GLYCOLIPID E-SELECTIN LIGANDS ON HUMAN GRANULOCYTES

炎症涉及大量粒细胞从血液外渗到感染或免疫激发部位的组织中。 粒细胞和血管壁内皮上的粘附分子和互补配体控制粒细胞在损伤部位进入组织的程度和身份。 E-选择素在活化的内皮细胞上从头表达，是一种选择素粘附蛋白，其通过结合白细胞表面糖缀合物在该免疫应答的早期介导细胞-细胞粘附。 鉴定负责E-选择素介导的细胞粘附的正常人粒细胞糖缀合物对于理解多种形式的炎症将是重要的，并可能为炎症控制提供新的途径。虽然已经鉴定了几种与选择素结合的糖缀合物，但人粒细胞上E-选择素的天然配体尚未完全阐明。 申请人和其他人已经获得了强有力的公开和初步数据，表明正常人嗜中性粒细胞和嗜酸性粒细胞上的鞘糖脂，而不是糖蛋白，是E-选择素的最重要的图例，并且这些鞘糖脂可能不同于先前在细胞系如HL-60中鉴定的鞘糖脂。 该提案结合了三个实验室在静态和生理流动条件下粒细胞粘附与鞘糖脂糖生物学的专业知识，以评估正常人粒细胞鞘糖脂在E-选择素结合和粒细胞功能中的作用。目的1测试的假设，鞘糖脂构成的主要配体E-选择素对完整的正常人中性粒细胞和嗜酸性粒细胞使用细胞粘附试验和高度特异性的酶，抗体和糖缀合物生物合成抑制剂。目的2采用鞘糖脂的提取、拆分和功能分析方法，从正常外周血中性粒细胞和嗜酸性粒细胞中分离和鉴定能够特异性结合E-选择素的鞘糖脂。 TLC分析、抗体、特异性酶和基质辅助激光解吸电离质谱将用于阐明和表征这些鞘糖脂的结构。 目标3将测试完整细胞上的鞘糖脂，以及从目标2中鉴定的鞘糖脂，它们在静态条件和生理流动下在粒细胞粘附中的作用，并将使我们能够测试这些鞘糖脂是否是正常人粒细胞上E-选择素的天然配体。从这些研究中获得的知识将促进改进的抗炎药物的设计，以及我们对鞘糖脂在白细胞粘附中的生理作用的理解。

Inflammation involves extensive granulocyte extravasation from the blood into tissues at a site of infection or immunologic challenge. Adhesion molecules and complementary ligands on granulocytes and on the blood vessel wall endothelium control the extent and identity of granulocytes entering the tissue at a site of insult. E-selectin, expressed de novo on activated endothelial cells, is a selectin adhesion protein which mediates cell-cell adhesion early in this immune response by binding to leukocyte surface glycoconjugates. The identification of normal human granulocyte glycoconjugates responsible for E-selectin-mediated cell adhesion will be important to the understanding of inflammation in its many forms, and may offer novel avenues for inflammation control. Although several glycoconjugates which bind to selectins have been identified, the natural ligands for E-selectin on human granulocytes have yet to be fully elucidated. The applicants and others have obtained strong published and preliminary data indicating that glycosphingolipids on normal human neutrophils and eosinophils, not glycoproteins, are the most important legends for E-selectin, and that these glycosphingolipids may differ from those previously identified in cell lines such as HL-60. This proposal combines expertise from three laboratories in granulocyte cell adhesion under both static and physiologic flow conditions with glycosphingolipid glycobiology to evaluate the roles of normal human granulocyte glycosphingolipids in E-selectin binding and granulocyte function. Aim 1 tests the hypothesis that glycosphingolipids constitute major ligands for E-selectin on intact normal human neutrophils and eosinophils using cell adhesion assays and highly specific enzymes, antibodies, and glycoconjugate biosynthesis inhibitors. Aim 2 uses methods for glycosphingolipid extraction, resolution and functional analysis to isolate and identify glycosphingolipids from normal peripheral blood neutrophils and eosinophils capable of specific binding to E-selectin. TLC analysis, antibodies, specific enzymes, and matrix assisted laser desorption ionization mass spectroscopy will be used to elucidate and characterize the structures of these glycosphingolipids. Aim 3 will test glycosphingolipids on intact cells, and glycosphingolipids identified from Aim 2, for their roles in granulocyte cell adhesion under static conditions and physiological flow, and will allow us to test whether these glycosphingolipids are the natural ligands for E-selectin on normal human granulocytes. Knowledge gained from these studies will advance the design of improved anti-inflammatory drugs, and our understanding of the physiological roles of glycosphingolipids in leukocyte adhesion.