Molecular Analysis Of Leukocyte Activation By Chemoattractants

该项目的目的是确定血液白细胞迁移到发炎或感染的特定组织部位的分子机制和生物学背景。我们已经集中在介导这一过程的趋化蛋白，并确定了一个大家族的趋化受体，部署在白细胞表面的成员。我们还鉴定了由病毒（包括疱疹病毒、痘病毒和HIV）产生的不同化学引诱物和化学引诱物受体模拟物。我们使用基因组学、分子生物学、细胞生物学和流行病学作为分析这些分子的主要方法。一个主要的目标是确定具体的疾病协会个别化学引诱物和化学引诱物受体，以确定潜在的新的治疗靶点。 一个关键的策略是分析疾病模型中基因敲除小鼠的表型以及人类疾病队列中相应人类基因中功能缺失突变的关联。 在2007财年，我们继续研究趋化因子对动脉粥样硬化的调节作用。 扩展我们在2006财年的先前报告，即致病性动脉粥样硬化相关氧化脂质能够间接调节趋化因子受体CCR 2的表达（减少）和CX 3CR 1在从血液单核细胞离体发育的原代人巨噬细胞上，在FY 07中，我们专注于CX 3CR 1配体CX 3CL 1，发现其表达也被这些致病脂质上调，对巨噬细胞和原代人冠状动脉平滑肌细胞（CASMC）的作用。 CX 3CL 1-CX 3CR 1配体-受体对是巨噬细胞与CASMC静态粘附的主要介质。 这些结果共同表明了斑块内泡沫细胞募集和保留的模型，其中CX 3CL 1-CX 3CR 1通过同型和异型粘附相互作用来协调巨噬细胞与血管壁中CASMC的粘附。 这提供了一种新的动脉粥样硬化模型，并确定CX 3CL 1-CX 3CR 1作为这种疾病的潜在新药物靶点。 此外，这些数据在分子和细胞水平上为我们之前报告的遗传和流行病学数据提供了解释，表明CX 3CR 1是一种致动脉粥样硬化因子。 在FY 07中，我们还定义了一种参与氧化脂质上调CX 3CL 1的信号转导途径，该途径涉及自分泌/旁分泌TNF依赖性途径和转录因子NF-κ B。 有趣的是，相同脂质通过涉及转录因子PPAR-gamma的不同机制上调巨噬细胞上的CX 3CR 1。 在2007财年，我们还定义了F2 L，一种在炎症部位发现的血红蛋白分解产物，作为甲酰肽受体同源物FPR 2的功能性配体。 作为该受体的首批内源性配体之一，这一新发现为分析FPR 2的生物学作用提供了重要的新方向。

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractant and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY07 we continued our study of chemokine regulation of atherosclerosis. Extending our previous report in FY06 that pathogenic atherosclerosis-associated oxidized lipids are able to reciprocally regulate expression of the chemokine receptors CCR2 (decreased) and CX3CR1 (increased) on primary human macrophages developed from blood monocytes ex vivo, in FY07 we focused on the CX3CR1 ligand CX3CL1 and found that its expression was also upregulated by these pathogenic lipids, on both macrophages and primary human coronary artery smooth muscle cells (CASMCs). The CX3CL1-CX3CR1 ligand-receptor pair is a major mediator of static adhesion of macrophages to CASMCs in this sytem. Together the results suggest a model for foam cell recruitment and retention within plaque in which CX3CL1-CX3CR1 act to coordinate adhesion of macrophages to CASMCs in the vessel wall through both homotypic and heterotypic adhesive interactions. This provides a novel model of atherogenesis and identifies CX3CL1-CX3CR1 as a potential new drug target in this disease. Moreover, the data provide an explanation at the molecular and cellular level for genetic and epidemiologic data we reported earlier showing that CX3CR1 is a proatherogenic factor. In FY07 we also defined a signal transduction pathway involved in CX3CL1 upregulation by oxidized lipids involving an autocrine/paracrine TNF-dependent pathway and the transcription factor NF-kB. Interestingly, upregulation of CX3CR1 on macrophages by the same lipids occurs through a different mechanism involving the transcription factor PPAR-gamma. In FY07 we also defined F2L, a breakdown production of hemoglobin found at sights of inflammation, as a functional ligand for the formylpeptide receptor homologue FPR2. One of the first endogenous ligands found for this receptor, this new finding provides an important new direction for analyzing the biological role of FPR2.