Immunoregulatory Defects in Inflammatory Bowel Disease

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Project I: TNBS-colitis is an IL-2-driven Th1 T cell-mediated inflammation that is rapidly reversed by the administration of an anti-IL-12; in the present study we sought to define the mechanism of this curative effect. In initial studies we found that anti-IL-12 administration was a more efficacious treatment of TNBS-colitis than anti-IFN-gamma administration in that its therapeutic effect was seen at a lower dose, it resolved colitis in a greater percentage of mice, and it led to more complete normalization of IFN-gamma production by stimulated spleen and lamina propria (LP) CD4+ T cells. In further studies, we found that anti-IL-12 treatment led to greatly increased numbers of apoptotic cells in the spleen and LP (by in situ TUNEL technique) whereas anti-IFN-gamma treatment led to only modestly increased numbers of apoptotic cells (as compared to untreated mice). These findings relating to apoptosis were corroborated by in vitro studies of freshly isolated dispersed spleen cells in which we showed that anti-IL-12 treatment was associated with increased numbers of apoptotic (TUNEL+) T cells and decreased numbers of Fas+ and CD4+ T cells. In addition, they were corroborated by in vivo studies in which we showed that MRL/lpr mice lacking Fas function administered TNBS per rectum developed a more severe colitis than control MRL/MPJ mice and responded poorly to treatment with anti-IL-12. Taken together, these studies show that a main mechanism of action of anti-IL-12 in TNBS-colitis is the induction of Fas-mediated apoptosis of the Th1 T cells causing the inflammation. Project II:. We previously demonstrated that TNP-OVA immunization leads to a transmural colitis in the IL-2-/- mouse that is caused by IL-12 driven CD4+ T cells and resembles closely human Crohn's disease. In order to evaluate whether the colitis was due to infiltrating peripheral T cells rather than local lamina propria (LP) T cell expansion, we administered mAb against Alpha-E-Beta7 to IL-2-/- mice which were immunized with TNP-OVA in CFA at the same time. This resulted in a significantly reduced colitis severity score (CSS); CSS: 0-2 vs. 3-4, respectively, which was associated with a significant reduction in CD4+ T cells lymphocytes within the IEL and LPL subpopulation (p<0.01). In contrast, such antibody treatment resulted in an increase of the total number of CD4+ T cells in the spleen of treated vs. untreated IL-2-/- mice (3.2 + 0.6 x 107 vs. 1.2 + 0.2 x 10(7)). Finally, in functional studies, we showed that anti-Alpha-E-Beta-7 treatment significantly reduced IFN-gamma production of LPL, but not of spleen T cells, isolated from IL-2-/- TNP-OVA immunized mice compared to IL-2-/- mice injected with TNP-OVA alone. These data demonstrated for the first time that the onset of colitis depends on the colonic localization of CD4+ lymphocytes expressing either Alpha-E-Beta- 7. In addition, they open the door to the possibility that Crohn?s disease can be treated by administration of antibodies directed against all homing/localization receptors. Project III: In these studies, we describe oxazalone-colitis, a new form of experimental colitis that displays many of the features of ulcerative colitis. This model is induced in SJL/J mice by the rectal instillation of oxazalone (6 mg in ethanol) and is characterized by a rapidly developing colitis involving the distal half of the colon exclusively that peaks at two to three days and resolves at ten days. The inflammation induced is superficial and marked by ulceration, mucin depletion, and a neutrophil infiltration and exudation; lymphocytic infiltration is present, but is also confined to the superficial layer. This distinctive histologic picture is accompanied by a distinctive cytokine profile. Isolated lamina propria T cells display a Th2 pattern characterized by a marked increase in unstimulated or stimulated IL-4 and IL-5 secretion and a decrease in stimulated IFN-gamma secretion (as compared to cells from ethanol-treated controls). In addition, the T cells produced greatly elevated amounts of TGF-Beta in the involved distal colon (20-30-fold above control secretion which was accompanied by an even greater elevation of TGF-Beta secretion in the uninvolved proximal colon (40-50-fold above control secretion). Upon resolution of the colitis, isolated T cells display baseline IL-4 secretion and persistent elevated IL-5 secretion; in addition, TGF-Beta secretion of the proximal, uninvolved colon remained high. To determine the role of the various cytokines in the pathogenesis of oxazalone-colitis we administered various anti-cyokine antibodies to mice at the time of colitis induction. Anti-IL-4 administration led to striking amelioration of the disease, whereas anti-IL-12 administration had no effect or exacerbated the disease; in addition, anti-TGF-Beta administration led to more severe inflammation affecting the entire colon. This finding establishes that oxazolone-colitis is strikingly different than that induced by another haptenizing agent (TNBS) in that it is a Th2-colitis that resembles ulcerative colitis rather than a Th1-colitis that resembles Crohn's disease. A distinctive feature of this model is the counter-regulatory role played by TGF-Beta which appears to delimit the disease to the distal half of the colon and is probably responsible for the rapid resolution of the inflammation. This model will clearly be useful in the further study of the pathogenesis and treatment of ulcerative colitis.