The goal of this proposal is to develop a detailed mechanistic understanding of the suppressive effects of IL-4 on IFNgamma-induced gene expression. IL-4 is well recognized as an important determinant of the character of immune response through promotion of humoral immunity at the expense of cell mediated immunity. The IL-4-dependent suppression of cell mediated immunity occurs in part through inhibition of IFNgamma- inducible gene transcription and requires the presence of the IL-4 activated signaling molecule STAT6. Preliminary data indicate that IL-4 and STAT6 can suppress IFNgamma-stimulated gene transcription in mononuclear phagocytes and endothelial cells using at least two distinct pathways. In primary mouse macrophages suppression does not diminish the activation of STAT1 by IFNgamma while in human monocytes IL-4 appears to act at this level. In consideration of these and other observations, we propose the following hypotheses: The requirement for STAT6 as a mediator of IL-4-dependent suppression of IFNgamma-induced gene expression may depend upon one or more of the following mechanisms: (l) competition between STAT6 and STAT1 for a STAT nucleotide binding site in promoters of sensitive genes. (2) interaction of STAT6 with other proteins (repressors or co-activators) through the transactivation domain or (3) the induction of genes which can suppress IFNgamma-activation of STAT1. We plan to test the importance of each of these mechanisms through performance of the following specific experimental aims. 1. Determine if DNA binding activity is necessary (and sufficient) for STAT6 mediated suppression. This will involve the analysis of STAT6 DNA binding activity using strategies involving mutagenesis and gene transfer and by evaluation of regulatory nucleotide sequence dependency. In addition we will attempt to assess the generality of these mechanisms by examining a broad spectrum of genes sensitive to induction by IFN gamma and suppression by IL-4. 2. Determine if protein-protein interaction domains of STAT6 are required for IL-4-mediated suppression. This will involve analysis of STAT6 structural domains which are required for suppressive function and identification of novel STAT6 interacting proteins. 3. Determine the mechanism(s) by which IL-4 suppresses IFNgamma- stimulated STAT1 activation in human monocytes. These experiments will determine the specific signaling components which are targets of IL-4 and attempt to link such function to one or more IL-4 inducible genes (e.g., SOCS gene family).
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