The objective of the proposed study is to understand the immunopathogenesis of the human inflammatory bowel diseases (IBD) by characterizing the regulated expression of interleukin-12 (IL-12). IL-12 is a heterodimeric cytokine with pleiotropic effects, including the induction and maintenance of T helper 1 (Th1) responses. In vivo, Th1 responses are critical in the development of immunity against intracellular microbes. In autoimmune disorders, Th1 cells may mediate tissue injury. T helper 2 (Th2) cells are deleterious in infections with intracellular organisms, but may inhibit the tissue damage elicited by Th1 responses in autoimmune and inflammatory diseases. Recently described animal models highlight the importance of Th1 and Th2 subsets in the pathogenesis of chronic intestinal mucosal inflammation. The secretion of IL-12 by antigen presenting cells (APCs) is an obligate signal for the generation of a stable Th1 phenotype. Therefore, the regulation of IL-12 gene expression is likely to be a primary event influencing Th subset differentiation and cytokine production Th vivo. Expression of the two IL- 12 genes will be characterized in APC lines by defining the effects of potential inducers, and determining the kinetics of mRNA induction. The 5 regulatory region of each gene will be analyzed from murine genomic DNA clones by transcription start site mapping, restriction mapping, and DNA sequencing. It will then be determined whether IL-12 expression is regulated through modulation of transcription initiation, mRNA stability, or attenuation of transcription. Finally, the promotor region for each lL- 12 gene will be functionally characterized by describing cis-acting control elements involved in initiation of IL-12 transcription in transient and stable transfection assays. Trans-acting factors involved in IL-12 gene-expression will be analyzed by electrophoretic mobility shift assays and DNAse I footprinting. Once these regulatory elements are identified, their contribution to IL-12 expression will be studied in specific disease models. By characterizing the regulated expression of IL- 12 in APC, molecular mechanisms that determine Th1 responses may be described. These mechanisms may then be exploited to alter host immunity in infectious and inflammatory disorders, including IBD.
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