ROLE OF LFA-1/MAC-1/P150,95 IN PMN LEUKOCYTE ADHERENCE

LFA-1/MAC-1/P150,95 在 PMN 白细胞粘附中的作用

• 批准号: 3135752
• 负责人: Donald C. Anderson
• 金额: $ 16.12万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3135752
• 关键词: cell adhesion; cell bank /registry; cell migration; collagen; density gradient ultracentrifugation; glycoproteins; human tissue; immunochemistry; inborn lysosomal enzyme disorder; inborn metabolism disorder; laboratory rat; membrane activity; molecular weight; monoclonal antibody; neutrophil; phase contrast microscopy; radiotracer; scanning electron microscopy; vascular endothelium

Understanding of polymorphonuclear leukocyte (PMNL) function has been greatly aided by the recognition of heritable abnormalities or absence of PMNL proteins. In this proposal, the discovery of eight patients (4 males and four females) with an absence of a high molecular weight glycoprotein family (LFA-1/Mac-1/p150,95) on both the cell surface and an intracellular pool are utilized to understand the molecular mechanism by which these proteins mediate cell adherence phenomena. Human endothelial cell monolayers will be examined for adherence, spreading, and penetration by normal and LFA-1/Mac-1/p150-95 deficient PMNL under resting conditions and after stimulation with chemotactic factors including F-Met-Leu-Phe. Three-dimensional type I collagen matrices will be investigated for adherence and invasion by normal and deficient PMNL under similar conditions to test the hypothesis that PMNL adherence is important for movement on endothelial cell surfaces, but is less important for penetration of collagen matrices. Direct observation with interference contrast microscopy as well as scanning and transmission electron microscopy will be used to document the adherence and invasion of the PMNL. The role of LFA-1/Mac-1/p150,95 in the modulation of PMNL adherence dependent function will be studied by determining the intracellular locations of LFA-1/Mac-1/p150,95 and certain related intermediates by a unique, quantitative immunoblotting assay utilizing monoclonal antibodies against LFA-1/Mac-1/p150,95 and the common Beta subunit. Subcellular fractionation will be accomplished on Percoll density gradients after nitrogen cavitation of the PMNL. Cell surface protease treatment, metabolic labeling with [3H]-galactosamine and [3H]-glucosamine and surface labeling with NaB3H4 and [125I] will be employed concurrently with the immunoblotting assay to determine whether cycling of LFA-1/Mac-1/p150,95 takes place. These studies will be complemented by detection of LFA-1/Mac-1/p150,95 in coated pits or vesicles by electron microscopy experiments utilizing colloidal gold or ferritin labeling experiments. Comparative assessments of surface Mac-1 subunit expression (flow cytofluoragraphy) and cell adherence properties will be performed employing chemotactic or secretory conditions designed to "up regulate" or "down regulate" Mac-1 proteins. These studies should provide new information relevant to the role of Mac-1 dependent PMNL adherence reactions in inflammation and host defense.

对多形核白细胞(PMNL)功能的了解 这在很大程度上有助于认识到可遗传的异常或缺乏 PMNL蛋白。在这项建议中，发现了8名患者(4名男性 和四名女性)，没有高分子量糖蛋白 家族(LFA-1/Mac-1/p150，95)在细胞表面和细胞内 POOL被用来理解这些 蛋白质是细胞黏附现象的中介。人内皮细胞 将检查单层膜的粘附性、扩散和渗透性 静息状态下正常和LFA-1/Mac-1/P150-95缺陷的PMNL和 经F-Met-Leu-Phe等趋化因子刺激后。 三维I型胶原基质将被研究 正常和缺陷PMNL在相似条件下的黏附和侵袭 检验PMNL依从性对以下假设的条件 内皮细胞表面的运动，但对 胶原蛋白基质的渗透性。有干扰的直接观察 对比显微镜以及扫描和透射式电子 显微镜将被用来记录黏附和侵袭 PMNL.LFA-1/Mac-1/p150，95在PMNL黏附调节中的作用 依赖功能将通过测定细胞内的 LFA-1/Mac-1/p150，95和某些相关中间体的位置 利用单抗进行独特的定量免疫印迹分析 针对LFA-1/Mac-1/p150，95和共同的Beta亚基。亚细胞 分级将在Percoll密度梯度上完成 PMNL的氮气空化。细胞表面蛋白酶处理， [~3H]-氨基半乳糖和[~3H]-氨基葡萄糖代谢标记及其表面 使用NaB3H4和[125I]标记将与 免疫印迹法检测LFA-1/Mac-1/p150，95细胞周期 发生了。这些研究将得到检测出 LFA-1/Mac-1/p150，95在包被坑或囊泡中的电子显微镜观察 利用胶体金或铁蛋白标记的实验。 表面Mac-1亚单位表达的比较评估(Flow 细胞荧光照相)和细胞粘附性将使用 趋化性或分泌性状态，旨在“上调”或“下调” 调节Mac-1蛋白。这些研究应该提供新的信息 与Mac-1依赖的PMNL黏附反应在 炎症和宿主防御。