Adhesion of Neutrophils to Fibronectin: Role of the CD66 Antigens

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 α4β1 and α5β1 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 β1 and β2 integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the αM subunit of the β2 integrin, thereby activating the integrin. The sugar α-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate–lectin interaction may exist between CD66 and αM integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that β2 integrin activation precedes β1 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 β2 integrin and in turn an alteration in the affinity of the β1 integrin, which enhances the adhesion of neutrophils to fibronectin.