Nitric oxide synthase induction,cGMP elevation,and biopterin synthesis in vascular smooth muscle cells stimulated with interleukin-1beta in hypoxia

In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Effects of nitric oxide and peroxynitrite on endotoxin-induced leukocyte adhesion to endothelium

Leukocyte accumulation has been shown to be increased in sepsis. Moreover, in inducible nitric oxide synthase (iNOS) knockout mice, a further increase in leukocyte accumulation has been observed during sepsis, suggesting that nitric oxide (NO) may affect leukocyte/endothelial interaction. Accelerated peroxynitrite formation also occurs during sepsis. In the present study, the effect of peroxynitrite or NO on leukocyte adhesion to nitric oxide synthase (NOS)-inhibited or endotoxin-treated endothelium was examined. Bovine aortic endothelial cells were treated with either L-NAME or lipopolysaccharide (LPS) and interferon-gamma for 4 hr and subsequent leukocyte adhesion was measured. Both L-NAME and LPS treatment resulted in increased leukocyte adhesion compared with control. Neither a peroxynitrite donor, SIN-1, nor a direct NO donor, DETA-NO, had any effect on leukocyte adhesion to untreated endothelium. However, when the L-NAME or LPS-treated endothelial cells were treated simultaneously with either SIN-1 or DETA-NO, there was a significant reduction in leukocyte adhesion. Moreover, at the concentrations used in the present study, neither peroxynitrite nor NO showed harmful effects on normal cultured endothelial cells. These data demonstrating inhibition of leukocyte adhesion to endotoxin-treated endothelium suggest that peroxynitrite or NO may exert a beneficial effect during sepsis.     

Synergistic cytokine-induced nitric oxide production in human alveolar epithelial cells.

Nitric oxide (NO) is an important mediator molecule in regulating normal airway function, as well as in the pathophysiology of inflammatory airway diseases. In addition, cytokines are potent messenger molecules at sites of inflammation. The specific relationship among IL-1beta, TNF-alpha, and IFN-gamma on iNOS induction and NO synthesis in human alveolar epithelial cells has not been determined. In addition, rigorous methods to determine potential synergistic action between the cytokines have not been employed. We exposed monolayer cultures of A549 cells to a factorial combination of three cytokines (IL-1beta, TNF-alpha, and IFN-gamma) and three concentrations (0, 5, and 100 ng/mL). TNF-alpha alone does not induce NO production directly; however, it does have a stimulatory effect on IL-1beta-induced NO production. IL-1beta and INF-gamma both induce NO production alone, yet at different concentration thresholds, and act synergistically when present together. In the presence of all three cytokines, the net effect of NO production exceeds the predicted additive effect of each individual cytokine and the two-way interactions. Several plausible mechanisms of synergy among IL-1beta, TNF-alpha, and IFN-gamma in NO production from human alveolar epithelial cells (A549) are proposed. In order to verify the proposed mechanisms of synergy, future experimental and theoretical studies must address several molecular steps through which the iNOS gene is expressed and regulated, as well as the expression and regulation of enzyme cofactors and substrates.     

Cytokines secreted by lymphokine-activated killer cells induce endogenous nitric oxide synthesis and apoptosis in DLD-1 colon cancer cells

IL-2-activated killer lymphocytes (LAK cells) secrete inflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNFalpha) that can induce nitric oxide (NO) synthesis. We evaluated whether LAK cells could activate NO synthesis in human cancer cells. LAK cells and their culture supernatants induced NO synthesis in DLD-1 colon cancer cells in a dose-dependent manner. NO synthesis was inhibited completely by blocking antibodies to IFN-gamma, demonstrating a key role for this LAK cell cytokine in regulating NO synthesis. The addition of TNFalpha antibodies resulted in partial inhibition. Induction of iNOS mRNA and protein expression in DLD-1 cells was detected. Endogenous NO production inhibited DLD-1 cell proliferation and induced apoptosis, processes that were inhibitable by the NO synthase inhibitor N(G)-monomethyl-l-arginine. Our study has identified a novel, non-contact-dependent LAK cell cytotoxic mechanism: induction of growth inhibition and programmed cell death due to endogenous NO synthesis in susceptible human cancer cells.     

GTP cyclohydrolase I is coinduced in hepatocytes stimulated to produce nitric oxide

GTP cyclohydrolase I is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide (NO) synthase. Here we show that GTP cyclohydrolase I mRNA was present in unstimulated hepatocytes and was up-regulated 2- to 3-fold concurrently with iNOS induction induced in vivo by LPS injection and in vitro by stimulation with LPS and inflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta, and interferon-gamma. Hepatocyte GTP cyclohydrolase I enzyme activity increased 2-fold in vivo after LPS. This coinduction of GTP cyclohydrolase I resulted in increased total intracellular biopterin which supported induced NO synthesis. The addition of a GTP cyclohydrolase I inhibitor to the stimulated hepatocytes decreased intracellular biopterin levels and resulted in a decrease in NO production. The results show that GTP cyclohydrolase I is up-regulated by certain acute inflammatory conditions. Further, the results indicate that biopterin is essential as a cofactor for induced NO synthase activity in hepatocytes.     

Cytokine-induced beta-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.

Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and tumour necrosis factor alpha (TNFalpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1beta and IFNgamma (IL-1beta/IFNgamma)induced apoptotic cell death. TNFalpha neither induced beta-cell death nor did it potentiate the effects of IL-1beta, IFNgamma or IL-1beta/IFNgamma . The cytotoxic effect of IL-1beta/IFNgamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1beta/IFNgamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1beta/IFNgamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X(L) blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X(L) as a potential target to combat beta-cell apoptosis.     

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.     

Sesquiterpene lactones inhibit inducible nitric oxide synthase gene expression in cultured rat aortic smooth muscle cells.

Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Sesquiterpene lactones are derived from Mexican-Indian medicinal plants and are known to have potent anti-inflammatory properties. The mechanisms by which sesquiterpene lactones exert their anti-inflammatory effects are not fully understood. In the current studies we determined if the sesquiterpene lactones, parthenolide and isohelenin, modulate iNOS gene expression in cultured rat aortic smooth muscle cells (RASMC) treated with lipopolysaccharide and interferon-gamma. Treatment with parthenolide or isohelenin inhibited NO production and iNOS mRNA expression in a concentration-dependent manner. Transient transfection studies with an iNOS promoter-luciferase reporter plasmid demonstrated that parthenolide and isohelenin also inhibited activation of the iNOS promoter. Inhibition of iNOS promoter activation was associated with inhibition of both I-kappaBalpha degradation and nuclear translocation of NF-kappaB. Neither parthenolide nor isohelenin induced the heat shock response in RASMC. We conclude that sesquiterpene lactones inhibit iNOS gene expression by a mechanism involving stabilization of the I-kappaBalpha/NF-kappaB complex. This effect is not related to induction of the heat shock response. The ability of sesquiterpene lactones to inhibit iNOS gene expression may account, in part, for their anti-inflammatory effects.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Is there a role for neuronal nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta cells?

Nitric oxide (NO), produced by the action of the inducible NO synthase, plays a crucial role in cytokine toxicity to pancreatic beta cells during type 1 diabetes development. It was the aim of this study to analyze the role of the neuronal NOS (nNOS) in proinflammatory cytokine-mediated beta cell toxicity. Expression of different isoforms of nitric oxide synthase in insulin-secreting INS1E cells and rat islets was analyzed by quantitative real-time PCR and Western blotting. The expression of nNOS in insulin-secreting INS1E cells was similar to that found in rat brain, while two other isoforms, namely the endothelial eNOS and inducible iNOS were not expressed in untreated cells. IL-1β alone or in combination with TNF-α and/or IFNγ induced iNOS but not eNOS expression. In contrast, nNOS expression was strongly decreased by the mixture of the three proinflammatory cytokines (IL-1β, TNF-α and IFNγ) both on the gene and protein level in INS1E cells and rat islet cells. The effects of cytokines on glucose-induced insulin-secretion followed the pattern of nNOS expression reduction and, on the other hand, of the iNOS induction. The data indicate that a low level of nitric oxide originating from the constitutive expression of nNOS in pancreatic beta cells is not deleterious. In particular since proinflammatory cytokines reduce this expression. This nNOS suppression can compensate for NO generation by low concentrations of IL-1β through iNOS induction. Thus, this basal nNOS expression level in pancreatic beta cells represents a protective element against cytokine toxicity.     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.     

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.     

Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide

Mesenchymal stem cells (MSCs) can become potently immunosuppressive through unknown mechanisms. We found that the immunosuppressive function of MSCs is elicited by IFNgamma and the concomitant presence of any of three other proinflammatory cytokines, TNFalpha, IL-1alpha, or IL-1beta. These cytokine combinations provoke the expression of high levels of several chemokines and inducible nitric oxide synthase (iNOS) by MSCs. Chemokines drive T cell migration into proximity with MSCs, where T cell responsiveness is suppressed by nitric oxide (NO). This cytokine-induced immunosuppression was absent in MSCs derived from iNOS(-/-) or IFNgammaR1(-/-) mice. Blockade of chemokine receptors also abolished the immunosuppression. Administration of wild-type MSCs, but not IFNgammaR1(-/-) or iNOS(-/-) MSCs, prevented graft-versus-host disease in mice, an effect reversed by anti-IFNgamma or iNOS inhibitors. Wild-type MSCs also inhibited delayed-type hypersensitivity, while iNOS(-/-) MSCs aggravated it. Therefore, proinflammatory cytokines are required to induce immunosuppression by MSCs through the concerted action of chemokines and NO.