Gastrointestinal permeability in interleukin-10 knockout mice

Gastrointestinal permeability in interleukin-10 knockout mice


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• G4565 EVIDENCE THAT IL-1 RECEPTOR ISOFORMS CONTRIBUTE TO THE IL-1 SYSTEM IMBALANCE FOUND IN IBD. J. Woraratanadharm and T.T. Pizarro. University of Virginia Health Sciences Center, Charlottesville, VA 22908. The biological effects of IL-1 are modulated by a complex system, including a natural antagonist (IL-lra) and its isoforms as well as cell bound and soluble receptors. It is hypothesized that the balance of the IL-1 system contributes to the regulation of immune and inflammatory responses. We have previously shown that a decreased ratio of IL-lra to IL-1 exists within the intestinal mucosa of inflammatory bowel disease (IBD) patients compared to those with self-limiting acute colitis and non-inflamed surgical controls (SC). These data indicated that Crohn's disease (CD) and ulcerative colitis (UC) patients produce lower levels of IL-lra needed to counteract the proinflammatory effects of IL-1. In the present study, the IL-IR isoforms (type I and II) were characterized in intestinal mucosal cell populations isolated from CD, UC and non-inflamed SC patients to determine their contribution to the IL-1 system's imbalance found in IBD. Semi-quantitative (multiplex) RT-PCR was utilized to identify which IL-1R isoform(s) were associated with intestinal epithelial ceils (IEC) and lamina propria mononuclear cells (LPMC). Densitometric tracings were evaluated from negative images of ethidium bromide-stained PCR products and results (relative area under the curve) reported as ratios of IL-1R type (I or II) to GAPDH. Results of quadruplicate experiments are shown below:

i::: In summary, our results demonstrate that IEC and LPMC express transcripts of both IL-1R isoforms, however: 1) increased mRNA levels of IL-1RI are detected in LPMC compared to IEC, whereas increased IL-1RII levels are detected in IEC compared to LPMC, and 2) IL-1RI mRNA levels in CD patients are elevated when compared to non-inflamed SC, while IL-1RII levels are decreased in IBD patients when compared to SC. In addition, protein measurements of IL-1Rs were shown to reflect changes observed in mRNA levels. Taken together, these data suggest that IL-1R isoforms may contribute to the imbalance in the IL-I system found in IBD and that IEC are active participants in the regulation of local immune responses within the gut mucosa. Supported by NIAID40303 (TFP). • G4566 INFLUENCE OF SMOKING HISTORY ON DISEASE COURSE IN ULCERATIVE COLITIS. DM Woseth, SB Hanauer. University of Chicago, Pritzker School of Medicine, Chicago, IL. Back~,round: Ulcerative colitis (UC) most commonly occurs in nonsmokers. Ex-smokers are at a greater risk of developing UC than patients who have never smoked. While clinical investigations have demonstrated that nicotine (transdermal patches and chewing gum) may improve the course of UC, the effect of smoking cessation on the natural history and severity of disease is not well known. Aim: This study examines differences in disease course among neversmokers, ex-smokers, and current smokers to better elucidate the long-term effects of smoking. Methods: A questionnaire regarding smoking history and disease course was mailed to 865 UC patients in the University of Chicago IBD Patient Registry and IBD Surgical Database. 347 of the individuals who responded were grouped according to smoking status as never-smokers, ex-smokers (with cessation prior to the diagnosis of UC), and current cigarette smokers (who began smoking prior to the diagnosis of UC). Differences in indicators of disease course were measured between groups with a two-tailed t-test. Results: No significant demographic differences (age, sex, duration of disease) were noted between the three patients groups. Age at diagnosis, however, was greater for ex-smokers. Characteristics of disease course are as follows: N= % COLONIC REsEcTIoN % TIME IMMUNOSUP/STEROIDS % W/SURGERY W/POUCHITIS % SCLEROSINGCHOLANGITIS

Never-Smokers Ex-Smoker~ CurrSmokers 16 209 122 31 30* 44* 31" ** 37* 40** 40 47* 30* 0* 2.4* 0.8

* and ** indicate p < 0.05 between corresponding patient groups

Conclusion: In addition to preventing ulcerative colitis, cigarette smoking alters the course of disease. Ex-smokers 1) develop UC at an older age than never- or current smokers and have a more virulent course requiring 2) colectomy more often than never smokers and 3) immunosuppressant and steroid therapy more often than current smokers. After colectomy, ex-smokers are less likely to develop pouchitis than never smokers. Sponsored by: CCFA Student Summer Research Program & The Reva and David Logan Center.

G4568 GASTROINTESTINAL PERMEABILITY IN INTERLEUKIN-10 KNOCKOUT MICE. J-N. Wu, D-M. McCafferty, K. A. Sharkey, R. N. Fedorak, and J. B. Meddings. Gastrointestinal Research Group, University of Calgary, Calgary, Alberta, Canada. Crohn's disease (CD), an idiopathic chronic inflammatory disorder of the gut, displays a characteristic profile of increased intestinal permeability during and prior to disease onset in man. However, it is still not clear whether increased permeability is a primary defect or secondary to intestinal inflammation. The aim of this study was to investigate whether changes in permeability precede enterocolitis in interleukin-10 knockout (IL-10-/-) mice, a CD model of spontaneously developing enterocolitis. Methods: Gastrointestinal permeability was monitored once per week from 3 to 16 weeks of age using sucrose, lactulose, mannitol, and sucralose. This allowed separate determination of gastric, small intestinal and colonic permeability in vitro. The severity of enterocolitis was assessed by both tissue myeloperoxidase (MPO) activity and morphology. Results: Evidence of colitis, in terms of macroscopic damage and increased MPO, was apparent by 3 weeks of age and persisted throughout the study. No increase in colonic permeability, which would suggest a diffuse process, was evident. Within the small intestine, transient increases in MPO activity were apparent at 3 weeks of age that quickly returned to normal. However, small intestinal permeability was grossly elevated by 6 weeks of age and persisted despite the absence of any other manifestation of disease in this region of the gut. No evidence of gastric inflammation was ever apparent, however, gastric permeability paralleled that of the small intestine and was markedly increased from 6 weeks of age. Conclusions: Increased colonic permeability does not precede colonic inflammation in this animal model of Crohn's disease. However, increases in small intestinal and gastric permeability clearly precede persistent disease in these locations. Whether increased small intestinal permeability contributes to colonic inflammation remains an intriguing question and is not ruled out by these results.

• G4569 MICE WITH DECREASED E-,P AND L-SELECTIN LIGAND BIOSYNTHESIS ARE MORE RESISTANT TO DEXTRAN SODIUM SULFATE-INDUCED COLITIS BUT HAVE MORE SEVERE TNBS COLITIS. RJ Xavier. PL Beck, Naifang Lu, DK Podolsky and B Seed. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease and Dept Molecular Biology. Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Background and Aims; Selectins are involved in the initial phase of leukocyte recruitment to sites of inflammation. Abnormal leukocyte trafficking and function have been implicated in the pathogenesis of inflammatory bowel disease. To determine the role of leukocyte adhesion molecules in two animal models of colitis; E-, P- and L- selectin ligand biosynthesis was blocked by targeted disruption of the mouse ~(1,3)fucosyltransferase Fuc-TVII (TVII) gene. Methods; TVII ko mice were produced using standard knockout technology. These ko mice have been found to have deficient leukocyte recruitment. Wild type (wt) animals were used as controls. Colitis was induced by adding dextran sodium sulfate (DSS) (2.5%) to drinking water or intrarectal administration of trinitrobenzene sulfonic (TNBS) acid in 40% ethanol (0.2 ml, of 2 mg/ml). Animals were sacrificed at day 1,3, 5 and 7 following TNBS and following 5 or 9 days of DSS. Body weight, the presence of fecal blood and diarrhea were followed and upon sacrifice macroscopic damage, and the extent of adhesions were score. Samples were processed for histology. Results; Following DSS exposure the TVII ko mice had significantly less weight loss, diarrhea and fecal occult blood than wt animals (p<0.05 to 0.01). The TVII ko mice also had lower damage scores at day 5 at 9 than wt mice (p<0.05 to 0.001). There was also less severe damage and inflammation noted on histologic exam at both days 5 and 9 i n the TVII ko mice versus wt. Following TNBS administration there were no significant differences in the severity of inflammation, extent of adhesions or degree of diarrhea in the TVII mice compared to wt animals when animals were sacrificed at days 1 and 3. However, when animals were sacrificed at days 5 and 7 following TNBS TVII ko mice more severe diarrhea (0-3 score; 2.6 _+0.2 vs 1.0 - 0.3 p<0.001), more adhesions (0-3 score; 2.5-+ 0.2 vs 0.07-+ 0.07 p, 0.0001), greater damage (0-5 score; 4.4 -+.0.4 vs 1.6 +_0.4 p<0.001). There was also more severe inflammation and greater increases in bowel wall thickness noted on histologic exam in the TVII ko mice vs wt at 5 and 7 days following TNBS.