Selective depletion of neutrophils by a monoclonal antibody, RP-3, suppresses dextran sulphate sodium-induced colitis in rats

Administration of dextran sulphate sodium to animals induces acute colitis characterized by infiltration of large numbers of neutrophils into the colonic mucosa, which histologically resembles human active ulcerative colitis. It has been reported that neutrophils and the reactive oxygen metabolites produced by them are involved in the progress of ulcerative colitis. This study was intended to clarify their roles by using this animal model. First, possible sources and species of reactive oxygen metabolites were determined using luminol-dependent chemiluminescence with addition of enzyme inhibitors and reactive oxygen metabolite scavengers. Next, to examine whether neutrophils and hypochlorous acid derived from them contribute to tissue injury, we administered RP-3, a monoclonal antibody capable of selectively depleting neutrophils, and taurine, a hypochlorous acid scavenger, to rats treated with dextran sulphate sodium. Addition of azide, taurine, catalase, superoxide dismutase and dimethyl sulphoxide into colonic mucosal scrapings significantly inhibited chemiluminescence production, but allopurinol and indomethacin had no effects. These results suggest that excessive hypochlorous acid, hydrogen peroxide, superoxide anion and hydroxyl radical are generated by the inflamed colonic mucosa. Intraperitoneal injections of RP-3 significantly suppressed bleeding, tissue myeloperoxidase activity, chemiluminescence production and erosion formation. On the other hand, administration of taurine tended to inhibit bleeding and erosion formation to some extent, although it could not significantly suppress them. These data suggest that neutrophils play an important role in the development of this colitis and that hypochlorous acid might be one of the causes of tissue injury induced by neutrophils.     

Increased production of luminol enhanced chemiluminescence by the inflamed colonic mucosa in patients with ulcerative colitis.

Reactive oxygen species have been implicated as mediators of inflammation in ulcerative colitis. Chemiluminescence is a reliable means of estimating reactive oxygen species in biological media. Increased reactive oxygen species values in the inflamed colonic mucosa in rats were seen by chemiluminescence. The aims of the study were to find out if chemiluminescence is raised in the colonic mucosa of patients with ulcerative colitis and correlates with disease activity, and to elucidate the sources of the chemiluminescence. It was found that reactive oxygen species, as measured by the chemiluminescence technique, are raised in inflamed colonic mucosa and correlates with symptom score, sigmoidoscopic score, disease activity, and activity of the neutrophil enzyme myeloperoxidase. Chemiluminescence was inhibited by a myeloperoxidase inhibitor (azide) and an H2O2 scavenger (catalase) but not by allopurinol, an inhibitor of the enzyme xanthine oxidase. Chemiluminescence was also inhibited by indomethacin, but this did not seem to be related to inhibition of cyclo-oxygenase. These findings suggest that a likely cellular source of reactive oxygen species in the inflamed colon of patients with ulcerative colitis is the neutrophil and that myeloperoxidase conversion of H2O2 to hypochlorous acid, contributes to the chemiluminescence signal and possibly, to the tissue injury. Neither cyclo-oxygenase nor lipoxygenase seem to play a part as sources for the chemiluminescence.     

Mucosal blood flow and generation of superoxide in rat experimental colitis induced by succinic acid

As we consider succinic acid to be an exacerbating factor in ulcerative colitis, we investigated its influence on rat colonic mucosa in terms of mucosal blood flow and superoxide generation. We measured mucosal blood flow by the hydrogen gas clearance method and superoxide generation by the chemiluminescence method, and observed histopathological findings to determine the effects of succinic acid. After the instillation of succinic acid of any concentration tested to the colon, mucosal blood flow decreased. Histopathologically, the higher the concentration of succinic acid, the greater was the erosion formation in the colonic mucosa, while significant polymorpho-nuclear cell infiltration and superoxide generation from colon tissue were observed with 0.01% succinic acid compared with higher or lower concentrations. Succinic acid, at fecal concentrations found in active stage ulcerative colitis, appears to be implicated in mucosal injury, mediated by a decrease in colonic mucosal blood flow and infiltration of superoxide-generating polymorpho-nuclear cells into the mucosa.     

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.     

Effect of gender on the manifestations of celiac disease: Evidence for greater malabsorption in men

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 duodenal expression of transforming growth factor-alpha and epidermal growth factor during experimental colitis in rats

OBJECTIVE/BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) protect gastrointestinal mucosa against injury. Having shown earlier, that TGFalpha but not EGF is locally increasingly expressed after mucosal injury in the colon, we now wanted to explore the pattern of expression of EGF and TGFalpha in the remaining gastrointestinal tract and to infer from the pattern of expression, to possible signals for the induction of the growth factor expression and further mechanisms for mucosal protection. DESIGN/METHODS: The trinitrobenzene sulfonic acid/ethanol-induced model of colitis in rats was used. TGFalpha-mRNA and EGF-mRNA expression was evaluated in inflamed and noninflamed colon, in the ileum, jejunum, duodenum, stomach, and in the submandibulary glands. RESULTS: A significant increase of TGFalpha-mRNA and EGF-mRNA expressions was detected in the duodenal mucosa and a significant increase in TGFalpha-mRNA expression was observed in the inflamed colonic mucosa after mucosal injury in the colon within the first hours of colitis. CONCLUSION: The increased expression of EGF and TGFalpha in the duodenum may lead to neutralization of gastric acid and proteolytic enzymes in the upper gastrointestinal tract during the course of colitis. Possible signals for the increased expression of EGF and TGFalpha presumably are fasting, parasympathetic, or adrenergic parts of the enteric nervous system or yet unknown mechanisms.     

Acetic acid-derived prostaglandin-dependent colonic adaptive cytoprotection is preserved in chronic colitis: role of cyclo-oxygenase

BACKGROUND AND AIMS: We recently reported the phenomenon of prostaglandin-dependent colonic adaptive cytoprotection (CAP) against acute colonic injury induced by acetic acid (AA) in the normal colon. This study investigated whether the CAP is preserved in the chronic inflamed colon. MATERIALS AND METHODS: Normal rats and a chronic colitis model, induced by trinitrobenzene sulfonic acid, received an intracolonal administration (0.5 ml) of saline or AA at low concentration (1%) followed by high concentration (8%) 30 min later. The distal colon was removed 48 h after 8% AA administration, and colitis was assessed by macroscopic scoring and measurement of the myeloperoxidase (MPO) activity. Indomethacin (5 mg/kg), a nonselective cyclo-oxygenase (COX) inhibitor, or N-[2-cyclohexyloxy-4-nitrophenyl] methane-sulfonamide (NS398, 1 mg/kg), a COX type 2 selective inhibitor, was injected intraperitoneally 1 h before pretreatment with 1% AA. RESULTS: Intracolonal administration of 8% AA induced colonic mucosal damage (macroscopic score 10.0+/-0.9) and elevated MPO activity (2.8+/-0.2 U/g), which were significantly reduced to 3.3+/-0.8 and 1.8+/-0.2 U/g by 1% AA pretreatment, respectively. Indomethacin abolished the gross mucosal protective effect by 1% AA pretreatment in 8% AA-derived colitis in normal rats while the NS398 had no effect. Both indomethacin and NS398 reversed the MPO activity reduction induced by 1% AA pretreatment. In chronic inflamed colon 8% AA treatment resulted in an increase in the macroscopic score to 11.5+/-0.4 from 4.7+/-0.4, but not the MPO activity, which was significantly reduced to 5.7+/-0.9 by 1% AA pretreatment. This gross mucosal protective effect by 1% AA pretreatment in chronic inflamed colon was reversed by indomethacin while the NS398 had no effect. CONCLUSION: These data show that COX-1 and COX-2 derived prostaglandins induced by low concentration AA pretreatment reduce the colonic mucosal injury and the increase in the MPO activity in colitis, respectively. The protective effect of COX-1 is preserved in chronic inflamed colon. These findings support the existence of a low concentration of AA-derived prostaglandin-dependent CAP and suggest that colonic AA, which is derived from bacterial breakdown of carbohydrate and protein in the colon, plays a crucial role in the endogenous defense mechanisms.     

Agents capable of eliminating reactive oxygen species

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorous acid have been implicated in the pathogenesis of inflammation and tissue injury in colitis. To determine whether or not anti-ROS agents can decrease the severity of colitis, we evaluated the effects of three known anti-ROS agents: catalase, WR-2721, and Cu(II)₂(3,5-DIPS)₄ on acetic acid-induced colonic inflammation in rats. Histologically, all three compounds significantly decreased the severity of colonic inflammation. The anti-ROS activity of these compounds was also tested using the luminol-enhanced chemiluminescence assay. Catalase, WR-2721, or Cu(II)₂(3,5-DIPS)₄ significantly inhibited luminol-enhanced chemiluminescence produced by inflamed colonic mucosa. These findings suggest that ROS, and in particular superoxide, hydrogen peroxide, and/or one of its secondarily derived species, may play an important role in acetic acid-induced colitis. Further studies are needed to determine the potential effectiveness of these compounds in human colitis.     

Mobilization of tissue kallikrein in inflammatory disease of the colon

Colonic tissue was taken at operation from 10 patients with active ulcerative colitis and three patients with uncomplicated diverticular disease but with severe symptoms. Levels of kininogen, kallikrein, and kallikrein precursor were measured in blood-free tissue samples. In normal colon tissue a kininogen occurred in the muscle and none was detected in the mucosa. Kallikrein and its precursor were found in mucosa but not in muscle. In acutely inflamed tissue from ulcerative colitis patients relatively high levels of active kallikrein were detected in the underlying colonic muscle. There was little change in the level of kallikrein in inflamed mucosa or of kininogen in the muscle of these patients. No kallikrein was found in colonic muscle from patients with diverticular disease and the mucosal kallikrein level in these patients was unchanged. The findings suggest a mechanism for the formation of kinins in the wall of the colon which is present in ulcerative colitis but not in diverticular disease.     

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.     

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.     

Inter-relationships between inflammatory mediators released from colonic mucosa in ulcerative colitis and their effects on colonic secretion.

Metabolites of arachidonic acid have been implicated in the pathophysiology of ulcerative colitis-they can stimulate intestinal secretion, increase mucosal blood flow, and influence smooth muscle activity. The influence on the mucosal transport function of culture medium in which colonic mucosal biopsy specimens had been incubated was investigated using rat stripped distal colonic mucosa in vitro as the assay system. Colonic tissue from patients with colitis and from control subjects was cultured. Medium from inflamed tissue contained more prostaglandin E2 (PGE2) and leukotriene D4 (LTD4) and evoked a greater electrical (secretory) response in rat colonic mucosa than control tissue medium. In inflamed tissue, cyclo-oxygenase inhibition (indomethacin) attenuated PGE2 but increased LTD4 production; conversely lipoxygenase inhibition (ICI 207968) inhibited LTD4 production but enhanced PGE2 output. Each inhibitor alone enhanced the electrical response in the rat colon. Inhibition of both enzymes (indomethacin plus ICI 207968) caused a fall in both PGE2 (82%) and LTD4 (89%) production and in the electrical response (57%). Inflamed tissue treated with a phospholipase A2 inhibitor (mepacrine) produced less PGE2, LTD4, and electrical responses when compared with inflamed tissue, either untreated (91%, 92%, and 79% respectively) or treated with cyclo-oxygenase and lipoxygenase inhibition. Incubation with bradykinin stimulated eicosanoid release and electrical response, while a bradykinin antagonist caused a modest inhibition. Analysis of these observations suggests that a combination of arachidonic acid derivatives accounts for about half the secretory response. Other products of phospholipase A2 activity are probably responsible for much of the remainder, leaving up to 20% the result of types of mediator not determined in this study.     

Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease.

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.     

Calpain inhibitor I reduces colon injury caused by dinitrobenzene sulphonic acid in the rat

BACKGROUND AND AIMS: Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leucocyte infiltration, upregulation of expression of intercellular adhesion molecule 1 (ICAM-1), and upregulation of P-selectin in the colon. The aim of the present study was to examine the effects of calpain inhibitor I in rats subjected to experimental colitis. METHODS: Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). RESULTS: Rats experienced haemorrhagic diarrhoea and weight loss. Four days after administration of DNAB, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology as well as by an increase in myeloperoxidase activity in the mucosa) was associated with upregulation of ICAM-1 and P-selectin as well as high tissue levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed intense staining in the inflamed colon. Staining of sections of colon obtained from DNBS treated rats with an anti-cyclooxygenase 2 antibody showed diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase was found mainly in macrophages located within the inflamed colon of DNBS treated rats. Calpain inhibitor I (5 mg/kg daily intraperitoneally) significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. Calpain inhibitor I also caused a substantial reduction in (i) degree of colon injury, (ii) rise in myeloperoxidase activity (mucosa), (iii) increase in tissue levels of malondialdehyde, (iv) increase in staining (immunohistochemistry) for nitrotyrosine and PARP, as well as (v) upregulation of ICAM-1 and P-selectin caused by DNBS in the colon. CONCLUSION: Calpain inhibitor I reduces the degree of colitis caused by DNBS. We propose that calpain inhibitor I may be useful in the treatment of inflammatory bowel disease.     

Expression of urokinase-type plasminogen activator in the mucosal lesions of inflammatory bowel disease

The expression of plasminogen activators was analysed in mucosal homogenates from inflammatory bowel disease patients to determine whether the urokinase-type (u-PA) is implicated in the pathogenesis of mucosal tissue injury. Homogenates of mucosal biopsy tissue from ulcerative colitis, Crohn's disease, infectious colitis and normal control patients were subjected to polyacrylamide gel electrophoresis. The types of plasminogen activator present were detected by zones of lysis in a fibrin-agarose gel overlay. All the tissues studied displayed tissue plasminogen activator activity (t-PA). In ulcerative colitis, 18 of the 19 diseased colon biopsies, but none of six biopsies from uninvolved areas of the same colon, showed u-PA activity. Similar results were found in 12 Crohn's disease patients. Biopsies from the infectious colitis group and from radiation colitis patients also showed both u-PA and t-PA activity. The age, sex, duration of disease, and presence and type of treatment did not affect u-PA expression in the inflamed mucosa. The results suggest that u-PA may be implicated in the mediation of tissue injury in the inflamed intestinal mucosa.     

高迁移率族蛋白B1抗体对结肠炎小鼠结肠黏膜的保护作用

背景:我国溃疡性结肠炎(UC)发病率明显上升,但目前尚无满意的治疗方法,因此研究其发病机制并寻求新的治疗途径具有重要意义。目的:研究高迁移率族蛋白B1(HMGB1)抗体对结肠炎小鼠结肠黏膜的影响。方法:36只BALB/c小鼠随机分为正常对照组、结肠炎模型组和HMGB1抗体治疗组,后两组以3%葡聚糖硫酸钠(DSS)制备小鼠结肠炎模型,HMGB1抗体治疗组小鼠腹腔注射HMGB1抗体。实验第7d,处死小鼠。行结肠组织学评分,测定结肠通透性,分别以RT-PCR和蛋白质印迹法检测结肠组织HMGB1 mRNA和蛋白表达。结果:与正常对照组相比,结肠炎模型组小鼠结肠组织学评分、结肠通透性以及HMGB1 mRNA和蛋白表达显著增高(P0.05)。与结肠炎模型组相比,HMGB1抗体治疗组小鼠结肠组织学评分、结肠通透性和HMGB1蛋白表达显著降低(P0.05)。结论:HMGB1参与了结肠炎小鼠的炎症反应过程,HMGB1抗体可有效抑制结肠炎小鼠结肠组织HMGB1表达,改善肠黏膜屏障功能,从而对UC结肠黏膜损伤起有明显保护作用。     

Decreased Total Antioxidant Capacity in Plasma, but Not Tissue, in Experimental Colitis

The aim of the present work was to compare colonic mucosa and plasmatic oxidative stress measured concomitantly and with different degrees of injury in rats with colitis induced by trinitrobenzene sulfonic acid. Three groups were studied: control group, colitis group, and colitis exacerbated by diclofenac. Enzymatic markers of colon injury showed enhanced activity in both groups with colitis. The colitis group treated with diclofenac presented higher colonic damage score than the other groups. In both groups with colitis, higher values of tert butyl hydroperoxide-initiated-chemiluminescence and thiobarbituric acid-reactive substances in tissue and decreased total radical-trapping antioxidant potential (TRAP) levels in plasma were found. In conclusion, independently of the degree of colonic mucosa injury and inflammation, oxidative stress in tissue occurs as a consequence of pro-oxidants increase, and is not explained by a reduction of antioxidant defenses. In both conditions, TRAP determination decreases in plasma, but not in tissue.     

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.     

Use of coculture of colonic mucosal biopsies to investigate the release of eicosanoids by inflamed and uninflamed mucosa from patients with inflammatory bowel disease.

Eicosanoid production was measured in cultured biopsies of colonic mucosa from control patients, with the irritable bowel syndrome, and from patients with proctosigmoiditis and with colonic Crohn's disease. Cultured inflamed colonic mucosa from patients with proctosigmoiditis and Crohn's disease produced more prostaglandin E2 and leukotrienes C4 than control tissues. In addition, eicosanoid production by macroscopically uninflamed or 'quiescent' mucosa from the right colon was examined in patients with proctosigmoiditis and between skip lesions in Crohn's disease patients. In the proctosigmoiditis group quiescent mucosa produced eicosanoids in similar quantities to control tissue. Coculture of quiescent plus inflamed tissue however, generated a marked increase in eicosanoid output in 12 of 20 of the patients and this was similar to the quantity obtained from two pieces of inflamed tissue. In the Crohn's disease group, quiescent mucosa produced more eicosanoids than control mucosa but production was markedly stimulated by coculture with inflamed mucosa in all patients. These findings suggest that in some patients with proctosigmoiditis and in all patients with Crohn's disease quiescent mucosa appears to be sensitised. A small but significant increase in the macrophage population may be partly responsible but it is likely that these and other cells are primed to release eicosanoids, and may be induced to do so by soluble mediators produced by actively inflamed tissue.     

Role of interleukin 1 in inflammatory bowel disease--enhanced production during active disease.

Interleukin 1 is a polypeptide cytokine produced by various cell types and has been shown to have a major role in inflammatory and immunological responses. In experimental colitis it proved to be a dominant mediator and a reliable marker of inflammation. The aim of the present study was to determine in vitro the extent of production and release of interleukin 1 from colonic mucosa of patients with active untreated inflammatory bowel disease. Colonic mucosal biopsy specimens were obtained during colonoscopy from 17 patients with ulcerative colitis, eight patients with Crohn's disease of the colon, and 16 normal control subjects. Interleukin 1 content was determined in fresh and 24 hour organ cultured mucosa as well as in cultured medium. Interleukin 1 content and release were significantly higher in the inflamed mucosa compared with that of control subjects. Prednisolone inhibited interleukin 1 release in a dose dependent fashion. We conclude that colonic mucosal interleukin 1 content and production is significantly raised in active inflammatory bowel disease and may have a role in the pathogenesis of the inflammatory response. Pharmacological suppression of tissue interleukin 1 production may have a beneficial therapeutic effect.