentiation. Both STAT1 and STAT3 are expressed in the central nervous system and are thought to be important for glial differentiation, as mainly demonstrated in vitro; however direct in vivo evidence is missing. We investigated whether STAT1 and STAT3 are essential for astrocyte development by testing the STAT responsiveness of astrocyte progenitors. STAT3 was absent in the ventricular zone where glial progenitors are born but begins to appear at the marginal zone at E16.5. At E18.5, both phospho-STAT1 and phospho-STAT3 were present in glial fibrillary acidic protein (GFAP)-expressing white matter astrocytes. Overexpression of STAT3 by electroporation of chicks in ovo induced increased numbers of astrocyte progenitors in the spinal cord. Likewise, elimination of STAT3 in Stat3 conditional knockout (cKO) mice resulted in depletion of white matter astrocytes. Interestingly, elimination of STAT1 in Stat1 null mice did not inhibit astrocyte differentiation and deletion of Stat1 failed to aggravate the glial defects in Stat3 cKO mice. Measuring the activity of STAT binding elements and the gfap promoter in the presence of various STAT mutants revealed that transactivation depended on the activity of STAT3 not STAT1. No synergistic interaction between STAT1 and STAT3 was observed. Cortical progenitors of Stat1 null; Stat3 cKO mice generated astrocytes when STAT3 or the splice variant Stat3β was supplied, but not when STAT1 was introduced. Together, our results suggest that STAT3 is necessary and sufficient for astrocyte differentiation whereas STAT1 is dispensable.
PLoS ONE 01/2014; 9(1):e86851. DOI:10.1371/journal.pone.0086851 · 3.23 Impact Factor
"When IL-6 binds to its receptor, homodimerization of gp130 occurs, followed by the activation of associated janus kinases (JAKs) , and the recruitment of signal transducer and activator of transcription (STAT) proteins to the nucleus, where they will modulate gene transcription . In vitro and in vivo studies showed that IL-6 signaling in the central nervous system is carried out by STAT3 that is phosphorylated by JAK at Tyr705 [34,35]. "
[Show abstract][Hide abstract] ABSTRACT: It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.
We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).
METH induced microglial cell death in a concentration-dependent manner (EC50 = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.
These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.
Journal of Neuroinflammation 05/2012; 9(1):103. DOI:10.1186/1742-2094-9-103 · 5.41 Impact Factor
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