MEMBRANE LIPID ORDER AND LYMPHOCYTE FUNCTION

血管内磷脂结构的改变 淋巴细胞膜可能影响多个淋巴细胞 功能。这项提案旨在评估 淋巴细胞与细胞相互作用中的磷脂堆积 网状内皮细胞与细胞溶解T细胞的靶点 淋巴细胞(CTL)。分离未成熟和活化的淋巴细胞 从组织中结合花青素540(MC540)，一种荧光 疏松脂类的亲脂性探针，而淋巴细胞 与外周循环隔离并不明显。这是不是 磷脂包装的不同是导致 这些淋巴细胞与其他细胞的相互作用将是 在几个方面进行了测试。使用离心法生产一种 可控制和可重现的力量，以区分粘附物和 非贴壁细胞，外周细胞相互作用的强度 带巨噬细胞的血淋巴细胞、胸腺细胞和脾细胞 血管内皮细胞将被测量。使用流式细胞术， 胸腺细胞和脾细胞用MC540染色 根据荧光强度和 测定了各组分的粘附性。这个 细胞相互作用对脂质组织的依赖将是 活体检查，通过以下清除从循环中 回输不同磷脂包装的淋巴细胞。二 生物物理方法将被用来研究这种机制。 改变的磷脂组织可能通过这种方式影响 坚持不懈。光漂白后的荧光恢复将是 用来评估是否增加了凝集性 脂肪堆积松散的胸腺细胞因增加 膜受体的流动性。荧光转移法 将被用来确定脂类的松散包装是否 活化的淋巴细胞伴随着一种局部聚集的 淋巴细胞黏附相关膜蛋白，LFA-1。最后， CTL中的磷脂组织及其靶点 通过MC540染色评估这一潜在的作用 细胞毒性T淋巴细胞抗自溶能力中的参数 靶细胞杀伤率。

Alterations in the organization of the phospholipids in the lymphocyte plasma membrane may affect several lymphocyte functions. This proposal is designed to assess the role of phospholipid packing in the interactions of lymphocytes with reticuloendothelial cells and with the targets of cytolytic T lymphocytes (CTL). Immature and activated lymphocytes isolated from tissues bind merocyanine 540 (MC540), a fluorescent lipophilic probe for loosely-packed lipids, whereas lymphocytes isolated from the peripheral circulation do not. Whether this difference in phospholipid packing is responsible for differences in the interactions of these lymphocytes with other cells will be tested in several ways. Using centrifugation to produce a controlled and reproducible force to distinguish adherent from nonadherent cells, the strength of the interactions of peripheral blood lymphocytes, thymocytes and splenocytes with macrophages and endothelial cells will be measured. Using flow cytometry, thymocytes and splenocytes stained with MC540 will be fractionated according to fluorescence intensity and the adherence properties of the fractions determined. The dependence of cellular interactions on lipid organization will be examined in vivo by following clearance from the circulation of reinfused lymphocytes which differ in phospholipid packing. Two biophysical approaches will be used to investigate the mechanisms by which altered phospholipid organization could affect adherence. Fluorescence recovery after photobleaching will be employed to assess whether increases agglutinability of thymocytes with loosely-packed lipids results from increased mobility of membrane receptors. Fluorescence transfer methods will be used to determine whether the loose packing of lipids in activated lymphocytes is accompanied by local aggregation of a lymphocyte adhesion-related membrane protein, LFA-1. Finally, phospholipid organization in CTL and their targets will be assessed by MC540 staining to evaluate the potential role of this parameter in the resistance of CTL to self-lysis and in the efficiency of killing of target cells.