Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Interleukin-1beta in the functional and structural luteolysis. Relationship with the nitric oxide system

The aim of the present report was to investigate the in vitro effect of interleukin-1beta(IL-1beta) on corpus luteum (CL) function and some aspects of this mechanism involved. Ovarian rat dispersates from mid-luteal phase were exposed to different doses of IL-1beta (1, 10, 20 ng/ml). Meanwhile 1, 10 and 20 ng/ml of IL-1beta decreased progesterone (P4) production, only the highest doses of IL-1beta increased prostaglandin F2alpha (PGF2alpha) levels. To investigate the possible relationship between PGs production and P4 synthesis, we incubated together IL-1beta (20 ng/ml) and indomethacin (0.1 mM) a potent inhibitor of cyclooxygenase pathway. We found that P4 inhibition induced by IL-1beta was completely prevented by addition of indomethacin. On the other hand, when ovarian rat tissue were exposed at 20 ng/ml of IL-1beta (doses that affected both PGF2alpha and P4 production) the nitric oxide synthase (NOS) activity was augmented. Moreover, IL-1beta effects on PGF2alpha and P4 levels were impaired when a NOS inhibitor N(W)-nitro- L -arginine methyl ester (L-NAME, 600 microM) was added to the incubation media. These data demonstrate that: (i) at the tested doses (1-20 ng/ml), IL-1beta is involved in CL function through the diminution of P4 production of whole ovarian dispersate culture; (ii) at the highest doses assayed (20 ng/ml) IL-1beta increased PGF2alpha production; (iii) at these doses, IL-1beta decreased P4 production by means of a cyclooxygenase pathway and (iv) the NO system would be a key intermediary second messenger in the IL-1beta actions.     

Cytokine- and Endotoxin-Induced Nitric Oxide Synthase in Rat Astroglial Cultures: Differential Modulation by Angiotensin II

Abstract: Recent studies have shown that the stimulatory effects of bacterial endotoxin [lipopolysaccharide (LPS)] on inducible nitric oxide (NO) synthase (iNOS) in astroglia are significantly reduced by the peptide angiotensin II (Ang II). In the present study we have compared the modulatory actions of Ang II on cytokine- and LPS-stimulated iNOS in astroglia cultured from adult rat brain. Incubation of astroglia with LPS (100 ng/ml; 24 h) and/or combinations of interleukin-1β (IL-1β; 10 ng/ml, 24 h), interferon-γ (IFN-γ; 100 U/ml, 24 h), or tumor necrosis factor-α (TNF-α; 100 ng/ml, 24 h) resulted in significant increases of iNOS mRNA, iNOS protein, and NO production, with the latter indicated by increased nitrite accumulation. The effects of LPS, IL-1β, and TNF-α were significantly decreased by coincubation with Ang II (100 ng/ml, 24 h). In contrast, Ang II did not alter the stimulation of iNOS mRNA levels and NO production elicited by IFN-γ. Therefore, Ang II differentially modulates the stimulatory actions of LPS and cytokines on iNOS, and subsequently NO production, in astroglia. These data suggest that Ang II may have an important modulatory role in intracerebral immune responses that involve production of NO by astroglia.     

Glutathione depletion inhibits IL-1 beta-stimulated nitric oxide production by reducing inducible nitric oxide synthase gene expression

L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, decreased IL-1 beta-induced nitrite release in rat islets and purified rat beta cells, nitrite formation and iNOS gene promoter activity in insulinoma cells, and iNOS mRNA expression in rat islets. The thiol depletor diethyl maleate (DEM) and an inhibitor of glutathione reductase 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) reduced IL-1 beta-stimulated nitrite release in islets. We conclude that GSH regulates IL-1 beta-induced NO production in islets, purified beta cells and insulinoma cells by modulation of iNOS gene expression.     

Juxtacrine effects of IL-1 alpha precursor promote iNOS expression in vascular smooth muscle cells

After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 alpha stimulates iNOS expression in vascular SMC, and assessed whether IL-1 alpha exerts autocrine effects on the cells producing IL-1 alpha or juxtacrine effects on cells that contact the IL-1 alpha producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 alpha precursor, which localizes to the plasma membrane, or mature IL-1 alpha, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 alpha precursor, and modestly elevated in SMC overexpressing mature IL-1 alpha, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 beta or TNF-alpha further stimulated iNOS gene expression in SMC producing IL-1 alpha; low levels of IL-1 beta (20 pg/ml) were effective in SMC transfected with IL-1 alpha precursor plasmid, whereas SMC transfected with mature IL-1 alpha plasmid or vector alone required higher concentrations of IL-1 beta (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 alpha precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 alpha precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 alpha precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 alpha themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 alpha precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.     

Suppression of interleukin-1 beta-induced nitric oxide production in RINm5F cells by inhibition of glucose-6-phosphate dehydrogenase

In rat pancreatic islets and insulin-producing cell lines, IL-1beta induces expression of inducible nitric oxide synthase and NO production leading to impairment of glucose-stimulated insulin release and decreased cell survival. NADPH is an obligatory cosubstrate for iNOS synthesis of NO. We hypothesized that IL-1beta stimulates an increase in activity of NADPH-producing enzyme(s) prior to NO production and that this increase is necessary for NO production. Using rat insulin-secreting RINm5F cells, we found that (1) IL-1beta caused a biphasic change in the NADPH level (increased by 6 h and decreased after prolonged incubation in the presence of 2 ng/mL IL-1beta); (2) IL-1beta stimulated increased activity of glucose-6-phosphate dehydrogenase (G6PD) in a time- and dose-dependent manner, and G6PD expression was increased by about 80% after exposure to 2 ng/mL IL-1beta for 18 h: (3) IL-1beta-stimulated NO production was positively correlated with increased G6PD activity; (4) IL-1beta did not cause any significant change in enzyme activity of another NADPH-producing enzyme, malic enzyme; (5) IL-1beta-induced NO production was significantly reduced either by inhibiting G6PD activity using an inhibitor of G6PD (dehydroepiandrosterone) or by inhibiting G6PD expression using an antisense oligonucleotide to G6PD mRNA; and (6) IL-1beta stimulated a decrease in the cAMP level. 8-Bromo-cAMP caused decreased G6PD activity, and the protein kinase A inhibitor H89 led to a increase in G6PD activity in RINm5F cells. In conclusion, our data show that IL-1beta stimulated G6PD activity and expression level, providing NADPH that is required by iNOS for NO production in RINm5F cells. Also, inhibition of the cAMP-dependent PKA signal pathway is involved in an IL-1beta-stimulated increase in G6PD activity.     

HIV gp120 enhances NO production by cardiac myocytes through p38 MAP kinase-mediated NF-kappaB activation

Human immunodeficiency virus (HIV) infection is associated with a surprisingly high frequency of myocardial dysfunction. Potential mechanisms include direct effects of HIV, indirect effects mediated by cytokines, or a combination. We have previously reported that interleukin-1beta (IL-1beta) (500 U/ml) alone induced nitric oxide (NO) production by neonatal rat cardiac myocytes (CM). Effects of the HIV-1 envelope, glycoprotein120 (gp120), on inducible NO synthase (iNOS) in CM have not been previously reported. Unlike IL-1beta, recombinant HIV-gp120 (1 microgram/ml) alone failed to enhance NO production in CM (0.5 +/- 0.4 vs. 0.4 +/- 0.5 micromol/1.25 x 10(5) cells/48 h, gp120 vs. control, respectively; n = 12, P = not significant). However, the addition of gp120 to IL-1beta significantly enhanced iNOS mRNA expression (70 +/- 1.5 vs. 26 +/- 2.4 optical units, IL-1beta + gp120 vs. IL-1beta, respectively; n = 3), iNOS protein synthesis (42 +/- 1.4 vs. 18 +/- 0.8 optical units, IL-1beta + gp120 vs. IL-1beta, respectively; n = 3), and NO production (NO(2)(-)) (6.6 +/- 0.6 vs. 4.1 +/- 0.8 micromol/1.25 x 10(5) cells/48 h, IL-1beta + gp120 vs. IL-1beta, respectively; n = 12, P 相似文献   

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

Inhibition of proinflammatory cytokine-induced invasiveness of HT-29 cells by chitosan oligosaccharide

The effect of chitosan oligosaccharide (COS, 1 kDa 相似文献   

Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis

This study examined the role of nitric oxide (NO) in cytokine-induced apoptosis in adult cardiac fibroblasts (CFbs). In cultured adult rat CFbs, IL-1beta (5 ng/ml), but not interferon-gamma (10 ng/ml) or tumor necrosis factor-alpha (10 ng/ml), induced inducible NO synthase (iNOS) expression and NO production that was associated with an increase in caspase-3 activity and apoptotic cell death. Apoptotic frequency was reduced by the iNOS inhibitor S-methylisothiourea (3 x 10(-5) M). Apoptosis in response to IL-1beta was attenuated by the caspase-3 inhibitor [Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DVED-FMK)] but not by inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). IL-1beta-induced CFb apoptosis was associated with an increase in p53 and Bax protein expression with no changes in Bcl-2 or Bcl-x(L). Nuclear condensation and fragmentation occurred when isolated nuclei were exposed to an NO donor [Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate) 10(-5) M], an effect that was not blocked by the peroxynitrite scavenger Mn(III)tetrakis(4-benzoic acid) porphyrin chloride. Moreover, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride attenuated but did not eliminate IL-1beta-induced CFb apoptosis, indicating that the proapoptotic effect of NO can occur independently of its conversion to peroxynitrite. Our results demonstrate that IL-1beta-induced iNOS expression can trigger NO-dependent apoptosis in adult CFbs, which appears to result from DNA damage and may be mediated by a p53-dependent apoptotic pathway.     

Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.     

Endogenous nitric oxide proves not to be involved in the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells.

Combined treatment of isolated rat gastric mucosal (RGM1) cells with interleukin (IL)-1beta and transforming growth factor (TGF)-alpha resulted in expression of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein 24 hr after the treatment. Constitutive NOS (nNOS) protein was not proved in the cells and not activated by IL-1beta+TGFalpha. Although IL-1beta and TGF-alpha alone exerted little or no effect on NO2 production, their combination gradually increased NO2- production from 12 to 24 hr following treatment. NO2- production stimulated by IL-1beta + TGFalpha was significantly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, yet not by D-NAME. S-nitroso-N-acetyl-D,L-penicillamine and sodium nitropruside significantly inhibited both spontaneous and TGF-alpha stimulated DNA synthesis. Nonetheless, L-NAME did not affect the inhibition by IL-1beta of TGF-alpha-stimulated proliferation of RGM1 cells, eliminating the possibility of involvement of NO in the underlying mechanisms.     

Strain-dependent difference in inducible nitric oxide synthesis (iNOS) expression in rat pancreatic islets correlates with interferon regulating factor 1 (IRF-1) and heat shock protein 70 (HSP70) expression

Nitric oxide (NO) may be a necessary but not sufficient mediator of cytokine-mediated, selective beta-cell destruction. Previously, we have described a difference in NO-dependent IL-1beta sensitivity in vivo and in vitro of pancreatic islets from two rat strains, Brown Norway (BN) and Wistar Kyoto (WK), the latter being the more sensitive strain. Here we investigated whether strain-dependent, differential islet iNOS expression was associated with differences in islet expression of the IL-1 receptor type 1(IL-1RI) or interferon regulating factor 1 (IRF-1), and/or caused differences in HSP70 expression, a marker of cell defence against oxidative stress. Methods: isolated islets from both rat strains were exposed to increasing concentrations of IL-1beta (0-150 pg/ml) for 24 hours or for varying culture periods (4-48 hours) to 15 pg/ml of IL-1beta. Measurements: accumulated insulin and nitrite release into incubation medium, and islet mRNA and protein expression of iNOS, IL-1RI, IRF-1 and HSP70 by semi-quantitative RT-PCR and Western blotting. Results: Higher insulin and lower nitrite release into the incubation medium were seen for BN compared to WK rats islets in both dose- and time-response experiments. IRF-1 expression preceded iNOS expression and was more pronounced in WK than in BN islets. No strain differences were observed for islet expression of IL-1RI. A strain-dependent HSP70 expression in response to IL-1beta with the highest levels in WK rat islets following iNOS expression was seen. Conclusion: There was a strain-dependent difference in iNOS expression which was associated with IRF-1 and HSP70 expression.     

Hydrogen sulfide potentiates interleukin-1beta-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells

Hydrogen sulfide (H(2)S) and nitric oxide (NO) are endogenously synthesized from l-cysteine and l-arginine, respectively. They might constitute a cooperative network to regulate their effects. In this study, we investigated whether H(2)S could affect NO production in rat vascular smooth muscle cells (VSMCs) stimulated with interleukin-1beta (IL-1beta). Although H(2)S by itself showed no effect on NO production, it augmented IL-beta-induced NO production and this effect was associated with increased expression of inducible NO synthase (iNOS) and activation of nuclear factor (NF)-kappaB. IL-1Beta activated the extracellular signal-regulated kinase 1/2 (ERK1/2), and this activation was also enhanced by H(2)S. Inhibition of ERK1/2 activation by the selective inhibitor U0126 inhibited IL-1beta-induced NF-kappaB activation, iNOS expression, and NO production either in the absence or presence of H(2)S. Our findings suggest that H(2)S enhances NO production and iNOS expression by potentiating IL-1beta-induced NF-kappaB activation through a mechanism involving ERK1/2 signaling cascade in rat VSMCs.