GLYCOLIPID E-SELECTIN LIGANDS ON HUMAN GRANULOCYTES

炎症包括感染或免疫挑战部位的大量粒细胞从血液渗入组织。粒细胞和血管壁内皮细胞上的黏附分子和互补配体控制着粒细胞在损伤部位进入组织的程度和特性。E-选择素在激活的内皮细胞上从头表达，是一种选择素黏附蛋白，在免疫反应早期通过与白细胞表面的糖偶联物结合来介导细胞与细胞的黏附。鉴定与E-选择素介导的细胞黏附有关的正常人类粒细胞糖偶联物对于理解多种形式的炎症具有重要意义，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偶联物，但人粒细胞上E-选择素的天然配体仍未完全阐明。申请人和其他人已经获得了强有力的已发表的初步数据，表明E-选择素最重要的传说是正常人中性粒细胞和嗜酸性粒细胞上的鞘糖脂，而不是糖蛋白，这些鞘糖脂可能不同于之前在HL-60等细胞系中发现的那些。这项建议结合了三个实验室在静态和生理流动条件下粒细胞黏附方面的专业知识和鞘糖脂糖生物学，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利用细胞黏附实验和高度特异性的酶、抗体和糖结合生物合成抑制剂，验证神经鞘糖脂是正常中性粒细胞和嗜酸性粒细胞上E-选择素的主要配体的假说。目的利用鞘糖脂的提取、拆分和功能分析方法，从正常人外周血中性粒细胞和嗜酸性粒细胞中分离鉴定能与E-选择素特异性结合的鞘糖脂。将使用TLC分析、抗体、特定酶和基质辅助激光解吸电离质谱仪来阐明和表征这些糖鞘糖脂的结构。AIM 3将测试完整细胞上的神经鞘糖脂，以及从AIM 2中鉴定的神经鞘糖脂，以确定它们在静态条件和生理流动下对粒细胞黏附的作用，并将允许我们测试这些神经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抗炎药物的设计，以及我们对神经鞘糖脂在白细胞黏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.