GENERATION AND CHARACTERIZATION OF CD45 NEGATIVE MACROPHAGE MUTANTS

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。CD45有 蛋白酪氨酸磷酸酶活性和似乎调节信号 信号转导与淋巴细胞活化 T和B淋巴细胞上的受体分子。我们之前已经表明， CD45表位参与中性粒细胞趋化 化学诱导剂、白三烯B4和重组人C5a(RHuC5a)。这个 本项目的目的是生成并表征稳定的CD45 阴性髓系细胞克隆及CD45在吞噬细胞中的作用 功能。Abelson-小鼠白血病病毒转化巨噬细胞 LINE，RAW 264，被选为这个项目是因为RAW 264巨噬细胞 对Rhuc5a的趋化作用呈剂量依赖性。原始264单元格 用烷化剂甲烷磺酸乙酯诱变24小时 在组织培养瓶中。诱变细胞被从烧瓶上刮下来， 复制到几个烧瓶中，并允许在最低限度的必需物质中生长 含10%胎牛血清的培养液。细胞是从 当它们达到融合生长时，并用 抗小鼠CD45的生物素标记的单抗。链霉亲和素- 共轭磁珠与CD45单抗处理的细胞孵育 将混合物在磁化柱上运行，以浓缩CD45 阴性细胞。经过多轮CD45阴性选择和 扩大培养中的丰富种群，75个CD45阴性克隆 经流式细胞仪分析鉴定。对20个克隆进行了筛选 对于Rhuc5a和&gt；的趋化反应，90%的克隆具有 有缺陷的趋化反应。目前的研究正在进行中，以 将CD45基因导入CD45阴性突变体，检测CD45基因的表达 CD45表达后即可恢复正常的趋化功能。这些结果 提示巨噬细胞质膜上CD45的表达具有重要意义 对Rhuc5a的正常趋化反应。CD45负向RAW 264 突变克隆提供了一种独特的细胞系统来探测功能 髓系细胞中CD45的活性。

The CD45 antigen family is a group of high molecular weight glycoproteins that are expressed on the plasma membranes of all leukocytes. CD45 has protein tyrosine phosphatase activity and appears to regulate signal transduction and lymphocyte activation by specific association with receptor molecules on T and B lymphocytes. We have previously shown that epitopes of CD45 are involved in neutrophil chemotaxis to the chemoattractants, leukotriene B4 and recombinant human C5a (rHuC5a). The purpose of this project is to generate and characterize stable CD45 negative myeloid cell clones and determine the role of CD45 in phagocyte function. The Abelson-murine leukemia virus transformed macrophage cell line, RAW 264, was selected for this project because RAW 264 macrophages exhibit dose-dependent chemotaxis to Rhuc5a. RAW 264 cells were mutagenized with the alkylating agent, ethyl methanesulfonate, for 24 hours in tissue culture flasks. Mutagenized cells were scraped off the flask, replated into several flasks, and allowed to grow in Minimal Essential Medium containing 10% fetal calf serum. Cells were harvested from the flasks when they reached confluent growth and were treated with a biotinylated-monoclonal antibody (mAb) to murine CD45. Streptavidin- conjugated magnetic beads were incubated with the CD45 Mab-treated cells and the mixture was run over a magnetized column to enrich for the CD45 negative cells. After multiple rounds of CD45 negative selection and expansion of the enriched population in culture, 75 CD45 negative clones were identified by flow cytometric analysis. Twenty clones were screened for chemotactic responsiveness to Rhuc5a and >90% of the clones had defective chemotactic responses. Current studies are in progress to transfect the CD45 negative mutants with CD45 cDNA and determine whether normal chemotactic function returns upon expression of CD45. These results indicate that CD45 expression on macrophage plasma membranes is important for normal chemotactic responsiveness to Rhuc5a. The CD45 negative RAW 264 mutant clones provide a unique cellular system to probe the functional activity of CD45 in myeloid cells.