ANTIINFLAMMATORY MECHANISMS OF INTERLEUKIN 4

本提案的目标是发展IL-4对ifngamma诱导的基因表达的抑制作用的详细机制理解。IL-4被认为是免疫反应的重要决定因素，它通过牺牲细胞介导的免疫来促进体液免疫。IL-4依赖性的细胞免疫抑制部分是通过抑制IFNgamma诱导的基因转录发生的，并且需要IL-4激活的信号分子STAT6的存在。初步数据表明，IL-4和STAT6可以通过至少两种不同的途径抑制单核吞噬细胞和内皮细胞中ifnγ刺激的基因转录。在原代小鼠巨噬细胞中，抑制ifnγ不会降低STAT1的激活，而在人单核细胞中，IL-4似乎在这个水平上起作用。考虑到这些和其他观察结果，我们提出以下假设：需要STAT6作为il -4依赖性抑制ifnγ诱导的基因表达的介质可能取决于以下一种或多种机制：(1)STAT6和STAT1在敏感基因启动子中竞争STAT核苷酸结合位点。(2) STAT6通过反活化结构域与其他蛋白（抑制因子或共激活因子）相互作用或(3)诱导抑制STAT1的ifngamma激活的基因。我们计划通过以下具体的实验目标来测试这些机制的重要性。1. 确定DNA结合活性对于STAT6介导的抑制是否必要（和充分）。这将涉及使用涉及诱变和基因转移的策略分析STAT6 DNA结合活性，并通过评估调节核苷酸序列依赖性。此外，我们将尝试通过检查对IFN γ诱导和IL-4抑制敏感的广泛基因谱来评估这些机制的普遍性。2. 确定STAT6的蛋白-蛋白相互作用域是否需要il -4介导的抑制。这将包括分析抑制功能所需的STAT6结构域和鉴定新的STAT6相互作用蛋白。3. 确定IL-4抑制人单核细胞中IFNgamma刺激的STAT1激活的机制。这些实验将确定作为IL-4靶点的特定信号成分，并试图将这些功能与一个或多个IL-4诱导基因（例如，SOCS基因家族）联系起来。

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).