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: 6161367
• 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/6161367
• 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

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 mutant cell line. In a CD45 knockout mutant, reversion will be negligible and the function of CD45 can be evaluated by transfection with wild-type and mutant CD45 cDNA. During the year, lambda clones comprised of various fragments of the CD45 gene were screened with an exon 12 oligonucleotide probe and two positive clones were obtained. Detailed restriction maps were derived for each lambda clone and were used in the construction of a CD45 knockout vector with exon 12 disrupted by a neomycin gene. In an exon 12 knockout mutant, protein synthesis should terminate before the transmembrane region and result in a nonfunctional protein (gene). Cells have been transfected with the CD45 knockout vector and a PCR screening procedure for the detection of a mutant cell has been developed. Studies are underway to isolate the CD45 deficient mutant cell from the transfectants. 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.

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。阐明CD45在巨噬细胞吞噬中的作用 趋化性，我们正试图分离CD45敲除突变细胞系。 在CD45敲除突变体中，回复将是可忽略的，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。在这一年里，lambda克隆体由各种片段组成， CD45基因外显子12寡核苷酸探针和两个 获得阳性克隆。详细的限制性酶切图谱被推导出， 每个λ克隆并用于构建CD45敲除 具有被新霉素基因破坏的外显子12的载体。12号外显子敲除 突变体，蛋白质合成应该在跨膜区之前终止 并导致无功能的蛋白质（基因）。细胞已被转染 使用CD45敲除载体和PCR筛选程序， 已经开发了突变细胞的检测。研究正在进行中， 从转染子中分离CD45缺陷型突变细胞。的 分离CD45缺陷细胞系将提供有价值的工具， 确定该分子在吞噬细胞趋化性中的功能作用 宿主防御