IMMUNOREGULATORY DEFECTS IN INFLAMMATORY BOWEL DISEASE

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Project 1: In previous studies we showed that a chronic colitis associated with a Th1 type cell response can be induced by the rectal administration of the hatptenizing reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS). We report here that oral administration of haptenized colonic proteins (HCP) before rectal administration of TNBS effectively suppresses the ability of the latter to induce colitis. CD4+ T cells from HCP-fed animals stimulated with anti-CD3/anti-CD28 had a 5-10-fold increase in production of TGF-beta and secreted increased amounts of IL-4 and IL-10 but lower levels of IFN-gamma in comparison to T cells from ovalbumin-fed control animals. In addition, the colons of HCP-fed mice showed strikingly increased TGF-beta secretion but decreased IL-12 expression by immunohistochemical studies and isolated mononuclear cells from HCP-fed animals secreted less IL-12 heterodimer. Finally, and most importantly, the suppressive effect of orally administered HCP was abrogated by the concomitant systemic administration of anti-TGF-beta or rIL-12 suggesting a reciprocal relationship between IL-12 and TGF-beta on tolerance induction in TNBS-induced colitis. These studies suggest for the first time that TGF-beta production can abrogate experimental granulomatous colitis even after such colitis is established, and thus, that regulation of TGF-beta levels may have relevance to the treatment of human inflammatory bowel disease. Project 2: In further studies of the immunologic mechanisms regulating TNBS-colitis we investigated the role of the CD40L-CD40 interaction in the presence of TH1 T cells and the production of IL-12 in this experimental disease. We found that the administration of anti-CD40L antibodies to mice in whom TNBS-colitis is being induced prevented interferon gamma production by lamina propria CD4+ T cells and also clinical and histological evidence of disease. In contrast, the secretion of IL-4, a Th2-type cytokine, was increased after anti-CD40L treatment. In further studies we showed that the prevention of disease activity was caused by an inhibition of IL-12 secretion and that the injection of recombinant IL-12p70 heterodimer into TNBS + anti-CD40L treated mice reversed the effect of anti-CD40L and resulted in severe disease. These studies show that the CD40L-CD40 interaction is critical for the development of TNBS-colitis. Project 3: In a further study, we investigated whether human inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease) is associated with altered lymphokine secretion profiles. In initial studies, we showed that LP T cells from inflamed Crohn's disease mucosa manifest increased IFN-gamma secretion compared with control LP T cells, particularly when stimulated via the CD2/CD28 pathway. In contrast, IL-4 and IL-5 production by Crohn's disease LP T cells was decreased compared with that of control LP T cells. In addition, IL-2 production by Crohn's LP T cells was decreased. In further studies, we showed that LP T cells from ulcerative colitis mucosa stimulated via either the TCR/CD3/CD28 or CD2/CD28 produced increased amounts of IL-5. Such increased IL-5 production was not associated with increased IL-4 secretion and, in contrast to Crohn's disease, ulcerative colitis LP T cell production of IL-2 and IFN-gamma was normal. Taken together, these studies provide strong evidence that the immunopathologic process characteristic of the two major forms of IBD is associated with very different cytokine secretion patterns: in Crohn's disease a Th1 cytokine pattern is present whereas in ulcerative colitis a Th2 cytokine pattern prevails. These different patterns may determine the type of inflammatory process present and may influence the kind of cytokine-based treatment needed.