Expression Of iNOS In Microglia Research Articles

GRK2 upregulates iNOS expression in microglia by mediating IRF1 and STAT1/3 activation

GRK2 upregulates iNOS expression in microglia by mediating IRF1 and STAT1/3 activation

A cannabigerol derivative suppresses immune responses and protects mice from experimental autoimmune encephalomyelitis.

Phytocannabinoids that do not produce psychotropic effects are considered of special interest as novel therapeutic agents in CNS diseases. A cannabigerol quinone, the compound VCE-003, has been shown to alleviate symptoms in a viral model of multiple sclerosis (MS). Hence, we studied T cells and macrophages as targets for VCE-003 and its efficacy in an autoimmune model of MS. Proliferation, cell cycle, expression of activation markers was assessed by FACs in human primary T cells, and cytokine and chemokine production was evaluated. Transcription was studied in Jurkat cells and RAW264.7 cells were used to study the effects of VCE-003 on IL-17-induced macrophage polarization to a M1 phenotype. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG35–55) immunization and spinal cord pathology was assessed by immunohistochemistry. Neurological impairment was evaluated using disease scores. We show here that VCE-003 inhibits CD3/CD28-induced proliferation, cell cycle progression and the expression of the IL-2Rα and ICAM-1 activation markers in human primary T cells. VCE-003 inhibits the secretion of Th1/Th17 cytokines and chemokines in primary murine T cells, and it reduces the transcriptional activity of the IL-2, IL-17 and TNFα promoters induced by CD3/CD28. In addition, VCE-003 and JWH-133, a selective CB2 agonist, dampened the IL-17-induced polarization of macrophages to a pro-inflammatory M1 profile. VCE-003 also prevented LPS-induced iNOS expression in microglia. VCE-003 ameliorates the neurological defects and the severity of MOG-induced EAE in mice through CB2 and PPARγ receptor activation. A reduction in cell infiltrates, mainly CD4+ T cells, was observed, and Th1 and Th17 responses were inhibited in the spinal cord of VCE-003-treated mice, accompanied by weaker microglial activation, structural preservation of myelin sheets and reduced axonal damage. This study highlights the therapeutic potential of VCE-003 as an agent for the treatment of human immune diseases with both inflammatory and autoimmune components.

IL‐12 p40 homodimer, the so‐called biologically inactive molecule, induces nitric oxide synthase in microglia via IL‐12Rβ1

Earlier we have demonstrated that IL-12 p40 homodimer (p40(2)) induces the expression of inducible nitric oxide synthase (iNOS) in microglia. This study was undertaken to investigate underlying mechanisms required for IL-12 p40(2)- and IL-12 p70-induced expression of iNOS in microglia. IL-12 p40(2) alone induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, the ERK pathway coupled p40(2) to iNOS expression via C/EBP beta, but not NF-kappaB, whereas the p38 pathway relayed the signal from p40(2) to iNOS expression via both NF-kappaB and C/EBP beta. Furthermore, by using microglia from IL-12R beta 1 (-/-) and IL-12R beta 2 (-/-) mice or siRNA against IL-12R beta 1 and IL-12R beta 2, we demonstrate that p40(2) induced the expression of iNOS in microglia via IL-12R beta 1-(ERK+p38)-(NF-kappaB +C/EBP beta) pathway. In contrast, both IL-12R beta 1 and IL-12R beta 2 were involved for IL-12 p70-induced microglial expression of iNOS. Although IL-12R beta 1 coupled p70 to NF-kappaB and C/EBP beta, IL-12R beta 2 was responsible for p70-mediated activation of GAS. This study delineates a new role of IL-12R beta 1 and IL-12R beta 2 for the expression of iNOS and production of NO in microglia that may participate in the pathogenesis of neuroinflammatory diseases.

Thrombin-induced microglial activation contributes to the degeneration of nigral dopaminergic neurons in vivo

To evaluate the role of thrombin-activated microglia in the neurodegeneration of nigral dopaminergic neurons in the rat substantia nigra (SN) in vivo. After stereotaxic thrombin injection into unilateral SN of rats, immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and biochemical methods were used to observe tyrosine hydroxylase (TH) immunoreactive positive cells, microglia activation, nitric oxide (NO) amount and inducible nitric-oxide synthase (iNOS) expression. (1) Selective damage to dopaminergic neurons was produced after thrombin injection, which was evidenced by loss of TH immunostaining in time-dependent manner; (2) Strong microglial activation was observed in the SN; (3) RT-PCR demonstrated the early and transient expression of neurotoxic factors iNOS mRNA in the SN. Immunofluorescence results found that thrombin induced expression of iNOS in microglia. The NO production in the thrombin-injected rats was significantly higher than that of controls (P < 0.05). Thrombin intranigral injection can injure the dopaminergic neurons in the SN. Thrombin-induced microglia activation precedes dopaminergic neuron degeneration, which suggest that activation of microglia and release of NO may play important roles in dopaminergic neuronal death in the SN.

Protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death in rat cortical neuron–glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron–glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-γ-induced neurotoxicity in rat cortical neuron–glia cultures with an EC 50 value of 0.09 μM. Radicicol inhibited the LPS/IFN-γ-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-γ-induced release of tumor necrosis factor-α (TNF-α) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-α was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death in neuron–glia cultures is mediated via the inhibition of TNF-α release, as well as the suppression of iNOS expression in microglia.

Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1α

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O 2 level, which is regulated by many physiological functions. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1α involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1α (HIF-1α). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1α in microglia.

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.

Induction of Nitric-oxide Synthase and Activation of NF-κB by Interleukin-12 p40 in Microglial Cells

Interleukin-12 (IL-12) is composed of two different subunits, p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the central nervous system of patients with multiple sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. However, the mode of action of p40 is completely unknown. Because nitric oxide produced from the induction of nitric-oxide synthase (iNOS) also plays a vital role in the pathophysiology of MS, the present study was undertaken to explore the role of p40 in the induction of NO production and the expression of iNOS in microglia. Both IL-12 and p40(2), the p40 homodimer, dose-dependently induced the production of NO in BV-2 microglial cells. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Induction of NO production by the expression of mouse p40 cDNA but not that of the mouse p35 cDNA suggests that the p40 but not the p35 subunit of IL-12 is involved in the expression of iNOS. In addition to BV-2 glial cells, p40(2) also induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 and p40(2) were unable to induce the production of NO in mouse primary astrocytes. Because activation of NF-kappaB is important for the expression of iNOS, we investigated the effect of p40(2) on the activation of NF-kappaB. Induction of the DNA binding as well as the transcriptional activity of NF-kappaB by p40(2) and inhibition of p40(2)-induced expression of iNOS by SN50, a cell-permeable peptide carrying the nuclear localization sequence of p50 NF-kappaB, but not by SN50M, a nonfunctional peptide mutant, suggests that p40(2) induces the expression of iNOS through the activation of NF-kappaB. This study delineates a novel role of IL-12 p40 in inducing the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.

Protein kinase C-mediated regulation of inducible nitric oxide synthase expression in cultured microglial cells

Nitric oxide (NO) has been implicated in a number of important brain functions, such as long-term potentiation (LTP) and long-term depression (LTD), and in events associated with neurodegeneration and neuroprotection. In response to brain injury or disease NO production is increased by an inducible enzyme (iNOS), which is only expressed under these conditions. Activated microglia are a major cellular source of iNOS in brain. Due to the important role of iNOS in brain injury and disease, a detailed understanding of intracellular events triggering the expression of iNOS in microglia would facilitate pharmacotherapeutic approaches. It is shown here, that iNOS mRNA, protein and NO product are induced in cultured microglia by lipopolysaccharide (LPS). This induction is reduced by a number of substances elevating intracellular cyclic AMP levels. It is unabated, however, in the presence of substances inhibiting cyclooxygenase-1 and/or cyclooxygenase-2 (e.g., acetyl salicylic acid, SC 58125, L 745337), but is decreased by approx. 50% with PDTC, a scavenger of reactive oxygen intermediates (ROI) that inhibits nuclear factor κB (NF-κB) activation. Furthermore, inhibitors of protein kinase C (PKC) strongly inhibit iNOS mRNA and protein induction. PKC, therefore, constitutes a major second messenger component (besides NF-κB) in the signaling pathway regulating iNOS expression in microglia.