MECHANISMS OF NEUTROPHIL ADHESION TO ENDOTHELIUM

这个项目的目标是定义 调节中性粒细胞(PMN，中性粒细胞， 粒细胞)向内皮细胞(EC)转化及其生物学后果 这种相互作用，例如激活(和随后的极化， 中性粒细胞迁移、脱颗粒等)。中性粒细胞黏附的调节 对于形成血管内膜的EC来说，是 血管生物学，因为它在生理性炎症中起着必要的作用 和东道主防守。此外，PMN与EC的不受管制的黏附， 随之而来的是颗粒酶、氧自由基和其他 由激活的白细胞介导的介质，导致血管损伤。 缺血再灌注损伤、心肌梗死、急性肺损伤 血管损伤，以及人类疾病中的其他综合征。我们已经描述了一种 中性粒细胞内皮依赖性黏附的新机制。胶粘剂的相互作用 当培养的人内皮细胞被激活时，迅速(在几分钟内)发生 激动剂，并且至少部分地由快速的、依赖于时间的、 血小板活化因子的合成及其在EC中的表面表达 (“1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine)，”；一种独特的甘油- 与中性粒细胞和其他类型的细胞相互作用的磷脂，通过与 表面受体。PMN黏附的这种依赖于EC的快速机制是 显然不同于细胞因子诱导的反应，后者发生在数小时内， 可能参与了PMN在特定地区的快速本地化 在生理性和病理性炎症中发生的内膜。这个 目前的项目有4个具体目标：1)我们将定义 粘合剂对PMN的激活响应的要求 相互作用和涉及的机制。这也是第一步 在确定PMN与激活的EC的粘附性如何影响后续 事件(如极化、酶和脂质介质的释放等 由PMN提供)。2)我们将确定PMN CD11/CD18粘合剂的作用 糖蛋白，假设它们发挥放大作用，而不是 有义务的角色。3)我们将描述其他EC分子的作用 在快速EC依赖的黏附中，最初集中在颗粒膜上 蛋白质140(GMP-140)--一种定位于分泌的新糖蛋白 EC的颗粒，可以快速转移到血浆中 薄膜。4)明确中性粒细胞与EC黏附的机制(S) 氧化剂激活与炎性血管损伤相关的模型 人类疾病。根据这一系列调查的结果，我们将 能够探索导致不受监管的PMN黏附和 在其他血管损伤模型中激活，并可能在体内激活。 实验将使用培养的EC、分离的本地EC、固定EC和 放射性标记的中性粒细胞，中性粒细胞，髓系细胞系，研究 由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，或在流体中，研究 脂类(PAF)和糖蛋白(GMP-140)介体的表面表达 利用EC对PAF和其他活化EC的产物进行代谢标记， 潜在地，分离和表征新的介体 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.