ANTIINFLAMMATORY MECHANISMS OF INTERLEUKIN 4

本研究的目的是对IL-4对IFN-γ诱导的基因表达的抑制作用进行详细的机制研究。IL-4通过以细胞介导的免疫为代价促进体液免疫而被公认为免疫应答特征的重要决定因素。细胞介导的免疫的IL-4依赖性抑制部分地通过抑制IFN γ诱导型基因转录而发生，并且需要IL-4活化的信号分子STAT 6的存在。 初步数据表明，IL-4和STAT 6可以抑制IFN γ刺激的基因转录单核吞噬细胞和内皮细胞使用至少两个不同的途径。在原代小鼠中，巨噬细胞抑制不会减少IFN γ对STAT 1的激活，而在人单核细胞中，IL-4似乎在该水平起作用。 考虑到这些和其他观察结果，我们提出了以下假设：STAT 6作为IL-4依赖性抑制IFN γ诱导的基因表达的介体的需要可能取决于以下机制中的一种或多种：（1）STAT 6和STAT 1之间竞争敏感基因启动子中的STAT核苷酸结合位点。(2)STAT 6通过反式激活结构域与其它蛋白质（阻遏物或共激活物）的相互作用，或（3）可抑制STAT 1的IFN γ激活的基因的诱导。我们计划通过以下具体实验目标的执行来测试这些机制中的每一个的重要性。1.确定DNA结合活性对于STAT 6介导的抑制是否是必需的（和足够的）。这将涉及使用涉及诱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STAT 6 DNA结合活性。此外，我们将尝试通过检查对IFN γ诱导和IL-4抑制敏感的广谱基因来评估这些机制的普遍性。2.确定STAT 6的蛋白质-蛋白质相互作用结构域是否是IL-4介导的抑制所需的。这将涉及STAT 6结构域的分析，这是需要抑制功能和新的STAT 6相互作用蛋白的鉴定。3.确定IL-4抑制人单核细胞中IFN γ刺激的STAT 1活化的机制。这些实验将确定作为IL-4靶的特异性信号传导组分，并试图将这种功能与一种或多种IL-4诱导型基因（例如，SOCS基因家族）。

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).