MECHANISMS OF NEUTROPHIL ADHESION TO ENDOTHELIUM

这个项目的目标是确定分子机制， 调节多形核白细胞（PMNs，嗜中性粒细胞， 内皮细胞（EC）和生物学后果 这种相互作用，例如激活（和随后的极化， 迁移、脱粒等）。 中性粒细胞粘附的调节 形成血管内膜的EC是一个中心问题， 血管生物学，因为它在生理炎症中的必要作用 宿主防御 此外，中性粒细胞对EC的不受调节的粘附， 随后释放颗粒酶、氧自由基和其他 介导的激活白细胞，有助于血管损伤， 缺血再灌注损伤，心肌梗死，急性肺动脉高压 血管损伤和其他人类疾病综合征。 我们已经描述了 中性粒细胞依赖内皮细胞粘附的新机制。 粘合剂相互作用 当培养的人EC被激活时，会迅速发生（几分钟内） 激动剂，并且至少部分地由快速的，时间依赖性的， 血小板活化因子的合成及EC法表面表达 （“PAF”; 1-0-烷基-2-乙酰基-sn-甘油基-3-磷酸胆碱），一种独特的甘油- 一种磷脂，通过结合一种 表面受体 这种快速的EC依赖性PMN粘附机制是 与细胞因子诱导的明显不同，细胞因子诱导的持续数小时， 并且很可能参与了PMNs在特定区域的快速定位 在生理性和病理性炎症中发生的内膜损伤。 的 本项目有4个具体目标：1）我们将定义 粘合剂的PMN激活响应要求 相互作用和相关机制。 这也是第一步 在确定中性粒细胞粘附到激活的EC如何影响随后的 事件（如极化、酶和脂质介质的释放等 的PMNs）。 2)我们将定义中性粒细胞CD 11/CD 18粘附的作用， 糖蛋白，假设他们发挥放大作用，而不是 一个义务性的角色。 3)我们将描述其他EC分子的作用 在快速EC依赖性粘附中，最初集中于颗粒膜 GMP-140蛋白是一种新的糖蛋白，定位于分泌型 EC的颗粒，并可迅速转移到血浆中 膜的 4)我们将明确中性粒细胞粘附EC的机制 氧化剂激活，与炎症性血管损伤相关的模型， 人类疾病 根据调查结果，我们将 能够探索导致不受调节的PMN粘附的机制， 在其他血管损伤模型中以及潜在地在体内激活。 实验将使用培养的EC、分离的天然的、固定的和未固定的EC。 放射性标记的中性粒细胞，中性粒细胞胞质，髓系细胞系， 中提呈的激动剂激活的中性粒细胞的信号转导机制 EC质膜，在模型膜中，或在流体相中，研究 脂质（PAF）和糖蛋白（GMP-140）介质的表面表达 通过EC，PAF和活化EC的其他产物的代谢标记， 潜在地，分离和表征新的介质， EC或PMN。

The goal of this project is to define the molecular mechanisms that regulate the adhesion of polymorphonuclear leukocytes (PMNs, neutrophils, granulocytes) to endothelial cells (EC) and the biologic consequences of this interaction, such as activation (and subsequent polarization, migration, degranulation, etc) of the PMNs. The regulation of PMN adhesion to the Ec that form the intima of blood vessels is a central issue in vascular biology because of its requisite role in physiologic inflammation and host defense. Furthermore, unregulated adhesion of PMNs to EC, followed by release of granular enzymes, oxygen radicals, and other mediators by the activated leukocytes, contributes to vascular damage in ischemia-reperfusion injury, myocardial infarction, acute pulmonary vascular injury, nad other syndromes in human disease. We have described a novel mechanism of EC-dependent adhesion of PMNs. The adhesive interaction occurs rapidly (within minutes) when cultured human EC are activated by an agonist and is mediated, at least in part, by the rapid, time-dependent, synthesis and surface expression by the EC of platelet-activating factor ("PAF"; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a unique glycero- phospholipid that interacts with PMNs and other cell types by binding to a surface receptor. This rapid EC-dependent mechanism of PMN adhesion is clearly different from that induced by cytokines, which occurs over hours, and likely is involved in the rapid localization of PMNs to specific areas of the intima that occurs in physiologic and pathologic inflammation. The current project has 4 specific objectives: 1) We will define the requirement for an activation response by the PMNs for the adhesive interaction and the mechanisms that are involved. This also the first step in determining how adhesion of PMNs to activated EC influences subsequent events (such as polarization, release of enzymes and lipid mediators, etc by the PMNs). 2) We will define the role of the PMN CD11/CD18 adhesive glycoproteins, with the hypothesis that they play an amplifying rather than an obligate role. 3) We will characterize the role of other EC molecules in rapid EC- dependent adhesion, focusing initially on granule membrane protein 140 (GMP-140), a novel glycoprotein that is localized in secretory granules of the EC and that can be rapidly translocated to the plasma membrane. 4) We will define the mechanism(s) of PMN adhesion to EC activated by oxidants, a model relevant to inflammatory vascular injury in human disease. Based on the results of this line of investigation, we will be able to explore the mechanisms that lead to unregulated PMN adhesion and activation in other models of vascular injury and potentially, in vivo. The experiments will employ cultured EC, isolated native, fixed, and radiolabeled PMNs, neutrophil cytoplasts, myeloid cell lines, studies of signal transduction mechanisms in PMNs activated by agonists presented in the EC plasma membrane, in model membranes, or in the fluid phase, studies of surface expression of lipid (PAF) and glycoprotein (GMP-140) mediators by Ec, metabolic labeling of PAF and other products of activated EC and, potentially, isolation and characterization of new mediators expressed by EC or PMNs.