Heparin-binding epidermal growth factor-like growth factor inhibits cytokine-induced NF-kappa B activation and nitric oxide production via activation of the phosphatidylinositol 3-kinase pathway

NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-kappaB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-kappaB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by approximately 2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-kappaB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-kappaB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-kappaB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.     

A novel potent inhibitor of inducible nitric oxide inhibitor, ONO-1714, reduces intestinal ischemia-reperfusion injury in rats.

The overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to the pathophysiology of intestinal injury induced by ischemia-reperfusion. The aim of the present study was to examine the effect of selective iNOS inhibition by a cyclic amidine analogue, ONO-1714, on reperfusion-induced small intestinal injury and inflammation in rats. Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion. The luminal nitrite concentration in the small intestine was measured by Griess reaction and the iNOS mRNA expression by RT-PCR. The severity of the intestinal mucosal injury and inflammation were evaluated by several biochemical markers and by the histological findings. The rats which were killed after ischemia-reperfusion had increased luminal concentrations of nitrite and iNOS mRNA expression, in addition to severe intestinal inflammation characterized by significant increases in myeloperoxidase activity, a marker of neutrophil infiltration, and by the mucosal content of CINC-1 cytokine, a neutrophil chemotactic cytokine. Administration with ONO-1714 significantly inhibited the luminal NO production. Reperfusion after 30-min ischemia resulted in an increase in luminal protein and hemoglobin concentrations, with levels reaching a maximum after 60 min of reperfusion. In contrast, pre-treatment with ONO-1714 2h before the ischemia inhibited the increases in luminal protein and hemoglobin concentration in a dose-dependent manner (0.001-0.1mg/kg). The contents of the thiobarbituric acid-reactive substances (a marker of oxidative lipid peroxidation) were significantly increased by ischemia-reperfusion, and this increase was reduced by ONO-1714. After reperfusion, the increase in tissue-associated myeloperoxidase activity, an index of neutrophil infiltration, was significantly inhibited by pre-treatment with ONO-1714. ONO-1714 also inhibited increases in intestinal CINC-1 protein and mRNA expression, as determined by ELISA and RT-PCR, respectively. In conclusion, the improvement of reperfusion-induced intestinal injury by ONO-1714 suggested that an excess of NO, produced by iNOS, may have contributed to the initiation/amplification of intestinal inflammatory injury by various mechanisms, including nitrosative and oxidative damage as well as the enhancement of inflammatory cytokine release.     

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

Background. Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up‐regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. Materials and Methods. Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT‐PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori‐infected epithelial cells, Caco‐2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. Results. iNOS mRNA levels were significantly up‐regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori‐induced iNOS and NO up‐regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL‐8 was released predominantly into basolateral compartment. The addition of IFN‐γ to H. pylori‐infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. Conclusion. These results suggest that apical NO production mediated by MAP kinase in H. pylori‐infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL‐8 may play a role in the tissue inflammation.     

Induced nitric oxide promotes intestinal inflammation following hemorrhagic shock

In hemorrhagic shock (HS), increased cytokine production contributes to tissue inflammation and injury through the recruitment of neutrophils [polymorphonuclear cells (PMN)]. HS stimulates the early expression of inducible nitric oxide synthase (iNOS) that modulates proinflammatory activation after hemorrhage. Experiments were performed to determine the contribution of iNOS to gut inflammation and dysmotility after HS. Rats subjected to HS (mean arterial pressure 40 mmHg for 2.5 h followed by resuscitation and death at 4 h) demonstrated histological signs of mucosal injury, impairment of intestinal smooth muscle contractility, extravasation of PMN, and increased gut mRNA levels of ICAM-1, IL-6, and granulocyte colony-stimulating factor (G-CSF). In addition, DNA binding activity of NF-kappaB and Stat3, an IL-6 signaling intermediate, was significantly increased. In shocked rats treated with the selective iNOS inhibitor l-N(6)-(1-iminoethyl)lysine at the time of resuscitation, histological signs of intestinal injury and PMN infiltration were reduced and muscle contractility was almost completely restored. Selective iNOS inhibition in shocked animals reduced the binding activity of NF-kappaB and Stat3 and reduced mRNA levels of ICAM-1, IL-6, and G-CSF. The results of studies using iNOS knockout mice subjected to HS were similar. We propose that early upregulation of iNOS contributes to the inflammatory response in the gut wall and participates in the activation of signaling cascades and cytokine expression that regulate intestinal injury, PMN recruitment, and impaired gut motility.     

Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression

Inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) play an important role in the pathology of ischemia-reperfusion. This study sought to determine if the proinflammatory effects of complement modulate iNOS and SOD in the rat after gastrointestinal ischemia and reperfusion (GI/R). An inhibitory or noninhibitory anti-complement component 5 (C5) monoclonal antibody (18A or 16C, respectively) was administered before GI/R. RT-PCR revealed a significant increase in intestinal iNOS mRNA compared with sham after GI/R that was attenuated significantly by 18A. Immunohistochemistry demonstrated increased iNOS protein expression within the intestinal crypts after GI/R. Cu/Zn SOD (mRNA and protein) was unaffected by GI/R, whereas Cu/Zn SOD activity was reduced significantly. Mn SOD protein expression was decreased significantly by GI/R. Anti-C5 preserved Cu/Zn SOD activity and Mn SOD protein expression. Staining for nitrotyrosine showed that anti-C5 treatment reduced protein nitration in the reperfused intestine. Immunohistochemistry demonstrated prominent phosphorylated (p) inhibitory factor-kappaB (IkappaB)-alpha staining of intestinal tissue after GI/R, whereas anti-C5 reduced p-IkappaB-alpha expression. These data indicate that complement may mediate tissue damage during GI/R by increasing intestinal iNOS and decreasing the activity and protein levels of Cu/Zn SOD and Mn SOD, respectively.     

Role of nitric oxide in liver ischemia and reperfusion injury

The present study was designed to assess the role of endothelial cell and inducible nitric oxide synthase (eNOS, iNOS)-derived NO in ischemia/reperfusion (I/R)-induced pro-inflammatory cytokine expression and tissue injury in a murine model of hepatic I/R. Forty-five min of partial hepatic ischemia and 3 h of reperfusion resulted in a significant increase in liver injury as assessed by serum alanine aminotransferase and histopathology which occurred in the absence of neutrophil infiltration. Both iNOS and eNOS deficient mice exhibited enhanced liver injury when compared to their wild type (wt) controls again in the absence of neutrophil infiltration. Interestingly, message expression for both tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12) were enhanced in eNOS, but not iNOS-deficient mice at 1 h post-ischemia when compared to their wt controls. In addition, eNOS message expression appeared to be up-regulated between 1 and 3 h ofreperfusion in wt mice while iNOS deficient mice exhibited substantial increases at I but not 3 h. Taken together, these data demonstrate the ability of eNOS and iNOS to protect the post-ischemic liver, however their mechanisms of action may be very different.     

Proinflammatory agents,IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1α, TNFα, and IFNγ. Inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGFβ) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption. © 1996 Wiley-Liss, Inc.     

Role of nitric oxide in liver ischemia and reperfusion injury

The present study was designed to assess the role of endothelial cell and inducible nitric oxide synthase (eNOS, iNOS)-derived NO in ischemia/reperfusion (I/R)-induced pro-inflammatory cytokine expression and tissue injury in a murine model of hepatic I/R. Forty-five min of partial hepatic ischemia and 3 h of reperfusion resulted in a significant increase in liver injury as assessed by serum alanine aminotransferase and histopathology which occurred in the absence of neutrophil infiltration. Both iNOS and eNOS deficient mice exhibited enhanced liver injury when compared to their wild type (wt) controls again in the absence of neutrophil infiltration. Interestingly, message expression for both tumor necrosis factor-alpha (TNF-) and interleukin 12 (IL-12) were enhanced in eNOS, but not iNOS-deficient mice at 1 h post-ischemia when compared to their wt controls. In addition, eNOS message expression appeared to be up-regulated between 1 and 3 h of reperfusion in wt mice while iNOS deficient mice exhibited substantial increases at 1 but not 3 h. Taken together, these data demonstrate the ability of eNOS and iNOS to protect the post-ischemic liver, however their mechanisms of action may be very different.     

Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-kappaB and p38 MAPK signaling pathways.

Mercury is well known to adversely affect the immune system; however, little is known regarding its molecular mechanisms. Macrophages are major producers of nitric oxide (NO) and this signaling molecule is important in the regulation of immune responses. The present study was designed to determine the impact of mercury on NO and cytokine production and to investigate the signaling pathways involved. The murine macrophage cell line J774A.1 was used to study the effects of low-dose inorganic mercury on the production of NO and proinflammatory cytokines. Cells were treated with mercury in the presence or absence of lipopolysaccharide (LPS). Mercury (5-20 microM) dose-dependently decreased the production of NO in LPS-stimulated cells. Concomitant decreases in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein were detected. Treatment of J774A.1 cells with mercury alone did not affect the production of NO nor the expression of iNOS mRNA or protein. Interestingly, mercury alone stimulated the expression of tumor necrosis factor alpha (TNFalpha), and increased LPS-induced TNFalpha and interleukin-6 mRNA expression. Mercury inhibited LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) but had no effect alone. In contrast, mercury activated p38 mitogen-activated protein kinase (p38 MAPK) and additively increased LPS-induced p38 MAPK phosphorylation. These results indicate that mercury suppresses NO synthesis by inhibition of the NF-kappaB pathway and modulates cytokine expression by p38 MAPK activation in J774A.1 macrophage cells.     

Intestinal myofibroblasts produce nitric oxide in response to combinatorial cytokine stimulation

Inflammatory bowel disease (IBD) patients display elevated levels of intraluminal nitric oxide (NO). NO can react with other molecules to form toxic compounds, which has led to the idea that NO may be an important mediator of IBD. However, the cellular source of NO and how its production is regulated in the intestine are unclear. In this study we aimed to determine if intestinal myofibroblasts produce NO in response to the IBD‐associated cytokines IL‐1β, TNFα, and IFNγ. Intestinal myofibroblasts were isolated from mice and found to express inducible nitric oxide synthase (iNOS) mRNA, but not endothelial NOS or neuronal NOS. Individual treatment of myofibroblasts with IL‐1β, TNFα, or IFNγ had no effect on NO production, but stimulation with combinations of these cytokines synergistically increased iNOS mRNA and protein expression. Treatment with TNFα or IFNγ increased cell surface expression of IFNγRI or TNFRII, respectively, suggesting that these cytokines act in concert to prime NO production by myofibroblasts. Impairment of NF‐κB activity with a small molecule inhibitor was sufficient to prevent increased expression of IFNγRI or TNFRII, and inhibition of Akt, JAK/STAT, or NF‐κB blocked nearly all NO production induced by combinatorial cytokine treatment. These data indicate that intestinal myofibroblasts require stimulation by multiple cytokines to produce NO and that these cytokines act through a novel pathway involving reciprocal cytokine receptor regulation and signaling by Akt, JAK/STAT, and NF‐κB. J. Cell. Physiol. 228: 572–580, 2013. © 2012 Wiley Periodicals, Inc.     

Epidermal growth factor and interleukin-1beta synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells

Epidermal growth factor (EGF) is one of the trophic factors for intestinal adaptation after small bowel transplantation (SBT). A recent report indicates that nitric oxide (NO) has cytoprotective effects on bacterial translocation (BT) after SBT. We hypothesized that EGF stimulates the expression of the inducible NO synthase (iNOS) gene in the graft after SBT, followed by increased production of NO, resulting in the decrease of BT. Intestinal epithelial cells (IEC)-6 were treated with EGF and/or IL-1beta in the presence and absence of phosphatidylinositol 3-kinase (PI3-kinase) and EGF receptor kinase inhibitors (LY-294002 and tyrphostin A25). The induction of NO production and iNOS and its signal molecules, including the inhibitory protein of NF-kappaB (IkappaB), NF-kappaB, and Akt, were analyzed. IL-1beta stimulated the degradation of IkappaB and the activation of NF-kappaB but had no effect on iNOS induction. EGF, which had no effect on the NF-kappaB activation and iNOS induction, stimulated the upregulation of type 1 IL-1 receptor (IL-1R1) through PI3-kinase/Akt. Simultaneous addition of EGF and IL-1beta stimulated synergistically the induction of iNOS, leading to the increased production of NO. Our results indicate that EGF and IL-1beta stimulate two essential signals for iNOS induction in IEC-6 cells: the upregulation of IL-1R1 through PI3-kinase/Akt and the activation of NF-kappaB through IkappaB kinase, respectively. Simultaneous addition of EGF and IL-1beta can enhance the production of NO, which may contribute to the cytoprotective effect of EGF against intestinal injury.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Inducible nitric oxide synthase mediates early epithelial repair of porcine ileum

Reports conflict regarding the effect of nitric oxide (NO) on intestinal epithelium. In chronic injury, NO appears detrimental by combining with reactive oxygen to form potent-free radicals. In contrast, inhibition of NO synthesis after acute injury exacerbates damage and inflammation. Recent studies have disclosed constitutive expression of inducible NO synthase (iNOS) by normal intestinal epithelia, yet little attention has been given to the role of iNOS in acute epithelial repair. We studied the local effects of iNOS on early epithelial repair of porcine ileal mucosa injured by deoxycholate within Ussing chambers. iNOS was constitutively expressed by the villous epithelium, and after deoxycholate injury, iNOS was expressed by injured and detaching enterocytes. Selective inhibition of iNOS abolished increases in NO synthesis and villous reepithelialization after injury. Exogenous L-arginine rescued baseline reepithelialization from NOS inhibitors but was only capable of stimulating additional repair in the presence of serum. These results demonstrate that iNOS-derived NO is a key mediator of early villous reepithelialization following acute mucosal injury.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

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.     

IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth

IFN-γ-mediated inducible nitric oxide synthase (iNOS) expression is critical for controlling chlamydial infection through microbicidal nitric oxide (NO) production. Interleukin-17A (IL-17A), as a new proinflammatory cytokine, has been shown to play a protective role in host defense against Chlamydia muridarum (Cm) infection. To define the related mechanism, we investigated, in the present study, the effect of IL-17A on IFN-γ induced iNOS expression and NO production during Cm infection in vitro and in vivo. Our data showed that IL-17A significantly enhanced IFN-γ-induced iNOS expression and NO production and inhibited Cm growth in Cm-infected murine lung epithelial (TC-1) cells. The synergistic effect of IL-17A and IFN-γ on Chlamydia clearance from TC-1 cells correlated with iNOS induction. Since one of the main antimicrobial mechanisms of activated macrophages is the release of NO, we also examined the inhibitory effect of IL-17A and IFN-γ on Cm growth in peritoneal macrophages. IL-17A (10 ng/ml) synergizes with IFN-γ (200 U/ml) in macrophages to inhibit Cm growth. This effect was largely reversed by aminoguanidine (AG), an iNOS inhibitor. Finally, neutralization of IL-17A in Cm infected mice resulted in reduced iNOS expression in the lung and higher Cm growth. Taken together, the results indicate that IL-17A and IFN-γ play a synergistic role in inhibiting chlamydial lung infection, at least partially through enhancing iNOS expression and NO production in epithelial cells and macrophages.