Heat-shock proteins (HSP) and HSP70 antibodies in the mucosa of inflammatory bowel disease

• DECREASE IN ORAL TOLERANCE TO OVALBUMIN IN INDOMETHACIN TREATED MICE. E.Louis, D.Franchimont, M.Deprez,A.Lamproye,N.Schaaf,P.Mahieu,J.Belaiche. CHU of Liege,4000 Liege,Belgium. NSAID enteropathy is characterized by ulcers and inflammation of the intestinal mucosa. The pathophysiology of these lesions is unknown although the prlmum movens seems to be an increase in intestinal permeability. The aim of our study was to test whether the indomethacin-induced increased intestinal permeability was associated With abnormalities in the systemic immune response toward a fed soluble antigen in mice. Mice were divided in 5 groups of I0:3 groups treated with oral indomethacin (0.5 mg/Kg; 2.5 mg/Kg; 5 mg/Kg), fed 10 mg ova 18 h later and immunized subcutaneously with 100 ug ova 8 days later; 1 group non treated with indomethacin but fed and immunized subcutaneously with ova; 1 group only immunized subcutaneously. 5 mice of each group were killed 3 b after being fed with ova for the measure of serum ova by RIA and for histology. The 5 other mice were killed i0 days after subcutaneous immunization for an evaluation of the humoral (by the serum anti-ova antibodies) and cellular (by a specific lymphocytes proliferation test) systemic immunune response to ova. Serum ova was increased in indomethacin treated mice and it was proportional to the indomethacin dosage. These mice had also intestinal mucosal lesions characterized by subepithelial oedema and epithelittm desquammation. The classical decrease in the systemic humoral and cellular response tO ova in ova fed mice (oral tolerance) was less marked in indomethacin treated mice and it was statistically significant for the humoral response. In conclusion, our study confirms the early mucosal lesions after oral administration Of indomethacin, shows that there is an increased intestinal permeability to a protein soluble antigen and that there is a decrease in oral tolerance to %his antigen which could be implicated in the pathogenesis of NSAID enteropathy. HEAT-SHOCK PROTEINS (HSP) AND HSP70 ANTIBODIES IN THE MUCOSA OF INFLAMMATORY BOWEL DISEASE D. Ludwio, M. Ibrahim, C. Pfister, K. Scbwarting, E. F. Stange Deportment of Internal Medicine, Division of Gastroenterology, Medical University of L0beck, Germany Heat-shock proteins or highly conserved immunogenic intracellular proteins which are directly involved in the biogenesis of ~)roteins and exert a eytoprotective function during metabolic stress. In view of a possible pathogenic role of stress proteins in inflammatory bowel disease (IBD) we examined different heat shock proteins and antibodies against HSPT0 at Ha mucosal level. Methods: Colonic mucosal biopsies Were obtained from controls (NC, n=42) and patients with Crohn's disease (CO, n=27) or ulcerative colitis (UC,~ n=24) by colonoscopy. The various HSPs of different molecular weight were identified by the western blot technique using specific rnonoclonal antibodies (Stress Gen). HSP-70 antibodies were identified in the mucoso by iooelectric focusing of the homogenate followed by a reverse blot using biotinor fluorescein-labelled human HSP70. The mucosal localization of human HSP-70 was assessed by the indirect immunoperoxidase technique. Results: Human HSP70 was found in the mucosa of nearly all patients with Crohn's disease (26/27) ulcerative colitis (18120) as well as in healthy controls (37142). Concerning the 60 kD protein all samples positive with the specific anti-human antibody LK-I reacted also with the bacterial er0ssreactive antibody LK-2. There was no evidence of an enhanced HSP induction in the inflamed mucoso compared to noninflamed areas in any disease. Antibodies directed against human HSP70 were detected more frequently in inflamed mucosal areas of patients with CD (25127) and CU (21/24) as compared to the control group (31/42, p<0.05). Significantly elevated antibody levels were found in areas of inflamed mucoso in diseased patients. Conclusions: Human HSP60 and HSPT0 are similory prevalent in both normal and inflamed intestinal mucosal areas. The elevated antibody level against HSP70 st the site of inflammation could be a factor inducing or sustaining the immune response in IBD. The induction of a local immune response by bacterial HSP cross-reacting with human HSP appears plausible but is not specific for IBD. • SPECIFIC ANERGY OR DELETION OF T CELLS THAT BEAR SPECIFIC T CELL RECEPTOR V13 SEQUENCES IN DIFFERENT PHASES OF CROHN'S DISEASE. N;Liiacrine, R.Stoll, T.Kucharzikl M.Fisahn, W:Domschke. Department of Medicine B, University of Miinster, Mfinster, Germany. Recent studies have shown an increase in V~8+ T cells in mesenteric lymph nodes of a subgroup of patients with Croha's disease (CD), suggesting a possible role for superantigens in inflammatory bowel disease (IBD). A comparison with results, from earlier reports is difficult due to the small number of samples and the fact that most previous reports have been published on materials where no data of dtiration and existence of extraintestinil manifestations were given. The present study characterizes TCR V~ gene expression among peripheral blood T cells of 19 CD patients and 15 healthy controls by means of semiquantitative PCR amplification of TCR VI3 cDNA prior to southern hybridization analysis. Being concerned that different patterhs max, be seen in different phases of the inflammatory d~sease process , we have also taken care to analyse Sequential samples from CD patients and from control subjects at variable times of our study. In order to look if mitogens alter the TCR repertoire, we further analysed our data with respect to unstimulated and phytohaemagglutinin (PHA)or anti-CD3stimulated T cells. Expression of all 22 VI 3 genes was found in each control tested and showed no variation over time. In contrast, the amount of the individual transcripts from CD patients differed considerably; three patients with mild disease and duration of disease longer than eighteen years revealed decreased V~19 and V~321 genes, although these two VI3-genes were detectable two months later. Three other patients with also longstanding disease expressed a restricted VI3 repertoire which predominantly showed decreased VI310 and VI321 genes; similarly, these two Vl~-genes were detected thr~ee months later. Notably, three patients, who experienced the most dramatic VI3-depletion (vI36,vI58,v~9,vI310,VI319,VI321), which was complete, suffered from strong extraintesfinal manifestations. Under the conditions used, stimulation of peripheral blood mononuelear Cells with anti-CD3 and PHA did not perturb the TCR VI3 repertoire. Our data indicate that in CD antigenic material exists that has properties consistent with those of T cell superantigens. The fact that in patients with CD different patterns of V[3 sequences could be found both at various time points and at various manifestations of disease implies that T cells in CD expressing different TCR gene products may interact with different superantigens or different regions of a superantigan, each of which interacts with a distinct spectrum of VI3-bearing T cells. SUSCEPTIBILITY TO 5-AMIND-SALICYLIC ACID (5-ASA)INDUCED NEPHROTOXIC1TY IN RATS IS STRAINAND SEXRELATED. C. Lundberg, I. ~sberg, C-::~Smedeg~rd, T. Berglindh. Dept. of Pharmacology, Pharmacia Pharmaceutidals Uppsala, Sweden. Introduction: Various m e s a l a z ~ fdi~mulations, in Contrast to olsalazine, cause a high and variable systemic exposure of 5-ASA tha t has been implied to give a potent ial r i sk for nephrotoxicity. Fur the r more, several cases of nephrotoxicity hove recently been reported in IBD pa t ien ts receiving mesalazine therapy. The purpose of the present in vestigation was to establish an experimenta 1 model in the ra t in which' 5-ASA-induced nephrotoxicity could be studied. Urine was analysed for micro albumin (glomerular damage), N-acetyl-[~ -D-glucosaminidase (NAG) and a lkal ine phosphatase (ALP) (tubular damage), and osmolority (overall kidney function). M e t h o d s : Fisher-344, Lister-Hooded, and Spraque Dawley ra t s of both sexes were housed individually in metabolic cages. After an adjustment phase of 3 days, 5 consecut ive24-hour urine samples were collected and analYsed for micro albumin, NAG, ALP, and osmolarity. Saline (negative control) or 5-ASA (600 mg/kg) was injected intraperi toneal ly (Lp.) immedia te ly following the first 24-hour urine sampl ing (time 0); a t 24, and 48 hours, respectively. HgC12 (2 p.mol/kg) was injected i.p. a t 48 hours as a positive control. Results: In female ra t s of al l three strains l i t t l e evidence of r e n a l damage after ei ther HgCI2 or 5-ASA was Observed. The male Spraque Dawley ra t s were the most susceptible showing stat is t ical ly significant increased ur ine levels Of NAG, ALP, and micro-albumin and decreased osmolarity a t 48 and 96 hours following HgC12 injection as compared to saline. In th is ra t s t ra in 5-ASA caused a reversible increase in al l va r i ables measured w i t h the exception for osmolarity were no s t a t i s t i ca l significant change was observed. Conclusion: We have shown tha t even a short te rm (3 days) systemic load of 5-ASA (600 mg/kg) in the ra t causes a reversible but significant renal glomerular and tubular damage tha t is s t r a in and sex-related. These resul ts imply tha t low p l a s m a levels of 5-ASA might be of importance to avoid potent ial nephrotoxicity in the t rea tment of ulcerative colitis pat ients . Further, th is experimental model will now be used for more detai led studies of 5-ASA induced-nephrotoxicity.