Cytokine-induced enhancement of calcium-dependent glutamate release from astrocytes mediated by nitric oxide

Cytokines are produced in the central nervous system (CNS) and exhibit various effects on neurons, microglia, and astrocytes. Astrocytes can release chemical transmitters, including glutamate, in a calcium-dependent manner, which may mediate communication between neurons and astrocytes. To date, no studies have been conducted on the effects of cytokines on calcium-dependent glutamate release from astrocytes. Here, we studied the effects of cytokines on calcium-dependent glutamate release. Cytokines enhanced glutamate release and induced the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO). The inhibition of iNOS eliminated the cytokine-induced enhancement of glutamate release, and treatment with a NO donor, even in the absence of cytokines, increased glutamate release. Thus, cytokines enhance glutamate release, and this enhancement is mediated by NO.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Inhibitory effect of Artemisia capillaris extract on cytokine-induced nitric oxide formation and cytotoxicity of RINm5F cells

Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. Cytokines stimulate an inducible form of nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, leading to insulin insufficiency. In the present study, the effects of Artemisia capillaris extract (ACE) on cytokine-induced beta-cell damage were examined. Treatment of RINm5F (RIN) rat insulinoma cells with interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) induced cell damage. ACE completely protected IL-1beta and IFN-gamma-mediated cytotoxicity in a concentration-dependent manner. Incubation with ACE resulted in a significant reduction in IL-1beta and IFN-gamma-induced NO production, a finding that correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which ACE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappaB binding activity and p65 subunit levels in the nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, ACE restored the cytokine-induced inhibition of insulin release from isolated islets. These results suggest that ACE protects beta-cells by suppressing NF-kappaB activation.     

Nitric oxide in the cerebral cortex of amyloid-precursor protein (SW) Tg2576 transgenic mice

Changes in the amyloid-peptide (Abeta), neuronal and inducible nitric oxide (NO)synthase (nNOS, iNOS), nitrotyrosine, glial fibrillary acidic protein, and lectin from Lycopersicon esculentum (tomato) were investigated in the cerebral cortex of transgenic mice (Tg2576) to amyloid precursor protein (APP), by immunohistochemistry (bright light, confocal, and electron microscopy). The expression of nitrergic proteins and synthesis of nitric oxide were analyzed by immunoblotting and NOS activity assays, respectively. The cerebral cortex of these transgenic mice showed an age-dependent progressive increase in intraneuronal aggregates of Abeta-peptide and extracellular formation of senile plaques surrounded by numerous microglial and reactive astrocytes. Basically, no changes to nNOS reactivity or expression were found in the cortical mantle of either wild or transgenic mice. This reactivity in wild mice corresponded to numerous large type I and small type II neurons. The transgenic mice showed swollen, twisted, and hypertrophic preterminal and terminal processes of type I neurons, and an increase of the type II neurons. The calcium-dependent NOS enzymatic activity was higher in wild than in the transgenic mice. The iNOS reactivity, expression and calcium-independent enzymatic activity increased in transgenic mice with respect to wild mice, and were related to cortical neurons and microglial cells. The progressive elevation of NO production resulted in a specific pattern of protein nitration in reactive astrocytes. The ultrastructural study carried out in the cortical mantle showed that the neurons contained intracellular aggregates of Abeta-peptide associated with the endoplasmic reticulum, mitochondria, and Golgi apparatus. The endothelial vascular cells also contained Abeta-peptide deposits. This transgenic model might contribute to understand the role of the nitrergic system in the biological changes related to neuropathological progression of Alzheimer's disease.     

Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases

Acute hypoxia increases the endogenous release of nitric oxide (NO) in rat carotid body and the expression of nitric oxide synthases is modulated by chronic hypoxia. The aim of the study was to examine hypoxia-induced NO generation in rat carotid body adapted to chronic hypoxia with inspired oxygen at 10% for 4 weeks. The concentration of NO was measured electrochemically with a Pt/Nafion/Pd-IrOx/POAP modified electrode inserted into the isolated carotid body superfused with bicarbonate-buffer saline at 35 degrees C. Acute hypoxia increased the concentration of NO by 471.3+/-71.4 nM in the carotid body of chronically hypoxic (CH) rats. The amount of NO release induced by hypoxia was significantly augmented when compared with that of the normoxic control (87.6+/-15.9 nM). The hypoxia-induced NO generation was markedly attenuated by pretreatment with L- NG-nitroarginine methylester (L-NAME; 500 microM), a non-selective nitric oxide synthase (NOS) inhibitor and also by removal of extracellular calcium with the calcium chelator EGTA (5 mM). Additionally, NO generation during hypoxia was reduced by 30% in the CH carotid body treated with S-methylisothiourea (SMT; 50 microM), a specific blocker of inducible NOS (iNOS). Immunohistochemical study revealed that positive iNOS protein immunoreactivity was detected in clusters of glomus cells in the carotid bodies of CH rats, but not in the normoxic group. Thus, chronic hypoxia enhances hypoxia-induced NO generation mediated by calcium-dependent NOSs and iNOS in the carotid body. Extracellular recording of sinus nerve activity of CH carotid bodies showed that L-NAME treatment enhanced the afferent discharge in response to hypoxia, confirming that the generation of NO suppresses the activities of carotid chemoreceptors. Taken together, our results suggest that hypoxia-induced NO production increases in the rat carotid body adapted to chronic hypoxia and that constitutive and inducible NOSs are involved in the NO generation. The enhancement of NO generation may play a physiological role in blunting the hypoxic chemosensitivity during chronic hypoxia.     

Release of nitric oxide from astroglial cells: A key mechanism in neuroimmune disorders

Astrocytes are glial cells able to release nitric oxide (NO) under basal conditions as well as following different neurochemical stimuli including cytokines, endotoxins and soluble antigens, thereby participating in neuroimmune responses. In particular, the inducible isoform of NO synthase seems to be activated during co-incubation of this cell type with cytokines as well as in the presence of the HIV coating gp120 glycoprotein, an effect which is associated with an enhancement of prostanoid release. This seems also to occur via activation of cyclooxygenase by NO. Thus, the l-arginine-NO pathway found in astrocytes may represent a novel approach in the treatment of neuroimmune disorders such as multiple sclerosis, Alzheimer's disease and AIDS.     

Lack of macrophage migration inhibitory factor in mice does not affect hallmarks of the inflammatory/immune response during the first week after stroke

Background

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA₂-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes.

Methods/Results

Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA₂-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression.

Conclusion

These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.     

Interferon-gamma-induced activation of nitric oxide-mediated antiviral activity of macrophages caused by a recombinant coxsackievirus B3

Cardiovascular disease is one of the major causes of human death and has been linked to many different risks including viral infections. Coxsackievirus B3 (CVB3) is one of the most important pathogens responsible for virus-induced myocarditis. Cytokines are normally involved in the control of CVB3 replication and pathogenesis. Among them, interferon-gamma (IFN-gamma) in particular is highly protective against CVB3. A novel strategy to circumvent virus-caused heart disease is based on the development of cytokine-expressing recombinant virus vectors. Using in vitro co-culture experiments, the release of IFN-gamma by the recombinant virus variant CVB3/IFN-gamma activates the expression of the inducible nitric oxide synthase (iNOS) in CVB3 non-susceptible murine macrophages and the release of nitric oxide (NO), which reduce coxsackieviral replication directly. In addition, the expression of IFN-gamma by CVB3/IFN-gamma contributes to protect mice from lethal infections by iNOS induction in murine peritoneal macrophages, viral load reduction, and pancreatic tissue protection.     

Soluble amyloid beta-peptide and myelin basic protein strongly stimulate, alone and in synergism with combined proinflammatory cytokines, the expression of functional nitric oxide synthase-2 in normal adult human astrocytes

The accumulation of amyloid beta (Abeta)-peptides and their collection in fibrillar plaques in the human brain are believed to be responsible for Alzheimer's disease. The major neuron killers in the Alzheimer brain include proinflammatory cytokines and NO made by NOS-2 (inducible nitric oxide synthase-2). We have determined the effect of a soluble Abeta peptide, Abeta(1-40), on the expression of NOS-2 in astrocytes using a novel model system consisting of pure cultures of cells from adult human brains that, after the first three passages in vitro, become stably locked into the normal astrocytic phenotype like their counterparts in the adult human brain. Abeta(1-40) alone stimulated quiescent astrocytes to start expressing functional NOS-2 and dumping NO into the culture medium during the next 4 days. But adding three of the proinflammatory cytokines commonly produced in the Alzheimer brain--IFN-gamma, IL-1beta, and TNF-alpha--along with Abeta(1-40) more than trebled NOS-2 expression and doubled NO production. In view of the possibility of myelin breakdown in the Alzheimer brain, we also tested the capability of myelin basic protein (MBP) to stimulate NO production using human astrocytes. We found that MBP mimicked the ability of Abeta(1-40) to induce cells to release NO and adding the cytokine triad along with MBP more than doubled NO production and release. Thus, it appears that Abeta peptides and MBP can join forces with proinflammatory cytokines to enhance the NO-mediated killing of neurons in the Alzheimer brain.     

Nitric oxide donors as potential additions to anti-alopecia areata armamentarium

Th1-type cytokines play a critical role in the pathogenesis of alopecia areata (AA) and a Th2 lymphocyte-like response may inhibit the tissue-damaging effects of Th1 lymphocytes. It is suggested that nitric oxide (NO) could induce a switch from Th1 to Th2 lymphocytes in the immune response. Additionally, decreased levels of calcitonin gene-related peptide (CGRP) in lesions have been proposed to play an important role in the pathomechanism of AA and NO augments both the release of CGRP and the vasorelaxation induced by it. Taken together, these data suggest that 1) NO donors could be useful in the treatment of AA through the induction of T cell switching towards Th2 cells and 2) induction of inducible nitric oxide synthase (iNOS) by both allergic and irritant contact dermatitis could provide a mechanism for the efficacy of contact dermatitis in the treatment of AA.     

Melatonin inhibits amphetamine-induced nitric oxide synthase mRNA overexpression in microglial cell lines

Amphetamine (AMPH) derivatives are the most commonly abused drugs. Chronic or intermittent AMPH abuse may create temporary or permanent disturbances in the dopaminergic system of the brain that may predispose individuals to Parkinsonism. AMPH induces a massive release of dopamine from synaptic vesicles and then generates reactive oxygen species (ROS). Furthermore, nitric oxide (NO), produced in the central nervous system (CNS) mediated by the activation of microglia, appears to play a critical role in stress-induced brain damage. In the present study, we examined the involvement of NO in the neurotoxic effects of AMPH, to investigate the hypothesis that altered nitric oxide synthase (NOS) function was involved. AMPH at a concentration of 0.4-3.2mM has a cytotoxic effect on highly aggressively proliferating immortalized (HAPI) cells, a rat microglial cell line. The effect of AMPH on increasing inducible NOS (iNOS) mRNA in HAPI microglial cells is concentration-dependent. Pretreatment with either S-methylisothiourea (S-MT), a selective iNOS inhibitor, or melatonin, a major secretory product of pineal gland, counteracted the over expression of iNOS induced by AMPH in a concentration-dependent manner. The induction of iNOS by AMPH in microglial cells could be an important source of NO in CNS inflammatory disorders associated with the death of neurons and oligodendrocytes. Administration of exogenous melatonin will be beneficial, as it reduces iNOS mRNA expression, and may, therefore, be able to be used as a neuroprotective agent in toxicity induced by AMPH or other immunogens.     

TGFβ inhibits IL-1 -induced iNOS expression and NO production in immortalized chondrocytes

Objective: The balance between anti-inflammatory (e.g. TGFβ) and proinflammatory cytokines (e.g. IL-1 and TNFα), regulates destructive processes in OA cartilage. IL-1 and TNFα enhance nitric oxide (NO) production in OA cartilage through the inducible nitric oxide synthase (iNOS) pathway and NO mediates many of the destructive effects of these cytokines. The aim of the present study was to investigate the effects of TGFβ on NO production in immortalized H4 chondrocytes exposed to IL-1. Results: IL-1 induced NO production in chondrocytes through nuclear factor kappa B (NF-κB) sensitive and dexamethasone insensitive expression of iNOS. TGFβ inhibited IL-1 -induced iNOS expression and NO production in chondrocytes, but it did not have any effect on iNOS mRNA levels. iNOS protein levels were similar in cells treated with IL-1 or IL-1 + TGFβ when measured after 8 h incubation, whereas when measured after 12 h and 24 h incubations, iNOS protein levels were 50% and 80% lower in cells treated with IL-1 + TGFβ than in cells treated with IL-1 alone. Conclusion: TGFβ suppressed IL-1-induced iNOS expression and NO production in chondrocytes, probably by enhancing iNOS protein degradation. This finding suggests an additional mechanism for TGFβ to counteract the destructive effects of IL-1 in OA. Received 24 March 2005; returned for revision 16 June 2005; accepted by J. Hamilton 7 July 2005     

Sudden infant death syndrome: is it a transepithelial transport disorder?

The first limiting factor of dietary zinc deficiency has been described as a loss of the protective role of zinc against auto-oxidation of membrane sulfhydryl (SH) compounds. It has now been established that the prohormones (nutriuretic peptides) of the intestinal guanylin family are activated extracellularly by conversion of cysteines in the peptide to disulfide bridges. The induction of uroguanylin mRNA is elevated in intestinal zinc deficiency and nutriuretic peptides regulate epithelial transport of salt and water. Nitric oxide (NO) is also a modulator of salt and water transport. The constitutive forms of nitric oxide synthase (cNOS) in neurons and endothelial cells are calcium-dependent. The inducible form of nitric oxide synthase (iNOS) is activated by bacterial entero-toxins and damaged mucosa with NO penetrating the cell and acting directly on guanylate cyclase. The activated receptor-guanylate cyclases initiating the intracellular cycle 3'-5' guanasine monophosphate (cyclic GMP) cascade in target cells results in a flux of chloride and water into the intestinal lumen. Most of the actions of NO are mediated by activation of cyclic GMP. High-altitude pulmonary edema (HAPE) is associated with a defect in transepithelial water transport. It is suggested that dietary zinc, by modulating thiol oxidation to disulfides in guanylin prohormones to active hormones, is associated with salt and water secretion such that the overworked heart in hypoxemia increases the production and release of natriuretic peptides to activate guanylate cyclase receptors in target tissue in sudden infant death syndrome.     

Protective effect of retinoic acid on interleukin-1 beta-induced cytotoxicity of pancreatic beta-cells

Cytokines produced by immune cells in pancreatic islets infiltrating are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of retinoic acid (RA) on cytokine-induced beta-cell dysfunction were examined. RA significantly protected interleukin-1 beta (IL-1) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of rat insulinoma cell (RINm5F), and also reduced in IL-1 and IFN-gamma-induced nitric oxide (NO) production, which correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism, by which RA inhibited iNOS gene expression, appeared to involve the inhibition of NF-kappa B activation. Our results suggest possible therapeutic value of RA for the prevention of diabetes mellitus progression.     

The metabolic coupling of arginine metabolism to nitric oxide generation by astrocytes

Arginine, the only known precursor of nitric oxide, enters the brain parenchyma from the blood through the endothelial cells or from the cerebral spinal fluid through the ependymal cells. Astrocytes, whose processes abut the endothelium and ependymum, take up arginine through cationic amino acid transporters and release arginine through this transport system to the synapses that astrocytes shield. Some of these synapses are excitatory, and liberate glutamate into the synaptic cleft. Glutamate induces arginine release from astrocytes, making it available to the neuron. Neurons can take up arginine to be used in nitric oxide-mediated processes, such as neurotransmission. Thus, neural and nonneural cells act in concert to affect neuron physiology in an elegantly integrated system. This review focuses on the components of the interaction between astrocytes and neurons in nitric oxide biology.     

炎性细胞因子及脂多糖诱导产生的一氧化氮对人血管内皮细胞的损伤作用

目的:探讨炎症过程诱生的一氧化氮(NO)对内皮细胞的损伤及其作用机制。方法:黄递酶法及Griess法检测可诱导性NO合酶(iNOS)活性、NO₂^-/NO₃^-水平,RT-PCR技术分析iNOSmRNA的表达;同时观察NO产生后对内皮细胞的损伤作用。结果:IL-1β2×10⁵U/L、TNF-α5×10⁵U/L、γ-INF2×10⁵U/L联用LPS(10mg/L)可诱导出高浓度NOS合成及NO产生,比单用这些细胞因子或LPS诱导的量高两倍多,iNOSmRNA的表达水平也显著增加;同时MDA及LDH释放率明显增加,细胞存活率下降,并伴随细胞受损的形态学改变。而单用上述细胞因子或LPS,以及降低剂量或缩短处理时间,其诱生的NOS及NO与正常对照相比P＞0.05;但MDA及LDH释放率仍增加明显。使用2倍剂量的这些炎性细胞因子或延长处理时间到48h,与标准剂量及24h组相比NOS和NO的增加虽不显著,但细胞生长受限更明显。NOS抑制剂能阻止NO的产生及其对细胞的损伤作用。结论:炎性细胞因子及脂多糖可激活iNOS大量表达诱生高浓度的NO,对内皮细胞具有明显的氧化损伤作用。