FUNCTIONAL ANALYSIS OF A PDGF INDUCIBLE CYTOKINE

描述（研究者摘要）：趋化因子是一种小蛋白质， 控制正常的白细胞运输，并负责白细胞 在炎症性疾病中的募集。这个蛋白质家族的生理学是 由于存在超过50种趋化因子配体而变得异常复杂， 20个感受器。为了理解趋化因子生物学并发现方法 为了治疗性地调节它们的活性，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，首先作为PDGF诱导的mRNA从小鼠中克隆， 成纤维细胞，在体外吸引单核细胞、NK细胞和记忆T细胞。下 在这项资助的支持下，我们使用转基因小鼠来证明 MCP-1在体内是一种有效的单核细胞特异性化学引诱物， 炎症中单核细胞募集的独特需要， 存在激活MCP-1受体的其它单核细胞化学引诱物， CCR 2;并且，它的缺失保护小鼠免受动脉粥样硬化。最近我们 显示MCP-1是诱导Th 2极化免疫所必需的， 应答这一发现令人惊讶，因为MCP-1的主要体内功能 被认为只涉及先天免疫，因为CCR 2缺陷小鼠 Th 1缺陷。 本申请中的工作旨在阐明MCP-1的细节 通过解决两个假设来研究体内作用机制：（1）MCP-1表达 通过次级淋巴器官中的细胞直接刺激Th 2极化 通过可能不依赖IL-4的途径;和（2）这种作用是特异性的 但可能通过CCR 2以外的受体起作用。测试这些 根据这些假设，我提出以下具体目标： 具体目标1：确定MCP-1影响Th 2的生理基础 极化这将通过重建极化免疫来实现 使用来自MCP-1 - 1-和MCP-1 + 1+小鼠的细胞的体外应答和测试 通过体内过继转移从这些实验中得出的推论 实验 具体目标2：检查MCP-1对T辅助细胞影响的分子基础 细胞极化我们将测试MCP-1诱导的Th 2细胞是否需要IL-4。 极化，MCP-1是否激活与IL-4相同的下游靶标，以及 BCL-6（一种抑制Th 2极化的转录抑制因子）是否 在MCP- 1通路中。 具体目标3：确定MCP-1介导的Th 2极化是否依赖于 特别是MCP-1和CCR 2。其他受体将进行测试， 通过MCP- 1和另一种CCR 2配体MCP-3影响Th 2极化的能力， 将被敲入MCP-1位点，以测试MCP-1是否特异性地 必需的.

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.