Molecular Analysis Of Leukocyte Activation By Chemoattractants

这个项目的目的是确定血液白细胞迁移到炎症或感染的特定组织部位的分子机制和生物学背景。我们一直专注于介导这一过程的趋化蛋白，并鉴定了部署在白细胞表面的趋化受体大家族的成员。我们还鉴定了一组不同的趋化物质和趋化物质受体模拟物，包括疱疹病毒、痘病毒和艾滋病毒。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单个趋化物质和趋化物质受体的特定疾病关联，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功能突变丧失的相关性。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。延续我们在06财年的报告，致病动脉粥样硬化相关的氧化脂质能够相互调节趋化因子受体CCR2(减少)和CX3CR1(增加)的表达，在07财年，我们专注于CX3CR1配体CX3CL1，发现CX3CR1配体CX3CL1在巨噬细胞和原代人冠状动脉平滑肌细胞(CASMCs)上的表达也受到这些致病脂质的上调。在这个系统中，CX3CL1-CX3CR1配体-受体对是巨噬细胞与CASMCs静态黏附的主要介质。综上所述，这些结果提出了一种泡沫细胞在斑块内募集和滞留的模型，在该模型中，CX3CL1-CX3CR1通过同型和异型黏附相互作用协调巨噬细胞与血管壁上CASMCs的黏附。这为动脉粥样硬化的发生提供了一种新的模型，并确认CX3CL1-CX3CR1是治疗该疾病的潜在新药靶点。此外，这些数据在分子和细胞水平上为我们早先报道的遗传和流行病学数据提供了解释，表明CX3CR1是一种致动脉粥样硬化因子。在2007财年，我们还定义了一个信号转导通路，涉及氧化脂质上调CX3CL1的信号转导通路，涉及依赖自分泌/旁分泌的肿瘤坏死因子途径和转录因子NF-kB。有趣的是，同样的脂类上调巨噬细胞上的CX3CR1是通过涉及转录因子PPAR-伽马的不同机制发生的。在07财年，我们还将F2L定义为甲酰肽受体同系物FPR2的功能配体。F2L是一种在炎症现场发现的血红蛋白的分解产物。这是最早发现的该受体的内源性配体之一，这一新发现为分析FPR2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.