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High expression of iNOS in colonic mucosa in ulcerative colitis
April 1 9 9 5
• HIGH EXPRESSION OF iNOS IN COLONIC MUCOSA 1N ULCERATIVE COLITIS. P.D. Revnolds. S.J. Middleton, J.O. Hunter. P. Facet, A Bishop, T. Evans. J.M. Polak. Department of Gastroenterology, Addenbrooke's Hospital, Cambridge CB2 2QQ and Department of Histocbemistry, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12, England Increased nitric oxide synthase ( N o s ) activity has been demonstrated in colonic biopsies in ulcerative colitis (UC) by an increase m citndline concentration. ] However. this method does not determine the cellular site of the NOS. Colonic tissue was obtained from eight patients with active UC, prior to treatment. Specnneus were prepared either from biopsies taken at colonoscopy or from colon which had been removed surgically and had been frozen in liquid nitrogen. Eight control biopsies were taken from patients at normal colonoscopic surveillance for polyps or in the investigation of idiopathic diarrhoea. Tissues were fixed in Zamboni's fluid or 1% paraformaldehyde. Frozen tissue was post fixed in formalin. iNOS expression was assessed by; (1} immunostaining with a rabbit antiserum to a peptide fragment of haman iNOS using the ABC method, and (2) by in situ hybridisation with an iNOS riboprobe which was transcribed with isotopic labelling (33p), from a linearised 200 base pair human iNOS cDNA. High expression of iNOS mRNA and translated iNOS protein luealised to the surface epithelium and crypts in the UC mucosa. The in situ hybridisation signal was of high intensity with the anusense iNOS riboprobe only in the UC specimens. This was patchy in distribution through the mocosa, tmmunostaining was also demonstrated m inflammatory cells in the mucosa, although this needs to be further elucidated. We conclude that the high activity of iNOS in UC, originates predominantly from the colonic epithelium. 1. Middleton SJ, Shorthouse M, Hunter JO. Increased nitric oxide synthesis in ulcerative colitis. Lancet 1993; 341:465-6.
• PROGRESSIVELY DIMINISHING INTESTINAL ANAPHYLACTIC RESPONSE AFTER REPEATED ORAL ANTIGEN CHALLENGE Kevin P. Rioux and John L. Wallace. Intestinal Disease Research Unit, The University of Calgary, Calgary, Alberta, Canada. This study compared intestinal immediate-type hypersensitivity responses to oral antigen in sensitized and control rats after single and repeated antigen challenge. Male Hooded Lister rats (100-125g) were sensitized to chicken egg albumin (EA) (10 mg i.p., alum adjuvant); control animals received only alum adjuvant. Serum EA-speciflc reaginic antibody titres were measured by passive cutaneous anaphylaxis. Animals with a positive reaction at 1:64 dilution were considered sensitized and used in subsequent experiments. After an overnight fast, animals were given EA (10 mg in 1.0 mL of saline) orally. Rat mast cell protease-II (RMCP-II) was measured by ELISA in sera taken immediately before each challenge and three hours later, and in proximal jejunum excised three days after challenge. Mucosal mast cell numbers were counted in Alcian Blue/Safranine-stained sections. In sensitized animals receiving a single oral challenge, there was a 33-fold increase in serum RMCP-II three hours after antigen (Ag) challenge (basa/: 181+15ng/mL; post-Ag: 6036+164, n=5), whereas no significant serum responses were detected in unsensitized controls. There was no significant difference in the number of mast cells detected histologically nor in RMCP-II content of the proximai jejunum of sensitized rats compared to controls three days after challenge. A separate group of control (n=5) and sensitized animals (n=6) was subjected to repeated oral antigen challenge on three day intervals for a total of 21 days. Three days after the last challenge, RMCP-II levels in proximal jejunum were significantly increased in sensitized rats compared to controls (sens: 374+43; ctr/: 207:t:38, p<0.05) but this was not reflected by the number of histologically detectable mast cells. Despite this, there was a progressive decline in the release of RMCP-II into serum in response to oral antigen during the course of repeated challenge (increase in serum RMCP-II after challenge: day O: 24-fold; day 12: 4-fold; day 18: 2-fold). These data suggest that, in sensitized rats, repeated exposure of the intestine to luminal antigen results in adaptive measures which reduce the release of mediators from mast cells, thereby lessening the potentially damaging effects of anaphylaxis. Whether this adaptation occurs at the level of the mast cell, the epithelium, or elsewhere remains to be determined.
Immunology, Microbiology, and Inflammatory Disorders
• INDUCIBLE NITRIC OXIDE SYNTHASE AND IDIOPATHIC COLITIS IN RHESUS MACAQUES. K.A. Ribbons, D.A. Clark, M.G. Carrie*, W.M. Moore* and M.I.S. Miller. Dept. Pediatrics Lcoisiana State University Medical Center, New Orleans, LA and *Moasanto/Searle, St. Louis, Me. INTRODUCTION: Chronic colitis in rhesus monkeys is associated with an elevation in nitric oxide release and induction of the inducible isoform of nitric oxide synthase (iNOS). The aims of the current study were to quantitate the clinical and morphological features of chronic colitis in rhesus monkeys and to compare the level of iNOS induction to that seen in human ulcerative colitis. Finally, the role of iNOS induction in rhesus colitis was assessed by administration of an inhibitor of iNOS, N-imincethyl-L-lysine (NIL). METHODS: Nitric oxide synthase activity was measured in the ascending colon of oolitic monkeys (u=5) and in a segment of inflamed bowel from an ulcerative colitis patient by the citruUine assay, isoform activity was evaluated with Ca++ chelation (EGTA) and specificity was determined by N Gmonomcthyl-L-arginine (NMA). 18 month old oolitic rhesus macaques were housed in metabolie cages and administered a lml bolus of 0.9% NaEI or 30mg/kg NIL twice daily via a nasogastric tube over a 10 day treatment period. The severity of diarrhea was quantitated by NIL-treated (n=2) animals to normal monkeys (n=4) over the treatment period on a daily basis. Morphological measurements were made on tissue segments collected at necropsy from the proximal, mid and distal colon, using quantitative image analysis techniques. RESULTS: iNOS activity in oolitic monkeys and human ulcerative colitis were comparable (16.2+6.9 vs 16.0 pmoles/min/mg protein, respectively). Moreover, the IC50 to NMA was simi!ar for both the monkey (20/.tM) and human (121.tM) enzymes. In rhesus colitis, daily fecal output and fecal fluid content were increased by 84% and 13% respectively, above normal values. NIL treatment produced a 50% reduction in total fecal output in colitic animals, however fecal fluid content was not affected. The Width of the colonic mucosa was increased in oolitic animals above normal values by 42-68% in all segments examined. A 10 day treatment regime With NIL had no effect on the mucosat thickness of eolitie monkeys. CONCLUSIONS: The level of iNOS activity in rhesus colitis is comparable to that seen in human ulcerative colitis. Administration of an inhibitor to iNOS results in an amelioration of some aspects of diarrhea associated with this model, although the dose regime and inhibitor used in this study did not alter the morphological features associated with the colitis.
• REGULATION OF ELECTROLYTE TRANSPORT WITH NITRIC OXIDE IN THE PROXIMAL COLON OF THE MOUSE. J. Rizzo, D. O'Neal, R. Burakoff and F.R. Homaidan. Division of Gastroenterolgy, Winthrop-University Hospital, Mineola, NY and Department of Medicine, Stony Brook School of Medicine, SUNY, Stony Brook, NY. Nitric oxide (NO) is produced from arginine by the action of the enzyme nitric oxide Sffnthase (NOS). In inflammatory bowel disease, NO levels have been shown to increase due to the increase of various inflammatory mediators such as IL-1. The aim of the study was to study the effect and the role of nitric oxide in the regulation of Na + and CI- transport in the proximal colon of Swiss-Webster mice. Methods: Ussing chamber voltage clamp technique was used to measure the short-cireuit current (Isc), a measure of all ion movement across the epithelial layer. Proximal colon pieces stripped from their muscle layer were mounted in Ussing chambers. Arginine was added to the serosal surface and NO levels was measured directly in the serosal bathing solution using an NO meter (WPI, Sarasota, F1). Results: Arginine (10-100 mM) caused a concentration dependent increase in Isc and in NO levels. A maximal increase of Isc of 119 + 27 p.A/cm2 was reached at an arginine concentration of 100 m M (n = 4). NO levels measured at the same concentration of arginine were increased by 142.7 + 53.5 nM (n = 4). To identify the ion responsible for causing the increase in Ise, ion substitution studies were performed. The effect of arginine on Ise was reduced from 119 + 27 ~tA/cm2 to 72 + 25 I.tA/cm2 in chloride free buffer (n = 5, p<0.05). Apical addition of amiloride at 10 ~M, a blocker of the Na ÷ channel and at 1 mM, a blocker of the Na+[H + exchanger, had no effect on arginine's-induced increase in Ise suggesting the lack of effect of arginine on Na + entry mechanisms. 36C1 and 22Na flux measurement were performed. Addition of serosal arginine resulted in a net ion flux of 10.4 rtA/cm2/hr and the net Chloride flux was 10.1 pA/cm2/hr and no net sodium flux. Conclusion: These results suggest that arginine, through production of NO, causes an increase in Isc due to increasing the activity of the apical CI- channel present on the crypt cells. Therefore, one pathway for diarrhea in IBD is the generation of NO in the colon causing increased secretion.
A903
• HIGH EXPRESSION OF iNOS IN COLONIC MUCOSA 1N ULCERATIVE COLITIS. P.D. Revnolds. S.J. Middleton, J.O. Hunter. P. Facet, A Bishop, T. Evans. J.M. Polak. Department of Gastroenterology, Addenbrooke's Hospital, Cambridge CB2 2QQ and Department of Histocbemistry, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12, England Increased nitric oxide synthase ( N o s ) activity has been demonstrated in colonic biopsies in ulcerative colitis (UC) by an increase m citndline concentration. ] However. this method does not determine the cellular site of the NOS. Colonic tissue was obtained from eight patients with active UC, prior to treatment. Specnneus were prepared either from biopsies taken at colonoscopy or from colon which had been removed surgically and had been frozen in liquid nitrogen. Eight control biopsies were taken from patients at normal colonoscopic surveillance for polyps or in the investigation of idiopathic diarrhoea. Tissues were fixed in Zamboni's fluid or 1% paraformaldehyde. Frozen tissue was post fixed in formalin. iNOS expression was assessed by; (1} immunostaining with a rabbit antiserum to a peptide fragment of haman iNOS using the ABC method, and (2) by in situ hybridisation with an iNOS riboprobe which was transcribed with isotopic labelling (33p), from a linearised 200 base pair human iNOS cDNA. High expression of iNOS mRNA and translated iNOS protein luealised to the surface epithelium and crypts in the UC mucosa. The in situ hybridisation signal was of high intensity with the anusense iNOS riboprobe only in the UC specimens. This was patchy in distribution through the mocosa, tmmunostaining was also demonstrated m inflammatory cells in the mucosa, although this needs to be further elucidated. We conclude that the high activity of iNOS in UC, originates predominantly from the colonic epithelium. 1. Middleton SJ, Shorthouse M, Hunter JO. Increased nitric oxide synthesis in ulcerative colitis. Lancet 1993; 341:465-6.
• PROGRESSIVELY DIMINISHING INTESTINAL ANAPHYLACTIC RESPONSE AFTER REPEATED ORAL ANTIGEN CHALLENGE Kevin P. Rioux and John L. Wallace. Intestinal Disease Research Unit, The University of Calgary, Calgary, Alberta, Canada. This study compared intestinal immediate-type hypersensitivity responses to oral antigen in sensitized and control rats after single and repeated antigen challenge. Male Hooded Lister rats (100-125g) were sensitized to chicken egg albumin (EA) (10 mg i.p., alum adjuvant); control animals received only alum adjuvant. Serum EA-speciflc reaginic antibody titres were measured by passive cutaneous anaphylaxis. Animals with a positive reaction at 1:64 dilution were considered sensitized and used in subsequent experiments. After an overnight fast, animals were given EA (10 mg in 1.0 mL of saline) orally. Rat mast cell protease-II (RMCP-II) was measured by ELISA in sera taken immediately before each challenge and three hours later, and in proximal jejunum excised three days after challenge. Mucosal mast cell numbers were counted in Alcian Blue/Safranine-stained sections. In sensitized animals receiving a single oral challenge, there was a 33-fold increase in serum RMCP-II three hours after antigen (Ag) challenge (basa/: 181+15ng/mL; post-Ag: 6036+164, n=5), whereas no significant serum responses were detected in unsensitized controls. There was no significant difference in the number of mast cells detected histologically nor in RMCP-II content of the proximai jejunum of sensitized rats compared to controls three days after challenge. A separate group of control (n=5) and sensitized animals (n=6) was subjected to repeated oral antigen challenge on three day intervals for a total of 21 days. Three days after the last challenge, RMCP-II levels in proximal jejunum were significantly increased in sensitized rats compared to controls (sens: 374+43; ctr/: 207:t:38, p<0.05) but this was not reflected by the number of histologically detectable mast cells. Despite this, there was a progressive decline in the release of RMCP-II into serum in response to oral antigen during the course of repeated challenge (increase in serum RMCP-II after challenge: day O: 24-fold; day 12: 4-fold; day 18: 2-fold). These data suggest that, in sensitized rats, repeated exposure of the intestine to luminal antigen results in adaptive measures which reduce the release of mediators from mast cells, thereby lessening the potentially damaging effects of anaphylaxis. Whether this adaptation occurs at the level of the mast cell, the epithelium, or elsewhere remains to be determined.
Immunology, Microbiology, and Inflammatory Disorders
• INDUCIBLE NITRIC OXIDE SYNTHASE AND IDIOPATHIC COLITIS IN RHESUS MACAQUES. K.A. Ribbons, D.A. Clark, M.G. Carrie*, W.M. Moore* and M.I.S. Miller. Dept. Pediatrics Lcoisiana State University Medical Center, New Orleans, LA and *Moasanto/Searle, St. Louis, Me. INTRODUCTION: Chronic colitis in rhesus monkeys is associated with an elevation in nitric oxide release and induction of the inducible isoform of nitric oxide synthase (iNOS). The aims of the current study were to quantitate the clinical and morphological features of chronic colitis in rhesus monkeys and to compare the level of iNOS induction to that seen in human ulcerative colitis. Finally, the role of iNOS induction in rhesus colitis was assessed by administration of an inhibitor of iNOS, N-imincethyl-L-lysine (NIL). METHODS: Nitric oxide synthase activity was measured in the ascending colon of oolitic monkeys (u=5) and in a segment of inflamed bowel from an ulcerative colitis patient by the citruUine assay, isoform activity was evaluated with Ca++ chelation (EGTA) and specificity was determined by N Gmonomcthyl-L-arginine (NMA). 18 month old oolitic rhesus macaques were housed in metabolie cages and administered a lml bolus of 0.9% NaEI or 30mg/kg NIL twice daily via a nasogastric tube over a 10 day treatment period. The severity of diarrhea was quantitated by NIL-treated (n=2) animals to normal monkeys (n=4) over the treatment period on a daily basis. Morphological measurements were made on tissue segments collected at necropsy from the proximal, mid and distal colon, using quantitative image analysis techniques. RESULTS: iNOS activity in oolitic monkeys and human ulcerative colitis were comparable (16.2+6.9 vs 16.0 pmoles/min/mg protein, respectively). Moreover, the IC50 to NMA was simi!ar for both the monkey (20/.tM) and human (121.tM) enzymes. In rhesus colitis, daily fecal output and fecal fluid content were increased by 84% and 13% respectively, above normal values. NIL treatment produced a 50% reduction in total fecal output in colitic animals, however fecal fluid content was not affected. The Width of the colonic mucosa was increased in oolitic animals above normal values by 42-68% in all segments examined. A 10 day treatment regime With NIL had no effect on the mucosat thickness of eolitie monkeys. CONCLUSIONS: The level of iNOS activity in rhesus colitis is comparable to that seen in human ulcerative colitis. Administration of an inhibitor to iNOS results in an amelioration of some aspects of diarrhea associated with this model, although the dose regime and inhibitor used in this study did not alter the morphological features associated with the colitis.
• REGULATION OF ELECTROLYTE TRANSPORT WITH NITRIC OXIDE IN THE PROXIMAL COLON OF THE MOUSE. J. Rizzo, D. O'Neal, R. Burakoff and F.R. Homaidan. Division of Gastroenterolgy, Winthrop-University Hospital, Mineola, NY and Department of Medicine, Stony Brook School of Medicine, SUNY, Stony Brook, NY. Nitric oxide (NO) is produced from arginine by the action of the enzyme nitric oxide Sffnthase (NOS). In inflammatory bowel disease, NO levels have been shown to increase due to the increase of various inflammatory mediators such as IL-1. The aim of the study was to study the effect and the role of nitric oxide in the regulation of Na + and CI- transport in the proximal colon of Swiss-Webster mice. Methods: Ussing chamber voltage clamp technique was used to measure the short-cireuit current (Isc), a measure of all ion movement across the epithelial layer. Proximal colon pieces stripped from their muscle layer were mounted in Ussing chambers. Arginine was added to the serosal surface and NO levels was measured directly in the serosal bathing solution using an NO meter (WPI, Sarasota, F1). Results: Arginine (10-100 mM) caused a concentration dependent increase in Isc and in NO levels. A maximal increase of Isc of 119 + 27 p.A/cm2 was reached at an arginine concentration of 100 m M (n = 4). NO levels measured at the same concentration of arginine were increased by 142.7 + 53.5 nM (n = 4). To identify the ion responsible for causing the increase in Ise, ion substitution studies were performed. The effect of arginine on Ise was reduced from 119 + 27 ~tA/cm2 to 72 + 25 I.tA/cm2 in chloride free buffer (n = 5, p<0.05). Apical addition of amiloride at 10 ~M, a blocker of the Na ÷ channel and at 1 mM, a blocker of the Na+[H + exchanger, had no effect on arginine's-induced increase in Ise suggesting the lack of effect of arginine on Na + entry mechanisms. 36C1 and 22Na flux measurement were performed. Addition of serosal arginine resulted in a net ion flux of 10.4 rtA/cm2/hr and the net Chloride flux was 10.1 pA/cm2/hr and no net sodium flux. Conclusion: These results suggest that arginine, through production of NO, causes an increase in Isc due to increasing the activity of the apical CI- channel present on the crypt cells. Therefore, one pathway for diarrhea in IBD is the generation of NO in the colon causing increased secretion.
A903