ANTIINFLAMMATORY MECHANISMS OF INTERLEUKIN 4

这项建议的目的是建立一个详细的机制了解IL-4对干扰素-γ诱导的基因表达的抑制作用。IL-4被公认为是以细胞免疫为代价促进体液免疫而导致免疫应答特征的重要决定因素。依赖IL-4的细胞免疫抑制作用部分是通过抑制IFN-γ诱导的基因转录而发生的，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可通过至少两条不同的途径抑制单核巨噬细胞和内皮细胞中由IFN-γ刺激的基因转录。在原代小鼠巨噬细胞中，抑制并不能减少干扰素-γ对STAT1的激活，而在人类单核细胞中，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，我们提出了以下假设：STAT6作为IL-4依赖的抑制IFNγ诱导的基因表达的中介可能取决于以下一种或多种机制：(L)STAT6和STAT1竞争敏感基因启动子中的STAT核苷酸结合部位。(2)STAT6通过反式激活结构域与其他蛋白质(抑制物或共激活物)的相互作用或(3)抑制STAT1的IFN-γ激活的基因的诱导。我们计划通过执行以下具体的实验目标来测试每一种机制的重要性。1.确定DNA结合活性对于STAT6介导的抑制是否必要(且充分)。这将涉及使用涉及突变和基因转移的策略以及通过评估调节核苷酸序列依赖性来分析STAT6 DNA结合活性。此外，我们将试图通过检测对干扰素-γ诱导和IL-4抑制敏感的广泛基因来评估这些机制的普遍性。2.确定IL-4介导的抑制是否需要STAT6的蛋白-蛋白相互作用结构域。这将包括分析抑制功能所需的STAT6结构域，并鉴定新的STAT6相互作用蛋白。3.确定IL-4抑制干扰素-γ刺激的人单核细胞STAT1活化的机制(S)。这些实验将确定作为IL-4靶标的特定信号成分，并试图将这种功能与一个或多个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).