IL-1 Beta-induced Nitric Oxide Production Research Articles

The breath ammonia measurement of the hemodialysis with a QCM-NH3 sensor

Osteoarthritis (OA) is a progressive joint disease which represents a combination of several disorders leading to cartilage degradation. The production of nitric oxide (NO) by iNOS, which is stimulated by proinflammatory mediators, is involved in cartilage catabolism and should therefore be considered a potential target for therapeutic interventions. Diacerein is a slow-acting drug that may slow down the breakdown of cartilage and relieve pain and swelling. Diacerhein, in contrast to an NSAID, is a potent inhibitor of IL-1beta induced NO production by chondrocytes and cartilage. This effect appeared to result from iNOS transcriptional and/or post-transcriptional events, maybe by the inhibition of the NF-kappaB transcription factor. This paper presents results on the influence of Diacerein on NO production.

Primary human articular chondrocytes, dedifferentiated chondrocytes, and synoviocytes exhibit differential responsiveness to interleukin‐4: Correlation with the expression pattern of the common receptor gamma chain

Interleukin (IL)-4, which exhibits potent anti-inflammatory activities, is of potential therapeutic value in destructive arthropathies. To further define the response of human joint cells to IL-4, we analyzed the ability of this cytokine to modulate the effects of IL-1beta and growth factors. Freshly isolated chondrocytes, dedifferentiated chondrocytes, and synoviocytes were treated with IL-4 before determination of nitric oxide (NO) and collagenase production in response to IL-1beta, or before proliferation assays in presence of IL-1beta, platelet-derived growth factor (PDGF), or transforming growth factor (TGF)-beta. IL-4 downregulated IL-1beta induced NO production in dedifferentiated chondrocytes and inhibited IL-1beta induced collagenase release, as well as IL-1beta and growth factor induced proliferation in dedifferentiated chondrocytes and synoviocytes. In contrast, IL-4 had no effect in freshly isolated primary chondrocytes and in cartilage explants. The lack of response to IL-4 in primary chondrocytes was associated with impaired signal transduction, as indicated by markedly decreased IL-4 dependent tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-6. It also correlated with differences in the expression pattern of IL-4 receptor (IL-4R) subunits during chondrocyte dedifferentiation. Indeed, whereas the IL-4Ralpha and IL-13Ralpha' subunits were expressed in all cell types, expression of the common receptor gamma chain was restricted to freshly isolated chondrocytes. In conclusion, IL-4 downregulated IL-1beta-induced catabolic events and cell proliferation in dedifferentiated chondrocytes and synoviocytes, but had no effects in freshly isolated chondrocytes. The difference in IL-4 responsiveness between primary and dedifferentiated chondrocytes correlated with changes in proximal signaling events and in the expression pattern of IL-4R subunits during cell dedifferentiation.

Effects of ageing and arthritic disease on nitric oxide production by human articular chondrocytes.

Nitric oxide (NO) has been considered as an important mediator in inflammatory phases and in loss of cartilage. In inflammatory arthritis, NO levels are correlated with disease activity and articular cartilage is able to produce large amounts of NO with the appropriate inducing factor such as cytokines. The old animals are shown to have a greater sensitivity to NO than young animals. This study evaluated the basal production of NO in normal and OA-affected chondroyctes from young and old patients and compared the levels of NO formation in response to IL-1beta. The results showed that the basal levels were 7-fold higher in old chondrocytes than those of young cells. However, the IL-1beta induced NO production was seen to decrease with age. Aminoguianidine (AG), a competitive inhibitor of iNOS, inhibited NO formation completely in both chondrocytes from young and old individuals. However, at the same concentration of AG it caused partial inhibition of NO and iNOS formation in chondrocytes from OA-affected individuals. In addition, although the IL-1beta induced NO production was much lesser than that of young chondrocytes, the inhibition of collagen production by IL-1beta was prominent in old chondrocytes and OA-affected chondrocytes. These results suggest that age-related differences in the regulation of NO production and collagen production, which may affect the ageing cells and osteoarthritic changes in some way.

The effect of erythropoietin on interleukin-1beta mediated increase in nitric oxide synthesis in vascular smooth muscle cells.

Recently, we observed that recombinant human erythropoietin (rHuEPO) inhibits the interleukin (IL)-1beta induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMC). The mechanisms of these inhibitory effects of rHuEPO were evaluated. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify a specific erythropoietin receptor (EpoR). Tyrosine phosphorylation of phospholipase C (PLC) was analyzed by combination of immunoprecipitation and Western blotting. Protein kinase C (PKC) activities were analyzed by phosphorylation assay of myelin basic protein (MBP4-14). VSMC were incubated with test agents for 24 h and nitrite as a stable NO metabolite was measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively. RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore, it might be alternatively spliced in VSMC. rHuEPO induced tyrosine phosphorylation of PLC-gamma1 and activation of PKC. rHuEPO inhibited not only IL-1beta induced nitrite production, but also the expression of iNOS mRNA and protein. These inhibitory effects of rHuEPO were reversed in the presence of PKC inhibitors, calphostin C (1 pmol/l) or staurosporine (10 nmol/l). PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1beta induced nitrite production was also eliminated in PKC depleted cells or in the existence of anti-EpoR antibody. rHuEPO inhibits IL-1beta induced NO production by suppressing iNOS mRNA and protein expressions through EpoR, and the PLC-gamma1 and PKC pathway may be involved.

Inhibition by lysophosphatidylcholine of nitric oxide production in interleukin 1 beta-stimulated vascular smooth muscle cells.

In vascular smooth muscle cells (VSMC), inflammatory cytokines such as interleukin 1 beta(IL-1 beta) stimulated nitric oxide (NO) production via the expression of an inducible type of NO synthase (iNOS). Lysophosphatidylcholine (LPC) is a major phospholipid component of atherogenic oxidized low density lipoprotein (Ox-LDL). In this study, we examined the effect of LPC on IL-1 beta-stimulated NO production in cultured. VSMC. LPC by itself did not stimulate the production of nitrite, a stable metabolite of NO, but dose-dependently inhibited IL-1 beta-stimulated nitrite production. LPC inhibited IL-1 beta-stimulated iNOS protein expression, whereas LPC did not inhibit IL-1 beta-stimulated iNOS mRNA expression. Analysis of iNOS protein degradation rate revealed that LPC had no effect on degradation rate of iNOS protein, suggesting that LPC inhibited iNOS translation. Moreover, Ox-LDL inhibited IL-1 beta-stimulated NO production by inhibiting iNOS protein expression without affecting iNOS mRNA expression. These results indicate that Ox-LDL negatively modulates IL-1 beta-induced NO production through the action of LPC, probably by blocking translation of iNOS mRNA.

Differences in cellular responses to mitogens in arterial smooth muscle cells derived from patients with moyamoya disease.

Moyamoya disease is a progressive cerebrovascular occlusive disease affecting primarily children. The etiology remains unknown. We examined the chemotactic and proliferative activities of inflammatory cell products from arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those from control subjects. We used 12 SMC strains from moyamoya patients and eight from control subjects. SMC migration was examined in a micro chemotaxis chamber. DNA synthesis was measured by an immunoperoxidase technique. Platelet-derived growth factor (PDGF)-BB markedly stimulated cell migration and DNA synthesis in control SMCs. PDGF-AA stimulated only DNA synthesis in control SMCs. In moyamoya SMCs, PDGF-AA and PDGF-BB stimulated cell migration but not DNA synthesis. Basic fibroblast growth factor had little migratory activity but stimulated DNA synthesis in moyamoya SMCs and control SMCs. Conversely, hepatocyte growth factor stimulated cell migration but not DNA synthesis in moyamoya SMCs and control SMCs. In contrast, interleukin-1 beta (IL-1 beta) significantly stimulated the migration and DNA synthesis of control SMCs, while it inhibited moyamoya SMC migration. The levels of IL-1 beta-induced nitric oxide production did not differ between moyamoya SMCs and control SMCs, suggesting that IL-1 beta inhibits the migration of moyamoya SMCs through a nitric oxide-independent pathway. The differences in responses to PDGF and IL-1 in moyamoya SMCs are involved in the mechanism by which intimal thickening develops in moyamoya disease.

Cytokine-stimulated astrocytes damage human neurons via a nitric oxide mechanism.

Astrocytes have been reported to play a neuropathogenic role within the brain, although little is known about the mechanism underlying astrocyte-mediated neuronal injury. We investigated the hypothesis that cytokine-stimulated astrocytes adversely affect neuronal cell survival via generation of the free radical nitric oxide (NO). Primary human astrocytes produced substantial amounts of NO in response to interleukin (IL)-1 alpha or IL-1 beta, which was blocked by the NO synthase inhibitor NG-mono-methyl-L-arginine (NMMA). IL-1 beta-induced NO production was markedly potentiated by interferon (IFN)-gamma. IL-1 receptor agonist protein (IRAP) totally blocked NO generation by cytokine-stimulated astrocytes. Using reverse transcription-polymerase chain reaction and sequencing analyses of the astrocyte NO synthase gene, we found a single band encoding for a 615 bp product that was identical to the corresponding sequence reported for human hepatocytes. Treatment of human fetal brain cell cultures with IL-1 beta plus IFN-gamma resulted in marked neuronal loss, as assessed by microscopic analysis and measurement of lactate dehydrogenase release. This cytokine-induced neuronal damage was blocked by simultaneous treatment of the brain cell cultures with NMMA or IRAP, suggesting a critical role of IL-1. These findings indicate that cytokine-stimulated astrocytes are neurotoxic via a NO-mediated mechanism and point to potential new therapies for neurodegenerative disorders that involve cytokines and reactive astrocytes.

Nitric oxide synthesis in cardiac myocytes and fibroblasts by inflammatory cytokines

The aim was to investigate nitric oxide (NO) synthase activity in cultured neonatal rat cardiac myocytes and fibroblasts upon treatment with inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), IL-2, IL-6, IL-8, transforming growth factor beta (TGF-beta) and gram negative bacterial lipopolysaccharide (LPS). NO and guanosine 3',5'-cyclic monophosphate (cGMP) synthesis was measured in cultured neonatal rat cardiac myocytes and fibroblasts, using Griess reagent and an enzyme immunoassay kit, respectively. The expression of inducible NO synthase (iNOS) mRNA and protein was assayed by northern and western blotting, respectively. Incubation of cardiac myocytes for 24 h with IL-1 beta (10 ng.ml-1) or LPS (1 microgram.ml-1) caused significant increases in NO and cGMP production. TNF-alpha, IL-2, IL-6, IL-8, and TGF-beta showed no significant effect on their production. IL-1 beta induced NO and cGMP production in a time and dose dependent manner. IL-1 beta also increased iNOS mRNA and protein accumulation in cardiac myocytes. Simultaneous incubation of IL-1 beta with NG-monomethyl-L-arginine, genistein, calphostin C, cycloheximide, or actinomycin D completely inhibited the IL-1 beta induced NO production by cardiac myocytes. TGF-beta, dexamethasone, or cyclosporin A also dose dependently inhibited the IL-1 beta induced NO production. Exposure to IL-1 beta for 12-24 h decreased the beating rate of cardiac myocytes, but addition of dexamethasone completely overcame this inhibition. In contrast to cardiac myocytes, incubation of cardiac fibroblasts for 24 h with IL-1 beta or LPS showed no significant effect on NO or cGMP production. These observations suggest that IL-1 beta/LPS responsive iNOS, which is an important regulator of contractile function of the heart, is present in cardiac myocytes but not in cardiac fibroblasts.

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide.

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.

Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide.

Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.

Interleukin-lβ-Induced Nitric Oxide Production in Isolated Rat Pancreatic Islets Requires Gene Transcription and May Lead to Inhibition of the Krebs Cycle Enzyme Aconitase*

The aim of this study was to characterize the dynamics and functional relevance of interleukin-1 beta (IL-1 beta)-induced nitric oxide production in isolated pancreatic islets. Thus, islets were isolated from adult rats, precultured for 3-5 days in medium RPMI-1640 plus 10% fetal calf serum, and then exposed to IL-1 beta for different time periods, after which islet nitrite production and aconitase activity were determined. IL-1 beta (5 ng/ml) did not increase islet nitrite production during the first hour of incubation. Moreover, the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (Meth-arg; 5 mM) failed to prevent the initial (90 min) IL-1 beta-induced increase in islet insulin release. After 4, 7, and 24 h, however, nitrite production was increased by 50%, 93%, and 139%, respectively. Islet aconitase activity and glucose oxidation rates were decreased by 70% after incubation for 24 h with IL-1 beta. Both Meth-arg and N alpha-p-tosyl-L-lysine chloromethyl ketone (0.1 mM), a protease inhibitor, could completely counteract the IL-1 beta-induced increases in nitrite production and inhibition of aconitase activity and glucose oxidation rates. In a separate series of experiments, islets were incubated for 60 min with or without IL-1 beta and the RNA synthesis inhibitor actinomycin-D (5 micrograms/ml) and subsequently incubated for another 9 h without any additions. The presence of actinomycin-D during the 1-h IL-1 beta incubation period prevented the IL-1 beta-induced rise in nitrite production and the IL-1 beta-induced inhibition of aconitase activity and insulin release. It is concluded that IL-1 beta-induced nitric oxide production is a late event which requires gene transcription and does not mediate the initial stimulatory effects of IL-1 beta on beta-cell function. However, the gradually augmented rate of nitric oxide production may inhibit the enzyme aconitase, leading to a suppressed mitochondrial activity and a defective insulin release in response to nutrient secretagogues.