Increased Nitric Oxide Production and Inducible Nitric Oxide Synthase Activity in Colonic Mucosa of Patients with Active Ulcerative Colitis and Crohn's Disease

It is postulated that an enhanced production ofnitric oxide by inflamed intestine plays a role in thepathophysiology of active inflammatory bowel disease. Inthis study, systemic NO_x concentrations and colonic nitric oxide synthase activity weredetermined in patients with ulcerative colitis orCrohn's disease. The relationship between these twoparameters and disease activity, as well as differences in nitric oxide synthase activity betweenulcerative colitis and Crohn's disease, were areas ofspecific focus. Patients with active ulcerative colitisand Crohn's disease had significantly elevated plasma NO_x concentrations; a positivecorrelation was found between NO_x values andinducible nitric oxide synthase activities in the activemucosa of these patients. In active ulcerative colitis,levels of inducible nitric oxide synthase were significantlyelevated in both normal and inflamed mucosa, althoughinducible nitric oxide synthase activity was higher inthe latter. These colonic inducible nitric oxidesynthase activities correlated well with the results ofendoscopic and histologic grading of inflammation. Therewas no increase in constitutive nitric oxide synthaseactivity in patients with active ulcerative colitis. However, constitutive nitric oxidesynthase activity was significantly increased in theinflamed mucosa in patients with Crohn's disease. InCrohn's disease, elevated inducible nitric oxidesynthase activity was found in both normal and inflamedmucosa, with no significant difference between thetissues. Such differences in nitric oxide production inthe colonic mucosa possibly reflect the significant differences in the pathophysiology andcharacteristic clinical features between ulcerativecolitis and Crohn's disease.     

Direct Determination of Colonic Nitric Oxide Level—A Sensitive Marker of Disease Activity in Ulcerative Colitis

Objective : In active ulcerative colitis, colonic nitric oxide (NO) generation is enhanced and probably has an important role in its pathogenesis. We tested the reliability of an NO electrode in monitoring colonic NO levels in ulcerative colitis patients and control subjects and its possible usage as a marker of disease activity. Methods : Colonic NO level was determined by the NO detection system model NO-501 (InterMedical, Nagoya, Japan). The working electrode was inserted into a 7-mm diameter polyvinyl tube and introduced at a distance 6 cm from the anus. In each subject sigmoidoscopy was performed and mucosal biopsies were obtained. NO synthase (NOS) activity was determined by monitoring the conversion of ³H-arginine to citrulline. Results : Colonic NO level is significantly increased in patients with active ulcerative or Crohn's colitis—more than 2-fold higher than in control subjects. There was good correlation between colonic NO level and NOS activity and the clinical and endoscopic indices of disease activity. Conclusion : Direct determination of colonic NO level is convenient, and reliable, and may help to monitor disease activity in ulcerative colitis.     

Greatly increased mucosal nitric oxide in ulcerative colitis determined in situ by a novel nitric oxide-selective microelectrode

Although current nitric oxide (NO) electrodes are simple, selective and sensitive, they are fragile and hard to use in clinical studies of patients. By preparing an improved NO electroneedle that overcomes these defects, we directly measured mucosal NO concentrations in 11 patients (six male, five female; mean 26.0 years old) with ulcerative colitis (UC) and five normal volunteers (three male, two female; mean 28.3 years old) in situ . An electroneedle was inserted into colonic mucosa through a biopsy channel during colonoscopy. The information concerning the concentration of NO generated and the appearances of the colonic mucosa at the same site were obtained simultaneously. In the ulcerative colitis patients, NO concentrations were significantly increased at all 24 mucosal sites tested. These included sites where: there was an absence of visible inflammation (five sites); the mucosa was mildly inflamed (eight sites); the mucosa was moderately inflamed (five sites); or severely inflamed (six sites). The NO concentrations in ulcerative colitis patients were 12–72 times higher than the NO levels in normal controls (10 sites). At the same 10 sites in four ulcerative colitis patients, the high NO concentrations were decreased by 53% after glucocorticoid treatment. These data are consistent with those of previous studies utilizing different NO electrodes. Excess mucosal NO is generated from inducible NO synthase in the inflamed mucosa itself and the invading inflammatory cells. Our results suggested that mucosal NO could be a marker for the extent of inflammation and its various actions correlated with the pathogenesis, natural history and prognosis of UC. Using the NO microelectrode system reported here, the concentration of NO generated can be monitored in real-time while observing the mucosal condition at the same site during endoscopy. This novel NO electrode may contribute to understanding the role of NO in colonic mucosal inflammation.     

Obliterative arteritis with nitric oxide synthase and HLA-DR expression in Crohn's colitis

BACKGROUND/AIMS: To cast light on whether inflammatory vascular injury is a possible pathogenic mechanism in Crohn's disease, the histological characteristics of vascular lesions were investigated. METHODOLOGY: Affected vessels in surgically resected colons from 23 patients with Crohn's disease, 20 with ulcerative colitis, 7 with ischemic colitis, and 9 normal controls were analyzed by Victoria blue and hematoxylin and eosin staining as well as immunohistochemistry for HLA-DR, nitric oxid synthase, vascular endothelial growth factor and E-cadherin. RESULTS: Inflammatory-cell infiltrates affecting arteries, accompanied by obliterative intimal thickening, were more frequent in Crohn's disease cases than in the other groups (P < 0.05-0.0001). Crohn's disease activity was positively correlated with the degree of obliterative arteritis. Granulomatous vasculitis was found exclusively in Crohn's disease (10 cases; 43.5%). In addition, focally enhanced endothelial staining of HLA-DR, with expression in granulomas adjacent to vessels was occasionally observed. In the endothelium of affected vessels, strong expression of HLA-DR was more prevalent in Crohn's disease and/or ulcerative colitis as compared with the ischemic colitis and controls (P < 0.05-0.01). In the involved arteries, enhanced endothelial nitric oxide synthase expression was most common in Crohn's disease among the groups (P < 0.05). A few cases of Crohn's disease, ulcerative colitis and ischemic colitis were positive for inducible nitric oxide synthase, vascular endothelial growth factor or E-cadherin in the vessel walls. CONCLUSIONS: The presence of characteristic obliterative arteritis and granulomatous vasculitis, a possible cause of ischemic injury, supports, in part, a vascular hypothesis for the pathogenesis of Crohn's disease. Enhanced expression of endothelial nitric oxide synthase and HLA-DR possibly reflects compensatory endothelium-mediated vasodilation and amplification of the immune response, respectively.     

Expression of nitric oxide synthases and effects of L-arginine and L-NMMA on nitric oxide production and fluid transport in collagenous colitis

BACKGROUND AND AIMS: 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. PATIENTS: Eight patients with collagenous colitis, nine with active ulcerative colitis, and 10 with uninflamed bowel were included. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.     

Expression of inducible nitric oxide synthase and effects of L-arginine on colonic nitric oxide production and fluid transport in patients with "minimal colitis"

OBJECTIVE: Some patients with idiopathic, chronic diarrhoea have minimal, non-specific colonic inflammation. As nitric oxide (NO) acts as a secretagogue in the colon, we studied the expression of inducible NO synthase (iNOS) in mucosal biopsies and the effects of NOS stimulation on colonic transfer of fluid and output of NO in patients with "minimal colitis". MATERIAL AND METHODS: Twelve patients with idiopathic, chronic diarrhoea and "minimal colitis" and 6 healthy volunteers were included in the study. Expression of iNOS in colonic mucosal biopsies was quantified by Western blot analysis and localized by immunohistochemistry. The effects of topical L-arginine or placebo on colonic net fluid transfer and nitrite/nitrate (NOx) output were assessed during "steady state" perfusion of the whole colon. Concentrations of NOx were measured by Griess' assay. RESULTS: The expression of iNOS was increased 10-fold (p<0.01) in patients with "minimal colitis" compared with that in healthy volunteers and localized to the colonic epithelium. Colonic absorption of fluid was impaired (mean (SEM) 1.5 (0.2) versus 3.0 (0.2) ml/min, p<0.001) and the output of NOx was increased (47 (4) nmol/min versus <37 nmol/min, p<0.05) in patients with "minimal colitis" compared with that in healthy volunteers. Luminal L-arginine (20 mM) reduced colonic absorption of fluid in both groups (95% confidence intervals (CIs) 21-50% in patients with "minimal colitis" versus 4-18% in healthy volunteers), but an increase in NOx output was detectable only in the group of patients (8-106%). In time control experiments, colonic net transfer rates of fluid and outputs of NOx were unaffected by placebo. CONCLUSIONS: In patients with idiopathic, chronic diarrhoea and histopathological evidence of "minimal colitis", colonic absorption of fluid is impaired, while epithelial expression of iNOS and mucosal production of NO is enhanced. It could be speculated that NO in excess contributes to the diarrhoea observed in "minimal colitis".     

Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity.

Enhanced nitric oxide (NO) generation by stimulated NO synthase (NOS) activity may, through its oxidative metabolism contribute to tissue injury in experimental colitis. In this study the possible amelioration of experimental colitis by NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS activity, was evaluated. Colitis was induced in rats by intracolonic administration of 30 mg trinitrobenzene sulphonic acid (TNB) dissolved in 0.25 ml 50% ethanol or by flushing the colon of capsaicin pretreated rats with 2 ml of 5% acetic acid. In several experiments, L-NAME 0.1 mg/ml was added to the drinking water at the time of colitis induction with TNB or seven days before acetic acid treatment. Rats were killed at various time intervals after induction of colitis. A 10 cm distal colonic segment was isolated, weighed, lesion area measured, and explants organ cultured for 24 hours for determination of NO generation by the Greiss reaction. The rest of the mucosa was scraped for determination of myeloperoxidase and NOS activities and leukotriene generation. In TNB treated rats mean arterial pressure was also determined up to 72 hours after damage induction, with or without cotreatment with nitroprusside. L-NAME significantly decreased the extent of tissue injury in TNB treated rats. Seven days after TNB treatment lesion area was reduced by 55%, colonic weight by 37%, and myeloperoxidase and NOS activity by 59% and 42%, respectively. Acetic acid induced colitis in capsaicin pretreated rats was also significantly decreased by L-NAME. Twenty four hours after acetic acid treatment lesion area was reduced by 61%, colonic weight by 21% and NOS activity by 39%. Mean (SEM) arterial blood pressure in TNB+L-NAME treated rats was 37.6 (8.1) mm Hg higher than in TNB treated rats, an effect that was only partially abolished by nitroprusside. These results show that inhibition of NO synthesis by an L-arginine analogue significantly ameliorates the extent of tissue injury in two models of experimental colitis, an effect that is not due only to its vasoconstrictor properties. Modulation of NO generation may be a novel therapeutic approach in inflammatory bowel disease.     

Interleukin-13 inhibits nitric oxide production in human colonic mucosa

BACKGROUND/AIMS: Nitric oxide synthesis is increased in rectal biopsies from patients with ulcerative colitis and colonic epithelial cells are considered to be a major source of nitric oxide in intestinal inflammation. METHODOLOGY: Human colonic biopsies from normal bowel mucosa and colonic epithelial cell line HT-29 were cultured in the presence of the inflammatory cytokines IL-1 alpha + TNF-alpha + IFN-alpha added after 1 hour pretreatment with vehicle or Interleukin-13. Nitrite levels were determined at 30 hours in culture supernatants by a fluorometric assay. RESULTS: Unstimulated human colonic biopsies and HT-29 cells produced a basal amount of nitrite. Stimulation with IL-1 alpha + TNF-alpha + IFN-alpha induced a significant (P < 0.001) increase of nitrite generation by both human colonic biopsies and HT-29 cells. The presence of Interleukin-13 produced a significant (P < 0.001) suppression of the cytokine-induced nitrite generation from both colonic biopsies and HT-29 cells. CONCLUSIONS: Nitric oxide generation in human colonic mucosa is susceptible to manipulation by proinflammatory cytokines. Interleukin-13 has an inhibitory effect on cytokine induced nitrite production in colonic mucosa and could play an anti-inflammatory role in intestinal inflammation.     

Oral administration of inducible nitric oxide synthase inhibitors reduces nitric oxide synthesis but has no effect on the severity of experimental colitis

BACKGROUND: Increased concentrations of nitrate and nitrite (the breakdown products of nitric oxide) in the serum and faeces of patients with inflammatory bowel disease (IBD) suggests that increased synthesis of nitric oxide occurs in IBD. The aim of this study was to assess aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase, with regard to its effectiveness as a nitric oxide inhibitor and as a modulator of inflammation in trinitrobenzene sulfonic acid (TNBS)-induced colitis. MATERIALS AND METHODS: Colitis was induced in Wistar rats. Selective (AMG) and non-selective (1-nitroso-arginine methyl ester (1-NAME)) inhibitors of nitric oxide synthase were given in the drinking water. Colonic citrulline and arginine concentrations were assessed using high-performance liquid chromatography. The severity of colitis was assessed by a macroscopic scoring system. RESULTS: Both 1-NAME and AMG successfully reduced nitric oxide synthesis. There was no evidence of substrate depletion in the colonic wall. Neither of the agents reduced the severity of colonic inflammation. CONCLUSIONS: Oral administration of nitric oxide synthase inhibitors reduced nitric oxide synthesis in the colonic wall. This study does not provide evidence to support a role for nitric oxide in the pathogenesis of colonic inflammation in TNBS colitis.     

Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis

OBJECTIVE: Increased lymphocyte activation and production of inflammatory cytokines are implicated in the pathogenesis of ulcerative colitis. Because antigen-presenting dendritic cells play a cardinal role in the activation and survival of activated lymphocytes, the aim of the present study was to characterize dendritic cells in ulcerative colitis. DESIGN: This study was designed to compare the phenotypes and functions of peripheral blood dendritic cells among healthy normal volunteers and patients with ulcerative colitis or Crohn's disease. Activated dendritic cells were also localized at the colonic mucosa. METHODS: Peripheral blood dendritic cells were generated from 15 patients with ulcerative colitis, 10 patients with Crohn's disease and 15 healthy control volunteers. The stimulatory capacities of dendritic cells were analysed in an allogenic mixed lymphocyte reaction. Nitric oxide was detected by the Griess method. Single- and dual-colour flow cytometry was employed to study the levels of maturation of dendritic cells. Activated dendritic cells were localized immunohistochemically in the colonic mucosa. RESULTS: In comparison to normal controls, peripheral blood dendritic cells from patients with ulcerative colitis showed significantly increased stimulatory capacities (P < 0.05) and produced significantly higher levels of nitric oxide (P < 0.05). The numbers of activated dendritic cells were also significantly higher in ulcerative colitis (P < 0.05). Mature and activated dendritic cells expressing the CD83 antigen were detected at the inflamed colonic mucosa in patients with ulcerative colitis and Crohn's disease. CONCLUSIONS: Activated and mature dendritic cells may have a role in the induction of an exacerbated immune response in ulcerative colitis. This study provides the scientific and logical basis for blocking the maturation and activation of dendritic cells in ulcerative colitis as a new therapeutic intervention.     

Expression of inducible nitric oxide synthase activity in human colon epithelial cells: modulation by T lymphocyte derived cytokines

Background—Nitricoxide (NO) synthesis and inducible nitric oxide synthase (iNOS)expression are increased in colonic biopsy specimens from patients withulcerative colitis, but the cellular source of NO production is not known.
Aims—To examine thedistribution of iNOS in human colonic mucosa and to explore the abilityof T lymphocyte derived cytokines to regulate iNOS expression andactivity in human colonic epithelial cells.
Methods—iNOSexpression was examined using immunohistochemistry in colonic biopsysamples from 12 patients with ulcerative colitis and three withinfectious colitis and compared with 10normal controls. In vitro iNOSexpression and activity were determined in HT-29 cell cultures; nitritelevels were measured using a fluorescent substrate, iNOS mRNAexpression by northern blot analysis, and iNOS protein expression bywestern blot analysis.
Results—No iNOSexpression was detected (10 of 10) in non-inflamed mucosa derived fromnormal controls. In 11 of 12 cases of newly diagnosed ulcerativecolitis, iNOS protein was expressed in the epithelial cells, while noother positive cells were found in the lamina propria. Similar iNOSlabelling was found in colonic biopsy samples from patients withinfectious colitis in the acute phase, but when re-examined in samplesfrom patients in total remission, no iNOS staining was observed. Bothinterleukin (IL)-13 and IL-4, but not IL-10, are potent inhibitors ofiNOS expression and activity induced by an optimal combination ofcytokines, namely IL-1α, tumour necrosis factor α and interferonγ.
Conclusions—The datasuggest that the epithelium is the major source of iNOS activity inulcerative colitis and that IL-13 and IL-4 may act as intrinsicregulators of NO generation in intestinal inflammation.

Keywords:interleukin 13; nitric oxide; inducible nitric oxidesynthase; colonic epithelial cells; ulcerative colitis

     

Increased production of vascular endothelial growth factor by intestinal mucosa of patients with inflammatory bowel disease.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeability-enhancing activities specific for endothelial cells. Recent studies have shown significantly increased VEGF serum levels in patients with active Crohn's disease and ulcerative colitis. The origin of the circulating VEGF is not yet completely described. The present investigation examines the VEGF production of colonic mucosa in consideration of mucosal disease activity in patients with inflammatory bowel disease. METHODOLOGY: Fifteen patients with inflammatory bowel disease were studied, 9 patients with Crohn's disease and 6 patients with ulcerative colitis. Biopsies were taken from endoscopically inflamed and non-inflamed colonic mucosa. Therefore, an analysis of the spontaneous VEGF production of cultured biopsies without stimulus and of the histological grade of inflammation scored on a scale of 0-3 (normal mucosa--severe chronic colitis) were performed. Eight patients with irritable bowel syndrome served as controls. VEGF levels in the supernatant of cultured mucosal biopsies were measured using an enzyme linked immunosorbent assay. RESULTS: VEGF production is expressed as pg/mg wet weight of the biopsies. Inflamed mucosa of patients with active ulcerative colitis (16.27 +/- 10.39, p = 0.003, n = 6) and active Crohn's disease (9.88 +/- 5.98, p < 0.012, n = 9) showed a significantly higher spontaneous production of VEGF by colonic mucosa than normal mucosa of controls (3.16 +/- 1.63, n = 8). In addition, there was an increased unstimulated VEGF production by cultured inflamed mucosa of patients with Crohn's disease compared with non-inflamed mucosa (3.88 +/- 3.66, p < 0.015, n = 9). In both Crohn's disease and ulcerative colitis, there was no significant difference between VEGF production by non-inflamed mucosa and normal mucosa of controls. CONCLUSIONS: The present study identifies the intestinal mucosa as one of the origins of the elevated VEGF serum levels in patients with active inflammatory bowel disease and verifies the findings of recent studies about the importance of VEGF in Crohn's disease and ulcerative colitis.     

Bile acid induced colonic irritation stimulates intracolonic nitric oxide release in humans.

AIM--To measure the intracolonic release of nitric oxide end products (nitrates plus nitrites) and eicosanoids in response to intraluminal irritation with deoxycholic acid (DCA). PATIENTS--Seven patients with irritable bowel syndrome. METHODS--The left colon was perfused with a solution with or without 3 mM deoxycholic acid. Aspirates were assayed for eicosanoids by specific radioimmuno-assay, and for nitrates plus nitrites by the Griess reaction. To confirm that stimulated colonic mucosa can produce nitric oxide (NO), ancillary studies were performed in vitro using samples of normal mucosa obtained from five surgically resected colons. Samples were incubated for 30 minutes in Kreb's solution, 3 mM DCA or DCA with 1 mM L-nitro-arginine-methyl-ester (L-NAME) to inhibit the NO synthase. Finally, NO synthase activity was measured in five samples of human colonic mucosa. RESULTS--Intracolonic release of nitrates plus nitrites was basally undetectable in six of seven patients. Bile acid considerably increased the release of prostaglandin E2 and nitrates plus nitrites (p < 0.01). By contrast, no increase in thromboxane and leukotriene was seen. In vitro mucosal incubation with DCA increased the production of NO synthase products, which was blocked by L-NAME. Activity of Ca+2 independent NO synthase was detectable in four of five samples of human colonic mucosa. CONCLUSION--The human colonic mucosa responds to bile acid induced irritation by a surge in NO generation via NO synthase.     

Budesonide treatment and expression of inducible nitric oxide synthase mRNA in colonic mucosa in collagenous colitis

OBJECTIVE: In collagenous colitis, the production of nitric oxide in the colon is found to be 50 to 100-fold higher than in healthy controls. The role of nitric oxide in collagenous colitis is debated and it has been suggested that nitric oxide has a causative role in diarrhoea. The aim of this study was to examine the possible effect of budesonide treatment on the level of inducible nitric oxide synthase mRNA. METHODS: In 20 patients with collagenous colitis, clinical activity was assessed by registration of the daily stool frequency and stool weight. Sigmoidoscopy was performed and biopsies for histological examination and one biopsy for determination of inducible nitric oxide synthase mRNA was obtained in 16 patients. RESULTS: Budesonide treatment was followed by a significant reduction of inducible nitric oxide synthase mRNA (P<0.01) whereas no change was observed after placebo treatment. Significant correlations between inducible nitric oxide synthase mRNA and the grade of inflammation (rho=0.47; P<0.01), the daily stool weight (rho=0.51; P<0.005) and the daily stool frequency (rho=0.49; P<0.005) were observed. No significant association was observed between inducible nitric oxide synthase mRNA and the thickness of the collagen layer. CONCLUSIONS: In patients with collagenous colitis, treatment with budesonide results in a reduction of inducible nitric oxide synthase mRNA. The level of inducible nitric oxide synthase mRNA in colonic mucosa correlates with the inflammatory and clinical activity. The results support that nitric oxide is a central factor in the pathogenesis of collagenous colitis.     

Increased group II phospholipase A2 in colonic mucosa of patients with Crohn's disease and ulcerative colitis.

The immunochemical protein content of group II phospholipase A2 (PLA2) and PLA2 enzymatic activity were measured for colonic mucosal biopsy samples obtained from patients with either Crohn's disease of the colon or ulcerative colitis, and control patients without inflammatory bowel disease. Immunoreactive group II PLA2 (IR-PLA2 II) content and PLA2 activity in actively inflamed colonic mucosa of Crohn's disease patients were significantly higher than those in inactively inflamed mucosa of Crohn's disease patients and the colonic mucosa of controls. IR-PLA2 II content and PLA2 activity in severely inflamed mucosa of ulcerative colitis patients were significantly higher than those in the colonic mucosa of the controls. Mucosal PLA2 enzymatic activity was closely correlated with mucosal IR-PLA2 II content in patients with Crohn's disease and ulcerative colitis. These results suggest that an increase in PLA2 enzymatic activity in inflamed colonic mucosa of Crohn's disease and ulcerative colitis was mainly attributed to increased protein content of group II PLA2, and that an increase in mucosal group II PLA2 may be involved in the pathogenesis of intestinal inflammation of Crohn's disease and ulcerative colitis.     

Colonic production of nitric oxide gas in ulcerative colitis, collagenous colitis and uninflamed bowel

BACKGROUND: Nitric oxide (NO) produced in excess by the inflamed human colon is generally considered a pathway of mucosal damage. In an attempt to quantify colonic mucosal production of NO in various forms of colitis we performed 'steady-state' gas perfusion of whole colon in 11 patients with ulcerative colitis, 10 patients with collagenous colitis and 20 controls with uninflamed mucosa. METHODS: The tip of a Teflon tube was placed in the caecum during colonoscopy. Subsequently, argon was infused at a constant rate for 70-180 min. Argon and NO in gas sampled from the rectum were measured by neutron activation analysis and the chemiluminescence technique, respectively. RESULTS: The use of argon as a marker of colonic NO output was justified by complete recovery (96%+/-2; mean +/- s(x); n = 5) of argon in gas collected from the rectum and a constant output of NO at varying perfusion rates (25, 50 and 75 ml/min coefficient of variation 21%; n = 6). In patients with ulcerative colitis, colonic output of NO was 10-fold higher (P < 0.001) than in controls and positively correlated (P < 0.01) to indices of disease activity. In patients with collagenous colitis, colonic output of NO was 50-fold higher (P < 0.01) than in controls during periods with watery diarrhoea (n = 6), but within the range observed in ulcerative colitis in the absence of diarrhoea (n = 4). CONCLUSIONS: Argon gas perfusion of whole colon using chemiluminescence technique for measurement of NO is a reliable method for quantification of colonic mucosal NO production. Increased colonic production of NO in collagenous colitis, which exceeds the output observed even in extensive ulcerative colitis, militates against the theory that NO per se is a cause of mucosal injury.     

Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis

Background—Increased production of reactivemetabolites of oxygen and nitrogen has been implicated in chronicinflammation of the gut. The object of this study was to examine themagnitude and location of nitric oxide synthase (NOS) activity andperoxynitrite formation in the colonic mucosa of patients withulcerative colitis in relation to the degree of inflammation.
Subjects—Thirty three patients with activeulcerative colitis (17 with mild or moderate inflammation, 16 withsevere inflammation).
Methods—Inducible NOS activity was determined inthe colonic mucosa by measuring the conversion ofL-arginine to citrulline in the absence of calcium. Thelocalisation of NOS and nitrotyrosine immunoreactivity was assessedimmunohistochemically using the labelled streptavidin biotin method.
Results—Inducible NOS activity increased inparallell with the degree of inflammation of the mucosa. Expression ofinducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed bynitrotyrosine staining was frequently observed in the lamina propria ofactively inflamed mucosa.
Conclusions—Nitric oxide and peroxynitriteformation may play an important role in causing irreversible cellularinjury to the colonic mucosa in patients with active ulcerative colitis.

Keywords:nitric oxide; peroxynitrite; nitricoxide synthase; ulcerative colitis; colonic mucosa

     

Homocysteine, Cysteine, and Glutathione in Human Colonic Mucosa: Elevated Levels of Homocysteine in Patients with Inflammatory Bowel Disease

The present study was performed to evaluate the levels of the amino thiols cysteine, homocysteine, and glutathione in the colonic mucosa of patients with various intestinal diseases, especially chronic inflammatory bowel disease. Colonic biopsies of macroscopically normal mucosa out of a proximal and distal segment were collected from 187 patients with various intestinal diseases. Protein was assayed in duplicate by the method of Lowry et al (1951), using bovine serum albumin as standard. Total glutathione, cysteine, and homocysteine were quantified by high performance liquid chromatography (HPLC) with fluorescent detection. Only in patients with inflammatory bowel disease were the homocysteine levels in the large bowel mucosa significantly elevated compared with the concentrations in patients with normal mucosa. No significant differences were seen for glutathione and cysteine concentrations in colonic mucosa among the different groups of diseases. No correlation was found between the age of the patients and levels of the amino thiols investigated. GSH content and concentrations of cysteine and homocysteine were similar in male and female subjects. In our study markedly elevated concentrations of homocysteine in the colonic mucosa were observed in patients suffering from ulcerative colitis and Crohn's disease. This finding has been reported already in the literature for plasma homocysteine levels. Increased homocysteine levels in the colonic mucosa and plasma of patients with inflammatory bowel disease may play a role in the pathogenesis of Crohn's disease and ulcerative colitis.     

Enhanced gastric nitric oxide synthase activity in duodenal ulcer patients.

Nitric oxide, the product of nitric oxide synthase in inflammatory cells, may have a role in tissue injury through its oxidative metabolism. Nitric oxide may have a role in the pathogenesis of duodenal ulcer and may be one of the mechanisms responsible for the association between gastric infection with Helicobacter pylori and peptic disease. In this study, calcium independent nitric oxide synthase activity was detected in human gastric mucosa suggesting expression of the inducible isoform. In 17 duodenal ulcer patients gastric antral and fundic nitric oxide synthase activity was found to be two and 1.5-fold respectively higher than its activity in the antrum and fundus of 14 normal subjects (p < 0.05). H pylori was detected in the antrum of 15 of 17 duodenal ulcer patients and only in 7 of 14 of the control subjects. Antral nitric oxide synthase activity in H pylori positive duodenal ulcer patients was twofold higher than in H pylori positive normal subjects (p < 0.05). In duodenal ulcer patients antral and fundic nitric oxide synthase activity resumed normal values after induction of ulcer healing with ranitidine. Eradication of H pylori did not further affect gastric nitric oxide synthase activity. These findings suggest that in duodenal ulcer patients stimulated gastric mucosal nitric oxide synthase activity, though independent of the H pylori state, may contribute to the pathogenesis of the disease.     

Changes in the expression and distribution of the inducible and endothelial nitric oxide synthase in mucosal biopsy specimens of inflammatory bowel disease

OBJECTIVE: The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel disease (IBD) is controversial. The aim of this study was to investigate the expression and localization of nitric oxide synthase isoforms (iNOS, eNOS) in IBD colonic mucosa. MATERIAL AND METHODS: Forty-four patients with IBD (24 ulcerative colitis (UC), 20 Crohn's disease (CD) and 16 controls) were investigated by colonoscopy. iNOS and eNOS in tissue sections was demonstrated by histochemistry (NADPH-diaphorase reaction) and immunohistochemistry. Cell type analysis and quantitative assessment of the iNOS immunoreactive (IR) cells and densitometry of iNOS in immunoblots were also performed. RESULTS: iNOS-IR cells were significantly numerous in inflamed mucosa of UC (64+/-4 cells/mm2) than in CD (4+/-2 cells/mm2). iNOS-IR/CD15-IR cells showed significant elevation in inflamed (i) versus uninflamed (u) UC mucosa (UCu 8+/-3%, UCi 85+/-10%) In CD, the percentage of iNOS-IR/CD68-IR cells was lower in inflamed sites (CDu 23+/-8%, CDi 4+/-3%). Immunoblot of biopsies revealed significant elevation of iNOS in active UC compared with uninflamed sites, whereas in CD no significant changes were detected. Differences were observed in eNOS and endothelial marker CD31 immunoreactivity. In patients with UC and in controls the ratios of eNOS/CD31-IR vessels were 82.3% and 92.0% respectively, whereas in CD the ratio was 8.3% with a concomitantly significant increase of CD31-IR vessels. The distribution and morphological characteristics of the NOS-IR inflammatory cells and endothelia were similar to those showing NADPH-diaphorase reactivity. CONCLUSIONS: Differences observed in the expression and distribution of NOS isoforms in immune and endothelial cells may contribute to better understanding of the structural and physiological changes in UC and CD.