ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

CD45抗原家族是一组高分子量的糖蛋白。 它们表达在所有白细胞的质膜上。我们有 先前研究表明CD45的表位与中性粒细胞有关。 白三烯B4和重组人对化学引诱剂的趋化作用 C5a(RHuC5a)。这个项目的目的是为了更好地了解 CD45在接尘过程中吞噬细胞信号转导中的作用 化学诱导剂或吞噬刺激物。两种不同的蜂窝系统 在这些研究中使用：来自正常捐赠者的人中性粒细胞和 转化的小鼠巨噬细胞系RAW264。年内，流量 用罗丹明标记的C5a和 荧光素标记的CD45单抗(MAb)与 吞噬细胞，随后评估细胞上的共定位 膜具有荧光共振能量转移技术。 初步数据表明，C5a受体和CD45关系密切 与人类中性粒细胞有关。能量转移研究正在进行中 用RAW264细胞和缺乏CD45的RAW264突变变体克隆 表情。建立了一种测定细胞内钙离子的流式细胞仪 UP使用荧光探针Fluo-3与细胞内钙变化 在人的中性粒细胞中被监测，其中Fc受体 用单抗交联或用化学诱导剂刺激。交联化 Fc受体导致细胞内钙离子增加。当 Fc受体与细胞内钙信号CD45发生交联 都减少了。由于CD45与Fc受体的关联似乎 改变中性粒细胞的钙反应，将会有类似的研究 在RAW264细胞和CD45突变体中进行，以确定CD45是否 正常调节Fc受体介导的钙离子 回应。

The CD45 antigen family is a group of high molecular weight glycoproteins that are expressed on the plasma membranes of all leukocytes. We have previously shown that epitopes of CD45 are involved in neutrophil chemotaxis to the chemo-attractants, leukotriene B4 and recombinant human C5a (rHuC5a). The purpose of this project is to better understand the role of CD45 in phagocyte signal transduction during exposure to chemoattractant or phagocytic stimuli. Two different cell systems are used in these studies: human neutrophils from normal donors and a transformed murine macrophage cell line, RAW264. During the year, flow cytometric studies were undertaken in which rhodamine-labeled C5a and fluorescein-labeled CD45 monoclonal antibodies (mAb) were exposed to phagocytes and subsequently evaluated for colocalization on the cell membrane with a fluorescence resonance energy transfer technique. Preliminary data suggest that the C5a receptor and CD45 are closely associated on human neutrophils. Energy transfer studies are in progress with RAW264 cells and mutagenized variant clones of RAW264 that lack CD45 expression. A flow cytometric assay for intracellular calcium was set up using the fluorescent probe fluo-3 and intracellular calcium changes were monitored in human neutrophils in which Fc receptors were crosslinked with mAbs or stimulated with chemoattractants. Crosslinking Fc receptors results in an increase in intracellular calcium. When the Fc receptors are crosslinked with CD45, the intracellular calcium signals are reduced. Since the association of CD45 with Fc receptors appears to alter the calcium responses in neutrophils, similar studies will be conducted in RAW264 cells and the CD45- mutants to determine whether CD45 normally functions in regulating the Fc receptor mediated calcium response.