The overall objective of this proposal is to determine the role of cytokines in acute and chronic colitis, specifically whether a particular pattern, sequence or quantity of cytokines, produced mainly by CD4+ T cells, is responsible for the development of chronic colitis. Colonic inflammation will be induced in inbred mice by direct instillation into the distal colon of the contact sensitizing agent trinitrobenzene sulfuric acid (TNBS) dissolved in 50% ethanol. This allows the establishment of an immune reaction to hapten modified self antigens. The resulting colitis has acute and chronic phases and disease """"""""flares"""""""" can be induced by repeat enemas. Moreover, there appears from initial studies, to be a genetic susceptibility among inbred strains of mice. A second model of colitis, with a different mechanism of induction, will also be employed. In this model, lethally irradiated mice are reconstituted with T cell depleted, syngeneic bone marrow followed by treatment with Cyclosporine A (CsA) for 6 weeks. Upon cessation of CsA treatment, multiple inflammatory lesions develop with particularly severe colon involvement. In the TNBS model the production of multiple cytokines, including Interleukins 1-7, neutrophil- activating factor (IL-8), interferon-gamma (IFNgamma), tumor necrosis factor (TNFalpha/TNFbeta) and transforming growth factor beta(TGFbeta) will be measured during acute and chronic colitis and compared to the same cytokines produced during an acute self-limited colitis induced by acetic acid enema. In the CsA/BMT-induced colitis, production of the same cytokines will be measured at weekly intervals following cessation of CsA treatment. Cytokine production will be quantitated by bioassay of supernatants of short-term organ culture of full thickness colon biopsies and of draining lymph node mononuclear cells; and by Northern blot analysis of RNA extracted from inflamed colon tissue and draining lymph nodes. We will also use PCR amplification to identify cytokine mRNA in mononuclear cells isolated from inflamed colon. Because CD4+ T cells are main producers of, and regulators of cytokine secretion, the effect of in vivo manipulation of T cell subsets on the induction and chronicity of colitis will also be investigated. This will be achieved by depletion of T cell subsets by in vivo administration of monoclonal antibodies to CD4 or CD8 T cells. In addition, mice will be tolerized by oral feeding of TNBS in oil prior to the induction of colitis. Using these models, we will test the hypothesis changes in the cytokine sequence, pattern or quantity regulate the severity and chronicity of murine colitis and that this alteration may be due to the preferential activation of a certain subset of CD4+ T cells.
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