ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

CD45 在吞噬细胞运动和信号转导中的作用

• 批准号: 6101309
• 负责人: L HARVATH
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6101309
• 关键词: CD antigens; antibody receptor; calcium flux; cell motility; chemoattractants; chemotaxis; flow cytometry; human tissue; immunoglobulin G; immunoprecipitation; intermolecular interaction; leukotrienes; neutrophil; phosphoprotein phosphatase; western blottings

Role of CD45 in Phagocyte Motility and Signal Transduction Liana Harvath The leukocyte common antigen (CD45) family is a group of high molecular weight single transmembrane glycoproteins with cytoplasmic protein tyrosine phosphatase domains expressed on the plasma membranes of all leukocytes. Recent information regarding CD45 function indicates that antibody reagents to certain CD45 isoforms have potent immunosuppressive activities and may contribute to the immunosuppressive action of therapeutic agents such as anti-thymocyte globulin which is administered to transplant recipients for the prevention of graft-versus-host-disease and graft rejection. We have previously shown that certain extracellular domain epitopes of CD45 are involved in human neutrophil chemotaxis. We have pursued these observations using a genetic approach and mutagenized a murine macrophage cell line to obtain variant clones with low or undetectable CD45 expression. We found that cell line variants lacking CD45 expression were defective in their chemotactic response to recombinant human C5a, however, the cells exhibited normal random motility. These observations suggested that CD45 may be involved in directional (chemotactic) migration which is an essential host defense response to inflammatory stimuli. To clarify the role of CD45 in phagocyte chemotaxis, we are attempting to isolate a CD45 knockout cell line. In a CD45 knockout, reversion will be negligible and the function of CD45 can be evaluated by transfection with wild-type and mutant CD45 cDNA. During the year, a CD45 targeting vector was constructed that utilized insertion of a neomycin gene driven by a heterologous promoter into exon 12 of CD45. Transfection experiments with the exon 12 CD45 targeting vector showed that random integration into the genome greatly exceeded targeted integration and indicated that it may be necessary to screen several thousand clones before a successful recombinant would be found. When this situation has arisen with other genes, a more favorable targeting to random integration ratio has been obtained with a promoterless targeting vector that utilizes the genomic promoter of the target gene. In an exon 12 knockout cell line, protein synthesis should terminate before the transmembrane region and result in a nonfunctional protein (gene). Studies are underway to construct a promoterless CD45 targeting vector, to investigate the targeting to random integration ratio, and to isolate a CD45 knockout. The isolation of a CD45 deficient cell line will provide a valuable tool for establishing the functional role of the molecule in phagocyte chemotaxis and host defense.

CD45在吞噬细胞运动和信号转导中的作用 利亚纳·哈瓦特 白细胞共同抗原（CD45）家族是一组高分子量的 单跨膜糖蛋白与胞浆蛋白的重量 酪氨酸磷酸酶域表达的质膜上的所有 白细胞 最近关于CD45功能的信息表明， 针对某些CD45同种型的抗体试剂具有有效的免疫抑制性， 活性，并可能有助于免疫抑制作用 治疗剂如抗胸腺细胞球蛋白 用于移植受者以预防移植物抗宿主病 和移植排斥反应我们以前已经证明，某些细胞外 CD45的结构域表位参与人嗜中性粒细胞趋化性。 我们 使用遗传学方法进行这些观察， 小鼠巨噬细胞系，以获得具有低或 检测不到CD45表达。 我们发现，细胞系变异缺乏 CD45表达在它们对CD45的趋化反应中有缺陷。 然而，重组人C5a，细胞表现出正常的随机性， 能动性 这些观察结果表明，CD45可能参与了 定向（趋化性）迁移，这是一种重要的宿主防御 对炎症刺激的反应。 为了阐明CD45在 吞噬细胞趋化性，我们试图分离出一个CD45敲除细胞 线 在CD 45敲除中，回复突变可以忽略不计，并且功能 可以通过用野生型和突变型CD45转染来评估CD45的表达 cDNA。 在这一年中，构建了一个CD45靶向载体， 利用由异源启动子驱动的新霉素基因的插入 插入CD45的第12外显子 用外显子12 CD45的转染实验 靶向载体显示，随机整合到基因组中， 超过了目标整合，并表示可能有必要 筛选几千个克隆，然后才能成功重组。 found. 当这种情况与其他基因一起出现时， 目标随机积分比已获得与 无启动子的靶向载体，其利用所述载体的基因组启动子。 靶基因 在外显子12敲除细胞系中，蛋白质合成应该 在跨膜区之前终止并导致非功能性的 蛋白质（基因）。 研究正在进行中，以构建无启动子的CD45 靶向载体，研究随机整合的靶向 比率，并分离CD45敲除。 一个CD45缺陷型的分离 细胞系将提供一个有价值的工具， 该分子在吞噬细胞趋化性和宿主防御中的作用。