IL-12 is a heterodimeric cytokine produced by macrophages and dendritic cells with pleiotropic effects that include the induction and maintenance of T helper 1 (Th1) responses (1,2). The IL-12 p40 gene is expressed in cells that make bioactive IL-12 and is highly inducible by bacteria that in vivo stimulate Th1 responses (6,7). A prominent role for IL-12 in the pathogenesis of chronic mucosal inflammation (3) and other chronic inflammatory disorders (4,5) has been established. In murine models of Th1-mediated mucosal inflammation, the inflammatory response can be triggered and perpetuated by enteric microbes or microbial products (18,21). Therefore, understanding the molecular events that lead to IL-12 gene expression will provide important insight into how antigen presenting cell-bacterial interactions influence T cell immune responses. Through an extensive analysis of the IL-12 p40 promoter, a C/EBP protein binding site at -96 to -88 with respect to the transcription start site has been identified that is critical for activation of gene expression by bacteria (8). We have recently identified an AP-1 site immediately downstream of the C/EBP site at -79 to -74 that interacts closely with the C/EBP element. This proposal will focus on this small but critical region of the IL-12 p40 promoter. Understanding activation of this promoter by C/EBP and AP-1 family members will uncover general mechanisms of regulation of the inflammatory response by macrophages. The expression of most other important inflammatory genes produced by macrophages including TNF-alpha, IL-1beta, and IL-8, appear to be regulated by similar mechanisms. Therefore, in depth analysis of IL-12 gene regulation will lead to a general understanding of host-bacterial interactions, macrophage activation, and macrophage specific gene expression. This series of experiments may ultimately elucidate novel molecular targets to alter IL-12 production in vivo. This project will describe functional DNA-protein interactions in a small but critical region, -96 to -72, of the IL-12 p40 promoter.
