Kinoshita T, Takeda J, Hong K, Kozono H, Sakai H, Inoue K
Department of Bacteriology, Osaka University Medical School, Japan.
J Immunol. 1988 May 1;140(9):3066-72.
mAb to murine C receptor type 1 (CR1) were produced and three of them were characterized. One antibody, designated as 8C12, immunoprecipitated a protein of 190,000 Mr from a detergent extract of surface-labeled spleen cells and stained spleen B but not T lymphocytes in fluorescent flow cytometry. It inhibited both CR1-mediated rosette formation and the cofactor activity of CR1 for factor I-mediated cleavage of C3b, suggesting that it recognizes the ligand-binding site of CR1. The two other antibodies, designated as 7G6 and 7E9, recognized different epitopes from that recognized by 8C12, and they cross-reacted with a protein of 150,000 Mr that is present in a spleen extract. The distribution of CR1 in murine hemopoietic cells was studied by binding experiments with radiolabeled 8C12 and fluorescent flow cytometry. When CR1 was not detected by 8C12 alone, the two other antibodies were used in combination with 8C12 to confirm the negative results. Almost all B lymphocytes from the spleen, lymph nodes, and peripheral blood were CR1 positive. Most of the Thy-1-positive lymphocytes from these tissues were CR1 negative. Thymus lymphocytes were also CR1 negative. Peritoneal macrophages and chemotactic factor stimulated but not unstimulated peripheral blood granulocytes were CR1 positive. In contrast to human E, mouse E were CR1 negative. This pattern of distribution was consistent with previous results obtained by rosette assays. Although mouse platelets cause immune adherence hemagglutination with C3b-bearing SRBC, they are CR1 negative. Three other lines of evidence also indicated that platelets are CR1 negative. First, no band of CR1 was demonstrated by immunoprecipitation with 8C12 of an extract of surface-labeled platelets. Second, 8C12, which inhibited rosette formation by lymphocytes, alone or in combination with 7G6 and 7E9, did not inhibit immune adherence between platelets and C3b-bearing SRBC. Third, polyclonal rabbit IgG prepared from anti-mouse CR1 antiserum did not inhibit immune adherence by platelets. These results strongly suggest that the C3b-binding factor(s) on mouse platelets is different from CR1 and that processing of C3b-bearing immune complexes in mouse blood may be mediated by a new and as yet unidentified C3b-binding factor(s).
制备了针对小鼠1型补体受体（CR1）的单克隆抗体（mAb），并对其中三种进行了特性鉴定。一种名为8C12的抗体，能从表面标记的脾细胞去污剂提取物中免疫沉淀出一种分子量为190,000的蛋白质，并且在荧光流式细胞术中可使脾B淋巴细胞而非T淋巴细胞染色。它既能抑制CR1介导的玫瑰花结形成，也能抑制CR1作为因子I介导的C3b裂解的辅助因子活性，这表明它识别CR1的配体结合位点。另外两种抗体，名为7G6和7E9，识别的表位与8C12不同，它们与脾提取物中存在的一种分子量为150,000的蛋白质发生交叉反应。通过用放射性标记的8C12进行结合实验和荧光流式细胞术研究了CR1在小鼠造血细胞中的分布。当单独用8C12未检测到CR1时，将另外两种抗体与8C12联合使用以确认阴性结果。来自脾、淋巴结和外周血的几乎所有B淋巴细胞均为CR1阳性。来自这些组织的大多数Thy-1阳性淋巴细胞为CR1阴性。胸腺淋巴细胞也为CR1阴性。腹腔巨噬细胞以及趋化因子刺激的而非未刺激的外周血粒细胞为CR1阳性。与人类红细胞（E）不同，小鼠E为CR1阴性。这种分布模式与先前通过玫瑰花结试验获得的结果一致。尽管小鼠血小板可导致与带有C3b的绵羊红细胞（SRBC）发生免疫粘附血凝反应，但它们为CR1阴性。另外三条证据也表明血小板为CR1阴性。第一，用8C12对表面标记的血小板提取物进行免疫沉淀未显示出CR1条带。第二，单独或与7G6和7E9联合使用时能抑制淋巴细胞玫瑰花结形成的8C12，并不抑制血小板与带有C3b的SRBC之间的免疫粘附。第三，由抗小鼠CR1抗血清制备的多克隆兔IgG不抑制血小板的免疫粘附。这些结果强烈表明，小鼠血小板上的C3b结合因子与CR1不同，并且小鼠血液中带有C3b的免疫复合物的处理可能由一种新的、尚未鉴定的C3b结合因子介导。
