3-nitrotyrosine and Colitis--Ulcerative

Ulcerative colitis is characterized by colonic mucosal bleeding and ulceration, often with repeated active and remission stages. One factor in ulcerative colitis development is increased susceptibility to commensal bacteria and lipopolysaccharide (LPS). LPS activates macrophages to release nitric oxide (NO) through Toll-like receptor 4 (TLR4) signaling. However, whether NO is beneficial or detrimental to colitis remains controversial. In this study, we investigated whether NO enhances the development of colitis in mice treated with dextran sulfate sodium (DSS) and inflammation in cells treated with low-dose LPS. An NO donor, NOC18, induced colitis and increased CD14 protein and nitrotyrosine levels in colonic macrophages from mice treated with DSS for 7 d (molecular weight: 5000). In the mouse peritoneal macrophage cell line RAW264.7 stimulated with 3 ng/mL LPS, NO activated the CD14-TLR4-nuclear factor kappa B (NF-κB) axis. Low-dose LPS stimulation did not change the levels of signal transducer and activator of transcription (STAT) 3 phosphorylation, CD14, inducible NO synthase, interleukin (IL)-6, or NF-κB. In addition, low-dose LPS increased phosphorylation of src homology protein tyrosine phosphatase 2 (SHP2), a negative regulator of STAT3 phosphorylation. However, NO decreased SHP2 phosphorylation and significantly activated the downstream signaling molecules. NO increased SHP2 nitration in LPS-stimulated RAW264.7 cells and DSS-treated mice. These results indicate that SHP2 nitration in macrophages might be involved in activation of the CD14-TLR4-NF-κB axis through STAT3 signaling in mice with DSS-induced colitis.

Topics: Animals; Colitis, Ulcerative; Dextran Sulfate; Disease Models, Animal; Inflammation; Intestinal Mucosa; Lipopolysaccharides; Mice; Nitric Oxide; Nitric Oxide Donors; Phosphorylation; RAW 264.7 Cells; SH2 Domain-Containing Protein Tyrosine Phosphatases; Signal Transduction; Toll-Like Receptor 4; Tyrosine

Little is known about the anti-colitis effect of tryptophan or its metabolites. Here, the protective effect and its mechanism of tryptophan administration on dextran sulfate sodium -induced colitis in mice was studied.. Twenty C57black6 female mice were equally divided into the control group, and treatment group. The control group received a standard CE-2 diet, while the tryptophan group received a CE-2 diet containing 0.5% l-tryptophan. After one week on this diet, all mice were orally administered a solution of 3.5% dextran sulfate sodium for 12 days to induce colitis. Changes in body weight and bloody stool frequency were monitored during dextran sulfate sodium administration. At 12 days post initial dextran sulfate sodium administration, all mice were sacrificed and the histology of their colonic tissue was examined. The nitrotyrosine levels in colonic tissues in both groups, and nitrate and nitrite levels in the urine of the control group, the tryptophan group and the group of mice without dextran sulfate sodium administration was measured.. The tryptophan group showed significantly attenuated body weight loss, bloody stool frequency and ameliorated histological changes of colitis. While tryptophan treatment significantly reduced nitrotyrosine level in the colonic tissues, there was no significant reduction in urine nitrate and nitrite levels compared with the (dextran sulfate sodium-induced) control group.. Tryptophan treatment ameliorated dextran sulfate sodium-induced colitis in this study. One of the anti-colitis mechanisms of tryptophan treatment is attributable to an anti-nitration effect, and may not be via the suppression of nitric oxide generation.

Topics: Animal Feed; Animals; Antidepressive Agents, Second-Generation; Body Weight; Colitis, Ulcerative; Dextran Sulfate; Disease Models, Animal; Female; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Nitric Oxide; Oxidative Stress; Random Allocation; Tryptophan; Tyrosine

Cyclooxygenase-2 (COX-2) and nitric oxide synthase 2 (NOS2) were detected and localized in 20 pigs with ulcerative colitis caused by natural infection with Salmonella typhimurium. Evidence of NOS2 activity was determined by the formation of nitrotyrosine, a reaction product of peroxynitrite, in NOS2-expressing ulcerative colons by immunohistochemistry. Transcript RNA of COX-2 and NOS2 was consistently detected in colonic tissues from the 20 pigs with ulcerative colitis by using reverse transcription-polymerase chain reaction. Immunohistochemical signals for COX-2 and NOS2 were detected in the ulcerated area of all 20 pigs. Expression of COX-2 and NOS2 was identified continuously within inflammatory intestinal lesions but was minimal in unaffected regions of the colon of S. typhimurium-infected pigs. The immunohistochemistry of serial sections of intestine indicated that the majority of colons containing numerous COX-2-positive cells also had numerous NOS2-positive cells. Localization of NOS2 and a nitrotyrosine antigen was prominent in neutrophils and macrophages in the periphery of the lesions. Simultaneous detection of COX-2 and NOS2 RNA and protein indicated functional activity of prostaglandin and NO production in vivo. This study suggested that COX-2 and NOS2 expression may play a role in the pathophysiologic processes in ulcerative colitis caused by S. typhimurium.

Topics: Animals; Colitis, Ulcerative; Cyclooxygenase 2; DNA Primers; Immunohistochemistry; Isoenzymes; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Polymerase Chain Reaction; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salmonella Infections, Animal; Salmonella typhimurium; Swine; Swine Diseases; Tyrosine

In ulcerative colitis (UC), inflammatory damage is associated with increased production of pro-inflammatory cytokines and nitric oxide through the inducible nitric oxide synthase (iNOS) pathway. In an animal model of acute experimental colitis we have previously shown amelioration of inflammation with the highly selective iNOS inhibitor 1400W. The aim of the present study was to investigate the effects of selective iNOS inhibition on the production of pro-inflammatory cytokines by the colon mucosa in UC.. Inflamed and uninflamed mucosa from patients with severe UC were incubated with a highly selective iNOS inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W), with a relatively selective cNOS inhibitor N(G)-nitro-L-arginine-methyl-esther (L-NAME), or with an NO-donor, S-nitroso-acetylpenicillamine (SNAP). Cytokine concentrations in the incubation medium were quantitated with ELISA.. Compared to uninflamed mucosa there was an increase in iNOS protein and nitrotyrosine levels in inflamed mucosal samples. Immunolocalization of iNOS and nitrotyrosine showed their expression in inflammatory cells in the lamina propria. Expression of iNOS was also found in the epithelial brush border. Selective inhibition of iNOS suppressed the release of tumour necrosis factor alpha (TNF-alpha, by 66%) and interleukin-6 (IL-6, by 27%). The NO-donor, SNAP, augmented the secretion of TNF-alpha, IL-6 and IL-1-beta (by 62%, 52% and 175%, respectively) and decreased the release of IL-1 receptor antagonist (IL-1Ra, by 34%) by the inflamed mucosa. Moreover, in uninflamed samples, 1400W suppressed the production of TNF-alpha (by 69%) and incubation with SNAP decreased IL-6 concentrations by 48%. The cNOS over iNOS selective inhibitor L-NAME had no significant effects on the accumulation of cytokines.. Selective inhibition of iNOS suppresses mucosal TNF-alpha and IL-6 release in active UC, whereas NO seems to exacerbate the inflammatory response. These results suggest that selective iNOS inhibition may have therapeutic promise in the treatment of UC.

Topics: Adult; Amidines; Benzylamines; Colitis, Ulcerative; Colon; Cytokines; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Humans; In Vitro Techniques; Inflammation Mediators; Interleukin-1; Interleukin-6; Intestinal Mucosa; Male; Middle Aged; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; S-Nitroso-N-Acetylpenicillamine; Tumor Necrosis Factor-alpha; Tyrosine

Overproduction of colonic oxidants contributes to mucosal injury in inflammatory bowel disease (IBD) but the mechanisms are unclear. Our recent findings using monolayers of intestinal cells suggest that the mechanism could be oxidant induced damage to cytoskeletal proteins. However, oxidants and oxidative damage have not been well characterised in IBD mucosa.. To determine whether there are increases in oxidants and in tissue and cytoskeletal protein oxidation in IBD mucosa.. We measured nitric oxide (NO) and markers of oxidative injury (carbonylation and nitrotyrosination) to tissue and cytoskeletal proteins in colonic mucosa from IBD patients (ulcerative colitis, Crohn's disease, specific colitis) and controls. Outcomes were correlated with IBD severity score.. Inflamed mucosa showed the greatest increases in oxidants and oxidative damage. Smaller but still significant increases were seen in normal appearing mucosa of patients with active and inactive IBD. Tissue NO levels correlated with oxidative damage. Actin was markedly (>50%) carbonylated and nitrated in inflamed tissues of active IBD, less so in normal appearing tissues. Tubulin carbonylation occurred in parallel; tubulin nitration was not observed. NO and all measures of oxidative damage in tissue and cytoskeletal proteins in the mucosa correlated with IBD severity. Disruption of the actin cytoarchitecture was primarily within the epithelial cells and paracellular area.. Oxidant levels increase in IBD along with oxidation of tissue and cytoskeletal proteins. Oxidative injury correlated with disease severity but is also present in substantial amounts in normal appearing mucosa of IBD patients, suggesting that oxidative injury does not necessarily lead to tissue injury and is not entirely a consequence of tissue injury. Marked actin oxidation (>50%)-which appears to result from cumulative oxidative damage-was only seen in inflamed mucosa, suggesting that oxidant induced cytoskeletal disruption is required for tissue injury, mucosal disruption, and IBD flare up.

Topics: Actins; Adult; Blotting, Western; Colitis, Ulcerative; Colon; Colonic Diseases, Functional; Crohn Disease; Cytoskeletal Proteins; Female; Free Radicals; Humans; Immunoblotting; Intestinal Mucosa; Luminescent Measurements; Male; Nitric Oxide; Oxidation-Reduction; Tubulin; Tyrosine

Intestinal inflammation is accompanied by excessive production of reactive oxygen and nitrogen metabolites. In order to counteract their harmful effects, the intestinal mucosa contains an extensive system of antioxidants. It has previously been shown that the levels of and the balance between the most important antioxidants are seriously impaired within the intestinal mucosa from inflammatory bowel disease (IBD) patients compared with normal mucosa. The present study investigated the consequences of this antioxidative imbalance by evaluating parameters of oxidative stress-related mucosal damage in the same tissue samples. The extent of apoptosis, peroxynitrite-mediated protein nitration (3-NT), and lipid peroxidation were assessed in relation to the expression of nitric oxide synthase (NOS) and the superoxide-producing enzyme xanthine oxidase (XO). In addition, bi- and multi-variate regression analyses were performed to associate these parameters with the levels of the antioxidants assessed previously. Apoptotic cell death was visualized by TUNEL staining in luminal epithelium of normal controls, and in IBD additionally in the inflammatory infiltrate and in deeper parts of the crypts, but its frequency was unrelated to the severity of inflammation. In Crohn's disease (CD), epithelial apoptosis levels were strongly associated with the expression of XO, implying a role for this enzyme in the regulation of epithelial cell homeostasis, although its levels were unaffected by intestinal inflammation and were comparable to those in normal control mucosa. 3-NT immunoreactivity was substantially increased in luminal crypt cells, neutrophils, and mononuclear cells in the inflamed mucosa of ulcerative colitis (UC) patients. The inflamed IBD luminal epithelium, but not the inflammatory cells, also contained increased amounts of NOS. The immunoreactivity of both 3-NT and NOS was significantly higher in UC than in CD. Unexpectedly, the increased 3-NT expression in UC was associated with neutrophilic myeloperoxidase and not with NOS, which suggests that 3-NT is formed in areas with a dense neutrophilic infiltrate via a peroxynitrite-independent oxidation pathway. Lipid peroxidation, as estimated by the malondialdehyde (MDA) concentration, was elevated in both the inflamed CD and the inflamed UC mucosa, and was identified in the luminal epithelium using a histochemical technique. In CD, lipid peroxidation was independently associated with the concentration of metallothio

Topics: Adult; Aged; Antioxidants; Apoptosis; Colitis, Ulcerative; Crohn Disease; Female; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Lipid Peroxidation; Male; Middle Aged; Nitric Oxide Synthase; Oxidative Stress; Tyrosine; Xanthine Oxidase

Luminal nitric oxide (NO) is greatly increased in the colon of patients with collagenous and ulcerative colitis. To define the source and consequence of enhanced NO production we have studied expression of NO synthase (NOS) isoforms and nitrotyrosine in mucosal biopsies from these patients. In addition, effects on colonic fluid transfer caused by manipulating the substrate of NOS were studied in patients with collagenous colitis.. Eight patients with collagenous colitis, nine with active ulcerative colitis, and 10 with uninflamed bowel were included.. Expression of NOS isoforms was quantified by western blotting. Inducible NOS (iNOS) and nitrotyrosine were localised by immunohistochemistry. Modulation of NOS activity by topical N(G)-monomethyl-L-arginine (L-NMMA) or L-arginine was assessed during perfusion of whole colon. Plasma and perfusate nitrite/nitrate (NOx) was measured by Griess' reaction.. Both in collagenous and ulcerative colitis, expression of iNOS was 10(2)-10(3) higher (p<0.001) than in uninflamed bowel and localised primarily to the epithelium. Endothelial NOS was evenly expressed in all groups while neuronal NOS was undetectable. Nitrotyrosine was markedly expressed in active ulcerative colitis but rarely detected in collagenous colitis and never in uninflamed bowel. In collagenous colitis, the output of NOx was markedly increased compared with uninflamed bowel (283 (58) v <37 nmol/min; p<0.01) and fluid was net secreted. L-NMMA reduced the output of NOx by 13-66% (95% confidence intervals) and secretion of fluid by 25-109% whereas L-arginine increased the output of NOx by 3-39% and secretion of fluid by 15-93%.. In collagenous colitis, as opposed to ulcerative colitis, upregulation of iNOS occurs in the absence of nitrotyrosine formation and mucosal damage. Excess generation of NO may be the primary cause of diarrhoea in this condition.

Topics: Adult; Aged; Arginine; Blotting, Western; Body Fluids; Case-Control Studies; Colitis, Ulcerative; Confidence Intervals; Female; Humans; Intestinal Absorption; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Statistics, Nonparametric; Tyrosine; Up-Regulation

Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation.. Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 with severe inflammation).. Inducible NOS activity was determined in the colonic mucosa by measuring the conversion of L-arginine to citrulline in the absence of calcium. The localisation of NOS and nitrotyrosine immunoreactivity was assessed immunohistochemically using the labelled streptavidin biotin method.. Inducible NOS activity increased in parallel with the degree of inflammation of the mucosa. Expression of inducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed by nitrotyrosine staining was frequently observed in the lamina propria of actively inflamed mucosa.. Nitric oxide and peroxynitrite formation may play an important role in causing irreversible cellular injury to the colonic mucosa in patients with active ulcerative colitis.

Topics: Acute Disease; Adolescent; Adult; Analysis of Variance; Antibodies, Monoclonal; Blotting, Western; Colitis, Ulcerative; Colon; Enzyme Induction; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Isoenzymes; Male; Middle Aged; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Tyrosine

Nitric oxide (NO) and reactive oxygen species (ROS) are important mediators in the pathogenesis of inflammatory bowel disease (IBD). NO in IBD can be either harmful or protective. NO can react with superoxide anions (O2.-), yielding the toxic oxidizing agent peroxynitrite (ONOO-). Peroxynitrite induces nitration of tyrosine residues (nitrotyrosine), leading to changes of protein structure and function. The aim of this study was to identify the cellular source of inducible nitric oxide synthase (iNOS) and to localize superoxide anion-producing cells in mucosal biopsies from patients with active IBD. Additional studies were performed to look at nitrotyrosine formation as a measure of peroxynitrite-mediated tissue damage. For this, antibodies against iNOS, endothelial NOS (eNOS), and nitrotyrosine were used. ROS-producing cells were detected cytochemically. Inflamed mucosa of patients with active IBD showed intense iNOS staining in the epithelial cells. iNOS could not be detected in non-inflamed mucosa of IBD patients and control subjects. eNOS was present in blood vessels, without any difference in the staining intensity between IBD patients and control subjects. ROS-producing cells were increased in the lamina propria of IBD patients; a fraction of these cells were CD15-positive. Nitrotyrosine formation was found on ROS-positive cells. These results show that iNOS is induced in epithelial cells from patients with active ulcerative colitis or Crohn's disease. Nitration of proteins was detected only on the ROS-producing cells at some distance from the iNOS-producing epithelial cells. These findings indicate that tissue damage during active inflammation in IBD patients is probably more related to ROS-producing cells than to NO. One may speculate that NO has a protective role when during active inflammation other mucosal defence systems are impaired.

Topics: Adult; Colitis, Ulcerative; Crohn Disease; Epithelial Cells; Female; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Male; Middle Aged; Nitric Oxide Synthase; Reactive Oxygen Species; Tyrosine