Nair K S, Zingde S M
Biochemistry and Molecular Biology Division, Cancer Research Institute, Parel, Mumbai, 400 012, India
Cell Immunol. 2001 Mar 15;208(2):96-106. doi: 10.1006/cimm.2001.1772.
Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.
中性粒细胞与底物的黏附由诱导外向内信号传导的受体-配体相互作用引发。内向信号以及表面分子之间的侧向相互作用进一步微调这一反应。本研究使用针对CD66的结构域定位单克隆抗体，探究CD66在中性粒细胞与纤连蛋白黏附中的作用。中性粒细胞在其表面表达CD66a、CD66b和CD66c。与纤连蛋白结合的中性粒细胞表面分子是α(4)β(1)和α(5)β(1)整合素。我们的结果表明，识别CD66a、b和c的AB结构域的单克隆抗体Kat4c以及多克隆抗CD66（抗癌胚抗原）可增强中性粒细胞与纤连蛋白的黏附，而针对单个CD66抗原的单克隆抗体、Kat4c的Fab片段以及针对CD66抗原的单个抗体的混合物均无法影响黏附。因此，CD66a、b和c的异源二聚化是促进中性粒细胞与纤连蛋白黏附所必需的。在存在Kat4c的情况下，黏附增加被β(1)和β(2)整合素的抗体所抑制。CD66抗原的抗体连接导致其聚集以及β(2)整合素的α(M)亚基随之共聚集，从而激活整合素。α-甲基甘露糖苷抑制抗CD66介导的聚集，表明CD66与α(M)整合素之间可能存在碳水化合物-凝集素相互作用。它还降低了中性粒细胞与纤连蛋白增加的黏附，提示β(2)整合素激活先于β(1)整合素激活。此外，抗CD66介导的与纤连蛋白的黏附伴随着Src家族激酶（lyn和hck）向细胞骨架的定位增加及其激酶活性的增强。这些结果表明，CD66a、CD66b和CD66c的交联促进β(2)整合素的激活，进而改变β(1)整合素的亲和力，增强中性粒细胞与纤连蛋白的黏附。
