3-nitrotyrosine and Crohn-Disease

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

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