April 1995 Immunology, Microbiology, and Inflammatory Disorders A775

O REGIONAL DIFFERENCES IN ENDOTOXIN-INDUCED NITRIC OXIDE SYNTHASE ACTIVITY IN THE RAT INTESTINE CAN BE INHIBITED BY AMINOGUANIDINE IN VIVO A.R. Aurora and W.K. MacNaughton, Defenee Research Establishment Ottawa and Digestive Diseases Research Group, Department of Physiology, University of Ottawa, Ottawa, Canada.

Nitric oxide (NO) is an important mediator o.f physiological and pathophysiological processes in the gut. In order to better understand the role of NO in intestinal inflammation, we measured NO production in rats injected with bacterial endotoxin (Salmonella t.vphimurinm LPS; 4 mg/kg i.v.). Segments of rat jejunum ileum and colon were removed 4 h after the injection ofendotoxin or saline and NO svnthase activity was measured using the t4C-L- arginine to *4C-L-citrulline conversion assav. Ca~-dependency of NOS activity was assessed by duplicate assays with EGTA (1 raM) present. In saline treated rats, no regional differences in total NOS activity were evident (0.629+0.055, 0.608+0.130 and 0.806+0.293 nmol/min/g tissue for jejunum, ileunt and colon, respectively). In endotoxin treated rats, all regions of intestine expressed a significant increase in total NOS activity, increasing 2-, 9- and 3.5-fold in jejunum, ileum and colon, respectively. Regional differences in total NOS activity of intestinal tissues from endotoxin treated rats were apparent (1.285_+0.159, 5.343_+0.890 and 2.766_+0.445 nmol/min/g tissue in the jejunum, ileum and colon; respectively). Total NOS activity in saline treated rats was primarily Ca~-dependent as it was largely inhibited by the addition of EGTA to the incubation buffer, whereas the endotoxin-induced increase in NOS activity was clearly due to the induction ofa Ca++-independent NOS as it was not inhibited by EGTA. The Ca~-independent component of total NOS activity was increased 3-, 13-, and 21-fold in the jejunum, ileum and colon of endotoxin treated rats, respectively. The non-specific NOS inhibitor, L-NAME (20 raM), when added to the incubation buffer, significantly inhibited NOS activity in all regions of the gut of both saline and endotoxin treated rats. To determine if iNOS activity could be specifically inhibited in vivo, aminoguanidine (AG) which preferentially inhibits iNOS, was injected (100 mg/kg i.v.) concomitantly with eodotoxin. AG significantly inhibited endotoxin-induced NOS activity in the rat ileum (62%) and colon (56%) but not in the jejunum (16%). No regional differences in total NOS activity were apparent following AG administration. The remaining NOS activity in all regions of AG treated rats was significantly inhibited by EGTA suggesting that it was Ca~-dependent. Conchtsions: These studies identify regional differences in endotoxin-induced total NOS activity in the rat intestine which are likely due to differences in the distribution or activation of a Ca ~- independent NOS. Furthermore, we demonstrated that systemic administration ofaminoguanidine can significantly inhibit the endotoxin-induced NOS activity in vivo.

I Dextran Sulfate Sodium Induced Colitis and Sulfasalazine Treatment in Bacteria- free Mice. L-G Axelsson, E Landstr~m, A-C Bylund-Felleuius, T Midtvedr Dept. of Pharmacolo£,/, Pharmacia Pharmaceuticals AB, U_p~sala; Dept. of Zoophysiology, Ufi~sa[a University, -Upp_sa_ la, and Laboratory of Medical ~crobial Ecology, Dept. of C~ll an;t Molecular BiologT', Karolimka Institute. Stockholm, Sweden. Dextran Sulfate Sodium (DSS) given orally to mice induces a colitis which success- fully can be treated with sutfasalazine (SASP), a molecule consisting of sulfapyridine and 5-amino salicylic acid (5-ASA), were 5-ASA is considered to he rhe active moiety in the clinical rherapy of inflammatory bowel disease. The intestinal bacterial flora has been proposed to be importanr for the parhogenesis of the DS8 colitis and for the splitting of SASP. Such dependencies can be studied in germ-free mice which lack the bacteria that could perpetuare colitis and to split SASP. The objective of this study was ro: 1) assess if the DSS colitis could be induced in germ-free (GF) mice, and, 2) if so, could this colitis be treated with sulfasalazine. GF and conventional (C) NMRI/KI mice were given sterile 2.5% DSS, 2.5% DSS with SASP (100 mg/kg/day) or placebo for 7 days. Clincal parameters were recorded and histopathological evaluation was oerformed on coded sections of ileum, cmcum and colon.

Substance n Colon Spleen Bod)r Animals with Well,at lenl~th weil~ht cecal inflam-

(cm) (rag) (g) mat;on Water 5 8.8 ±0.4 70.0 ± 6.5 22.1 ±0.6 0/5 DSS 10 6.7 ±0.5*** 98~6 ±27.9* t9.4 ±1.2"** 7/8 DSS+SASP 10 7.0 ±0.9 79.2 ± 7.1 • 20.0 ±2.0 217 Mean ±S.D., DSS vs. water P<0.05 *, <0.001 ***; DSS+SASP vs. DSS P<O.05 •.

Clinical and histological parameters showed that DSS-coliris could be induced in GF mice. The intestinal histology in these mice corresponded essentially to that seen in C NMRI-mice, with toss of crypts, mixed inflammatory cells and macrophages, h involved the colon atul the cecum hut trot the ileum. In C mice, features of DSS- colitis are loss of body weight, enlargement of the spleen, and shortened colon length. Of these features the SASP treated group showed a significantly lowered spleen weight. Histologically, SASP treated mice had less cecal inflammation in comparison to untreated mice given DSS. Sulfasalazine is normally split by intestinal bacteria which are absent in GF mice and the effects of SASP in these mice could possibly impliy that the intact SASP molecule has effects on DSS colitis in the GF animals. In conclusion: DSS colitis can be induced in GF mice and resembles this coliris seen in C mice. The modullating effect of SASP in this model indicates that nor only 5- ASA, bur also unsplit SASP, cmdd be active in DSS-induced colitis.

Lipopolysaccharides are not essential for the mortality seen in DSS induced colitis. L-G Axelsson, E Larutstr&n and A-C Bylund-Fellenius. Dept. of Pharmaeolo~, Pharmacia Pharmaceuticals AB, Uppsala; Dept, of Zoophysiolog~, Uppsa[a University, Zlppsala, Sweden. Dextran sulfate sodium (DSS) colitis can be indnced in mice and can be treated with olsalazine (OLZ). The exact pathogenesis of this colitis is not known. In severe di- sease this colitis results in death. Bacterial products like lipopolysaccharides (LPS) might play a role for the terminal outcome of the coilitls. Therefore C3H/HEJ LPS d mice, which show low sensitivity towards LPS, was subjected to DSS colitis. The response to OLZ treatment was also studied. Female C3H/HEJ LPS" and control C3H/HEN mice were given 0, 2.5, 5 and 10% DSS or 5 and 10% DSS with OLZ /100 mg/kg/day) in rite drinking water for 21 days, deaths were recorded and evaluated with Survival Analysis Log-Rank test.

Percentage of survivlng mice: Strain: Substance: Day: 4 7 10 14 17 21 HEJ 2.5% DSS 100 100 100 100 90 90

5% DSS 100 100 50 10 0 5% DSS+OLZ* 100 100 90 50 20 0 10% DSS 80 10 0 10%DSS+OLZ** 100 90 50 0

HEN 2.5% DSS 100 100 90 90 90 90 5% DSS 100 100 50 0 10% DSS 100 10 0

"P~0.0026 (vs. 5% DSSI, ** P=0.0010 (vs. 10% DSS) There was no mortality in the water control group and all DSS-doses had a signifi- cant mortality compared ro water (p<0.001), with 2.5% DSS it was to low for the study of treatment effects. Survival in mice given 5 and 10% DSS was significantly higher with OLZ treatment, as compared to DSS alone. At comparable doses of DSS the mortality was not statistically significantly altered between HEJ LPS °- attd HEN- mice, indicating that bacterial LPS was nor responsible for rhe death in severe DSS- colitis.The hematocrir on day 10 was significantly lowered in both 5% DSS-groups, 28.4 and 25.8% (P<O.001, Student's t-test), but was normal Ii.e. 45%) in both 2.5% DSS groups as well in the 5% DSS+OLZ group (44.9%).Thus it seems reasonable that the mortality seen in DSS-colitis is caused by loss of blood and disturbance in the water-regulatory propernes of the colon, ultimately leading to a circtdatoty conapse.The effect of OLZ trearment in these mice. taken together with the earlier found effect in other strains of mice points to a common mechanism, for OLZ in this experimental colitis. In conclusion: The DSS-colitis in the genetically aberrant HEJ LPS ° mouse is comparable to that of conventional mice in regard to mortality and to its response to rreatmenr with OLZ.

O T R A N S F O R M I N G G R O W T H F A C T O R S c¢ AND [3 ARE D I F F E R E N T I A L L Y E X P R E S S E D IN C R O H N ' S D I S E A S E AND U L C E R A T I V E COLITIS . M.W.Babyatskv, G. Rossitcr, D.K. Podolsky. GI Unit, Mount Sinai Medical Center, New York, NY; Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, MA.

Transforming growth factors o~ and 13 (TGFot and 13) are expressed in the gastroir!testinal epithelium and modulate epithelial proliferation and other key

functions which may play a role in inflammatory disorders. While TGF~t is

expressed exclusively in epithelial ceils, TGF13 may be expressed by a variety of cell populations within the epithelium and lamina propria. The aim

of this study was to compare and localize TGFQt and 13 expression in inflamed and non-inflamed mucosa of patients with inflammatory bowel disease and controls.

RNA was prepared by cesium chloride gradient from endoscopic biopsies of colon tissue from patients with active Crohn's disease (CD) and ulcerative colitis (UC), inactive CD and UC, and non-inflamed colon, mRNA purified by oligo-dT cellulose chromatography was hybridized with ant;sense cDNA

probes for TGFcx and TGF131. In situ hybridization was performed on lx3 cm segments of inflamed and inactive segments of surgical specimens from patients with active UC, Crohn's colitis and ileitis, diverticulitis, and colon cancer. Tissues were fixed, paraffin-embedded, sectioned, and hybridized

with ant;sense and sense cRNA 35S-UTP probes for TGFct or [31.

By Northern blot analysis, TGF~x mRNA expression was increased in mucosa obtained from patients with inactive UC compared to inactive CD, active CD or UC or normal controls. In situ hybridization demonstrated

maximal TGF~ mRNA expression at the villus tip in the small intestine, and in the surface epithelium of the colonic mucosa in both non-inflamed and

inflamed tissues. Further, TGFct expression increased in association with

active inflammation; expression remained confined to the epithelium. TGF13 mRNA was expressed particularly in the crypts of non-inflamed mucosal samples, but also in individual cells of the lamina propria and the submucosa

subjacent to the crypts. TGF13 mRNA expression was markedly increased in all samples demonstrating active inflammation: this increased expression occurred specifically in cells of the lamina propria and correlated with the

degree of inflammation. These results support the concept that TGFo~ and 13 play an important role in modulating mucosal inflammation and healing in the gastrointestinal tract.