Cytokine-mediated Nitric Oxide Research Articles

Glycogen synthase kinase 3 regulates IL-1β mediated iNOS expression in hepatocytes by down-regulating c-Jun.

Excessive nitric oxide from the inducible nitric oxide synthase (iNOS) increases shock-induced hepatic injury, hepatic dysfunction, inflammation, and mortality in animal models. Cytokines increase the expression of iNOS in hepatocytes, but the signaling mechanisms involved are not completely understood. We have previously demonstrated that Akt mediates the inhibitory effect of cAMP and insulin on cytokine-induced hepatocyte iNOS expression. We hypothesized that glycogen synthase kinase 3 (GSK3), a target of Akt phosphorylation, would regulate hepatocyte iNOS expression. In cultured rat hepatocytes, GSK3 inhibitors decreased IL-1β mediated nitric oxide (NO) production and iNOS protein expression, while the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002 increased the cytokine-mediated NO production and iNOS expression. Over-expression of the constitutively active form of GSK3β enhanced IL-1β-mediated iNOS expression. GSK3 catalyzes the phosphorylation of c-Jun at the c-terminal Thr239 that facilitates c-Jun degradation. Inhibition of GSK3 with SB216763 and lithium chloride significantly reduced, whereas blocking PI3K/Akt increased phosphorylation of c-Jun at Thr239. The levels of total-c-Jun and c-Jun phosphorylated at Ser63 inversely correlated with c-Jun phosphorylated at Thr239, GSK3 activation and iNOS expression. Over-expression of a dominant negative c-Jun not only caused an increase in IL-1β-mediated iNOS promoter activity and iNOS protein expression but was also able to reverse the SB216763-mediated suppression of iNOS. These results demonstrate that GSK3, a downstream target of Akt, regulates IL-1β-stimulated iNOS expression in hepatocytes by directly phosphorylating c-Jun in an inhibitory manner.

Therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats

Chronic pulmonary hypertension in the neonate and infant frequently presents with right-ventricular (RV) failure. Current clinical management may include protracted treatment with inhaled nitric oxide (iNO), with the goal of reducing RV afterload. We have previously reported that prolonged exposure to iNO causes RV systolic dysfunction in the chronic hypoxia-exposed juvenile rat, which was prevented by a peroxynitrite decomposition catalyst. Given that inhalation of CO2 (therapeutic hypercapnia) may limit oxidative stress and upregulated cytokine expression in the lung and other organs, we hypothesized that therapeutic hypercapnia would attenuate cytokine-mediated nitric oxide synthase (NOS) upregulation, thus limiting peroxynitrite generation. Sprague–Dawley rat pups were exposed to chronic hypoxia (13% O2) from postnatal day 1 to 21, while receiving iNO (20ppm) from day 14 to 21, with or without therapeutic hypercapnia (10% CO2). Therapeutic hypercapnia completely normalized RV systolic function, RV hypertrophy, and remodeling of pulmonary resistance arteries in animals exposed to iNO. Inhaled nitric oxide-mediated increases in RV peroxynitrite, apoptosis, and contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and NOS-2 were all attenuated by therapeutic hypercapnia. Inhibition of NOS-2 activity with 1400W (1mg/kg/day) prevented iNO-mediated upregulation of peroxynitrite and led to improved RV systolic function. Blockade of IL-1 receptor signaling with anakinra (500mg/kg/day) decreased NOS-2 content and had similar effects compared to NOS-2 inhibition on iNO-mediated effects, whereas blockade of TNF-α signaling with etanercept (0.4mg/kg on alternate days) had no effects on these parameters. We conclude that therapeutic hypercapnia prevents the adverse effects of sustained exposure to iNO on RV systolic function by limiting IL-1-mediated NOS-2 upregulation and consequent nitration. Therapeutic hypercapnia also acts synergistically with iNO in normalizing RV hypertrophy, vascular remodeling, and raised pulmonary vascular resistance secondary to chronic hypoxia.

Apolipoprotein CIII Reduces Proinflammatory Cytokine-Induced Apoptosis in Rat Pancreatic Islets via the Akt Prosurvival Pathway

Apolipoprotein CIII (ApoCIII) is mainly synthesized in the liver and is important for triglyceride metabolism. The plasma concentration of ApoCIII is elevated in patients with type 1 diabetes (T1D), and in vitro ApoCIII causes apoptosis in pancreatic β-cells in the absence of inflammatory stress. Here, we investigated the effects of ApoCIII on function, signaling, and viability in intact rat pancreatic islets exposed to proinflammatory cytokines to model the intraislet inflammatory milieu in T1D. In contrast to earlier observations in mouse β-cells, exposure of rat islets to ApoCIII alone (50 μg/ml) did not cause apoptosis. In the presence of the islet-cytotoxic cytokines IL-1β + interferon-γ, ApoCIII reduced cytokine-mediated islet cell death and impairment of β-cell function. ApoCIII had no effects on mitogen-activated protein kinases (c-Jun N-terminal kinase, p38, and ERK) and had no impact on IL-1β-induced c-Jun N-terminal kinase activation. However, ApoCIII augmented cytokine-mediated nitric oxide (NO) production and inducible NO synthase expression. Further, ApoCIII caused degradation of the nuclear factor κB-inhibitor inhibitor of κB and stimulated Ser473-phosphorylation of the survival serine-threonine kinase Akt. Inhibition of the Akt signaling pathway by the phosphatidylinositol 3 kinase inhibitor LY294002 counteracted the antiapoptotic effect of ApoCIII on cytokine-induced apoptosis. We conclude that ApoCIII in the presence of T1D-relevant proinflammatory cytokines reduces rat pancreatic islet cell apoptosis via Akt.

Signaling by IL-1β+IFN-γ and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic β-cell apoptosis

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic beta-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1beta+IFN-gamma and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in beta-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for beta-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1beta+IFN-gamma induce beta-cell death and the nature of the downstream signals by which ER stress 'convinces' beta-cells to trigger apoptosis.

Inhibition of Cytokine-Mediated Nitric Oxide Synthase Expression in Rat Insulinoma Cells by Scoparone

Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. Scoparone (6,7-dimethoxycoumarin) is known to have a wide range of pharmacological properties in vitro. In this study, the effects of scoparone on cytokine-induced beta-cell dysfunction were examined. Presence of scoparone significantly protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of RINm5F, a rat insulinoma cell line, and preserved glucose-stimulated insulin secretion in rat pancreatic islets. Scoparone also resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which scoparone inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. These results revealed the possible therapeutic value of scoparone for the prevention of diabetes mellitus progression.

Statin blocks Rho/Rho-kinase signalling and disrupts the actin cytoskeleton: relationship to enhancement of LPS-mediated nitric oxide synthesis in vascular smooth muscle cells.

We previously demonstrated statins to enhance cytokine-mediated nitric oxide (NO) synthesis in vascular smooth muscle cells (VSMC). To clarify the mechanism by which this occurs, we evaluated the effects of fluvastatin in lipopolysaccharide (LPS)-stimulated VSMC. NO production induced by LPS was dose-dependently enhanced by fluvastatin, as were iNOS mRNA levels and iNOS protein expression. Exogenous mevalonate and geranylgeranylpyrophosphate (GGPP) dampened the stimulatory effect of fluvastatin. A pull-down assay demonstrated fluvastatin to decrease levels of GTP-bound Rho A. Moreover, a Rho-kinase inhibitor, Y-27632, was observed to enhance LPS-induced NO production. We recently demonstrated that disrupting F-actin formation dramatically potentiates the ability of LPS to induce iNOS mRNA and protein expression. In the present study, staining of F-actin with nitrobenzoxadiazole (NBD)-phallacidin demonstrated that fluvastatin significantly impairs F-actin stress fiber formation. In light of these results, the ability of statins to increase NO production is due, at least in part, to their ability to block the biosynthesis of mevalonate, thereby preventing isoprenoid biosynthesis. This inhibits Rho/Rho-kinase signalling and, in turn, disrupts the actin cytoskeleton. Further analysis of the signalling pathway by which the actin cytoskeleton affects iNOS expression might yield new insight into mechanisms of regulation of NO production.

Role of nitric oxide, endothelin-1, and inflammatory cytokines in blood pressure regulation in hemodialysis patients

Background: Altered regulation of blood pressure (BP) in hemodialysis patients is associated with increased morbidity and mortality. Regulation of BP is dependent in part on such vasoactive agents as nitric oxide (NO) and endothelin-1 (ET-1). Cytokine-mediated NO synthase activation during dialysis previously has been reported. The purpose of this study is to investigate the relationship between cytokine-mediated activation of the NO and ET-1 systems and BP regulation in hemodialysis patients. Methods: Nine patients with chronic hypotension (predialysis systolic BP < 110 mm Hg, duration > 1 month), nine patients with hypertension (predialysis systolic BP ≥ 180 mm Hg), and nine age- and sex-matched normotensive controls were enrolled. Results: Predialysis NO end product levels in the hypotensive group were greater than in controls (17.63 ± 5.9 versus 11.06 ± 2.12 μm/mL; P = 0.01), whereas the hypertensive group showed lower levels (4.76 ± 2.33 μm/mL; P < 0.01). The hypotensive group had low postdialysis levels (3.45 ± 1.11 μm/mL; P = 0.01). Predialysis ET-1 levels in the hypotensive and hypertensive groups were greater in comparison to the normotensive group (7.54 ± 4.52 and 8.95 ± 3.52 versus 4.41 ± 0.6 pg/mL; P < 0.01). Postdialysis endothelin levels increased in both the control and hypertensive groups (P < 0.01). Interleukin-1 and tumor necrosis factor-α levels increased postdialysis in all groups, but not significantly. Conclusion: High levels of NO end products in hypotensive patients and low levels in hypertensive patients suggest a critical influence of NO in BP control. In addition, elevated ET-1 levels in hypertensive patients may contribute to systemic vasoconstriction and may suggest vascular dysfunction in this patient population. © 2002 by the National Kidney Foundation, Inc.

Nitric oxide-independent suppression of P450 2C11 expression by interleukin-1β and endotoxin in primary rat hepatocytes

Hepatic expression of multiple cytochrome P450 genes is suppressed in the livers of rats undergoing an inflammatory response. Nitric oxide (NO) released during inflammation has been implicated in the decreased activities and expression of several cytochrome P450 isozymes. We examined the role of cytokine-mediated NO release on cytochrome P450 2C11 expression in rat hepatocytes cultured on Matrigel. Lipopolysaccharide (LPS), interleukin-1β (IL-lβ), IL-6, and tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ), suppressed the expression of P450 2C11 mRNA. Neither IL-6 nor IFN-γ caused NO release into the medium or induction of inducible nitric oxide synthase (iNOS) mRNA. IL-1β and LPS were the most effective in causing NO release and iNOS induction, and in down-regulating P450 2C11 mRNA expression. Combinations of the cytokines, IFN-γ, and LPS produced an additive release of NO but did not synergize to further suppress P450 2C11 mRNA. To investigate the role of NO in the IL-1β- or LPS-mediated suppression of P450 2C11, N-monomethyl- l-arginine (NMA) was administered at concentrations ranging from 30 to 300 μM. Three hundred micromolar NMA returned NO release back to control levels, but did not affect the IL-1β- or LPS-mediated down-regulation of P450 2C11 mRNA or protein expression. Our results suggest that NO is not required for IL-1β- or LPS-mediated down-regulation of P450 2C11 expression in cultured hepatocytes.

Nitric oxide-associated regulation of hepatocyte glutathione synthesis is a guanylyl cyclase-independent event

In a system of rat hepatocytes in primary culture, inhibition of cytokine-mediated nitric oxide (NO) production has been shown to be protective in states of oxidative stress. In the absence of oxidative injury, inhibition of NO synthesis has been associated with decreased intracellular levels of reduced glutathione. To further characterize the role of NO in hepatocyte glutathione metabolism, cytokine-mediated NO synthesis was inhibited by addition of a competitive substrate inhibitor. Reduced glutathione, NO metabolites, and enzyme activity and steady-state mRNA levels of the rate-limiting enzyme for reduced glutathione (GSH) synthesis, gamma-glutamylcysteine synthetase, were determined in the presence and absence of the substrate inhibitor. A diffusible cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP, was added in selected instances to determine the potential role of soluble guanylyl cyclase in glutathione metabolism. Inhibition of cytokine-induced NO synthesis was associated with depletion of glutathione. These levels were restored in the presence of pharmacologic concentrations of a NO donor. Along with decreased glutathione levels, gamma-glutamylcysteine synthetase enzyme activity and steady state mRNA levels were also decreased with inhibition of NO synthesis. Addition of 8-bromo-cGMP did not alter glutathione content or gamma-glutamylcysteine synthetase enzyme activity and steady-state mRNA levels. In this system of cultured rat hepatocytes, cytokine-mediated NO synthesis may be protective in states of oxidative stress through regulation of glutathione synthesis.

Serum Nitrite Plus Nitrate in Infection with Human Immunodeficiency Virus Type-1

To get a measure of the extent of induction of nitric oxide synthase in infection with human immunodeficiency virus type 1 (HIV-1) in vivo, we estimated serum nitrite plus nitrate concentrations in 110 HIV-1 infected individuals compared to 76 blood donors. To monitor cytokine action and to measure induction of pteridine synthesis, we determined in parallel neopterin, biopterin, soluble tumor necrosis factor-alpha receptor 55 and 75, β2-micro globulin. Serum nitrite plus nitrate concentrations were elevated in patients as compared to blood donor controls. In sera of patients, nitrite plus nitrate levels correlated significantly with neopterin, soluble tumor necrosis factor receptor 55 and 75, β2-microglobulin. Nitrite plus nitrate levels were higher and correlations were stronger in groups of patients with lower CD4+ cell count. These results suggest that cytokine-mediated nitric oxide synthesis occurs in individuals with HIV-1 infection.

Nitric Oxide Decreases Oxidant-Mediated Hepatocyte Injury

Nitric oxide (NO) is a readily diffusible, short-lived free radical with a multitude of organ-specific regulatory functions. Within the hepatocyte, NO production is associated with inhibition of mitochondrial electron transport enzyme activity, activation of soluble guanylyl cyclase, and inhibition of glyceraldehyde-3-phosphate dehydrogenase. However, while NO can regulate a number of hepatocyte functions, it is unknown whether NO production is hepatoprotective or hepatotoxic. Using isolated rat hepatocytes in primary short-term culture, we investigated the role of cytokine-mediated NO production in toxin-induced hepatocyte injury. In a model of acetaminophen (AM) hepatotoxicity, inhibition of cytokine-mediated NO production potentiated AM injury. In the presence of an inhibitor of NO synthesis, NG -monomethyl-l-arginine (l-NMMA), hepatocyte release of aspartate aminotransferase was increased twofold in the presence of 4.0 and 8.0 mM AM (P < 0.01). In addition, in the presence of AM, cytokine-mediated NO production was increased by 75% over baseline (P < 0.01). Maximum NO synthesis occurred at an AM concentration of 2 mM. A potential mechanism for the hepatoprotective effect of NO centers on its role in glutathione (GSH) homeostasis. In the presence of increasing concentrations of AM, hepatocyte GSH stores decreased in parallel in both control and cytokine-stimulated hepatocytes (ANOVA, P < 0.01). When cytokine-stimulated hepatocytes were exposed to 50 μMl -NMMA, NO release was ablated, while glutathione levels decreased by threefold in comparison to controls (P < 0.01). In the presence of increasing concentrations of AM, cytokine-treated cells exposed to 50 μM l -NMMA exhibited significant decremental decreases in GSH levels (P < 0.05). These data suggest that inhibition of cytokine-mediated NO production potentiates AM hepatotoxicity by modulation of hepatocyte glutathione stores.

The hepatoprotective effect of cytokine-mediated nitric oxide production is guanylyl cyclase-dependent

The hepatoprotective effect of cytokine-mediated nitric oxide production is guanylyl cyclase-dependent

Nitric oxide-mediated neuronal injury in multiple sclerosis

Although several explanations have been proposed for destruction of myelin and oligodendrocytes in multiple sclerosis, there is no proven mechanism of injury. We postulate that the autoimmune response seen in multiple sclerosis results in a cytokine-mediated increase in nitric oxide production by macrophages/microglia, smooth muscle cells and/or endothelium of the central nervous system. 3 mechanisms of cellular damage due to nitric oxide are proposed: 1. direct nitric oxide cytotoxicity; 2. injury due to peroxynitrite formation from superoxide anion and nitric oxide; and 3. nitric oxide-mediated elevations of cellular cGMP that enhance tumor necrosis factor-alpha toxicity. In support of these hypotheses, the anti-inflammatory effectors, dexamethasone and transforming growth factor-β, ameliorate symptoms seen in clinical multiple sclerosis and experimental allergic encephalitis, respectively. These 2 immunomodulators also inhibit induction of cytokine-mediated nitric oxide production by macrophages. An experimental design and therapeutic interventions which will evaluate the role of nitric oxide in the pathophysiology of experimental allergic encephalitis are presented.