FUNCTIONAL ANALYSIS OF A PDGF INDUCIBLE CYTOKINE

描述(研究人员摘要)：趋化因子是一种小蛋白， 控制正常的白细胞运输，并负责白细胞 在炎症性疾病中招募。这个蛋白质家族的生理学是 由于50多个趋化因子配体和 20个受体。为了了解趋化因子生物学并发现方法 为了从治疗上调节它们的活性，我们研究了一种趋化因子 深度，即单核细胞趋化蛋白-1(MCP-1)。 MCP-1，一种CC趋化因子，首次从小鼠中克隆为PDGF诱导的mRNA 成纤维细胞，在体外吸引单核细胞、NK细胞和记忆T细胞。在……下面 在这笔赠款的赞助下，我们使用转基因小鼠来证明 那就是：MCP-1在体内是一种强大的单核细胞特异性趋化因子；它是 在炎症中单核细胞募集是唯一必需的，尽管 是否存在其他激活单核细胞趋化蛋白-1‘S受体的单核细胞趋化因子 CCR2；而且，它的缺失可以保护小鼠免受动脉粥样硬化的影响。最近，我们有 表明MCP-1是诱导Th2极化免疫所必需的 回应。这一发现令人惊讶，因为单核细胞趋化蛋白-L的主要体内功能 被认为只涉及先天免疫，而且因为CCR2缺陷的小鼠 有Th1缺陷。 本申请中的工作旨在阐明MCP-1的细节，即S 解决两个假说的体内作用机制：(1)MCP-1的表达 次级淋巴器官中的细胞直接刺激Th2极化 通过一条可能不依赖于IL-4的途径；以及(2)这种效应是特异的 MCP-1，但可能通过CCR2以外的受体起作用。为了测试这些 根据假设，我提出以下具体目标： 具体目标1：确定单核细胞趋化蛋白-1 S影响Th2的生理基础 极化。这将通过重建极化免疫来实现 单核细胞趋化蛋白-1和单核细胞趋化蛋白-11小鼠细胞的体外反应及检测 通过体内收养转移从这些实验中得出的推论 实验。 特异性目标2：检测单核细胞趋化蛋白-1的分子基础S对T辅助细胞的影响 细胞极化。我们将测试MCP-1诱导的Th2是否需要IL-4 极化，MCP-1是否激活与IL-4相同的下游靶点，以及 BCL-6(一种抑制Th2极化的转录抑制因子)是否 在MCP-1途径中。 具体目标3：确定MCP-1介导的Th2极化是否依赖于 特别是在MCP-1和CCR2上。其他受体将接受测试，以确定其 MCP-1和另一种CCR2配体MCP-3影响Th2极化的能力， 将被敲入MCP-1基因座以检测MCP-1是否具有特异性 必填项。

DESCRIPTION (investigator's abstract): Chemokines are small proteins that control normal leukocyte trafficking and are responsible for leukocyte recruitment in inflammatory disorders. The physiology of this protein family is made extraordinarily complex by the existence of over 50 chemokine ligands and 20 receptors. In order to understand chemokine biology and to discover methods for therapeutically modulating their activity, we have studied one chemokine in depth, namely monocyte chemoattractant protein-1 (MCP- 1). MCP-1, a CC chemokine that was first cloned as a PDGF-inducible mRNA from mouse fibroblasts, attracts monocytes, NK cells, and memory T cells in vitro. Under the auspices of this grant, we used genetically modified mice to demonstrate that: MCP-1 is a potent monocyte-specific chemoattractant in vivo; it is uniquely required for monocyte recruitment in inflammation despite the existence of other monocyte chemoattractants that activate MCP-1's receptor, CCR2; and, its absence protects mice from atherosclerosis. Recently, we have shown that MCP-1 is required for the induction of Th2 polarized immune responses. This finding is surprising because MCP-l's primary in vivo functions were thought to involve only innate immunity, and because CCR2-deficient mice have a Th1 defect. The work in this application is designed to elucidate details of MCP-1's mechanisms of action in vivo by addressing two hypotheses: (1) MCP-1 expression by cells in secondary lymphoid organs directly stimulates Th2 polarization through a pathway that may be IL-4-independent; and (2) This effect is specific for MCP-1 but may work through a receptor other than CCR2. To test these hypotheses, I propose the following specific aims: Specific Aim 1: Determine the physiological basis for MCP-1's influence on Th2 polarization. This will be approached by reconstituting polarized immune responses in vitro using cells from MCP-1 -I- and MCP-1 +1+ mice and testing inferences drawn from these experiments by in vivo adoptive transfer experiments. Specific Aim 2: Examine the molecular basis for MCP-1's influence on T helper cell polarization. We will test whether IL-4 is required for MCP-1-induced Th2 polarization, whether MCP-1 activates the same downstream targets as IL-4, and whether BCL-6 (a transcriptional repressor that suppresses Th2 polarization) is in the MCP- 1 pathway. Specific Aim 3: Determine whether MCP-1-mediated Th2 polarization depends specifically on MCP-1 and CCR2. Other receptors will be tested for their ability to effect Th2 polarization by MCP- 1, and another CCR2 ligand, MCP-3, will be knocked into the MCP-1 locus to test whether MCP-1 is specifically required.