THE ROLE OF NEUTROPHIL MEMBRANE GLYCOPROTEIN-GP-150 IN NEUTROPHIL ADHERENCE TO ENDOTHELIUM INVITRO

Abstract

Two patients were with a congenital defect in neutrophil function were characterized by an inability to form pus. The patients'' neutrophils lack a membrane glycoprotein of MW 150,000 daltons (GP-150) on analysis by SDS-PAGE [sodium dodecyl sulfate-polyacrylamide gel electrophoresis]. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, the interaction of the patients'' neutrophils and normal neutrophils treated with MoAB 60.3 with cultured endothelium was examined. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated or laminin-coated plastic was similar to that in normal neutrophils (55-84% adherence with normal neutrophils vs. 73-78% adherence with the patient''s neutrophils and 63-82% adherence with MoAB 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAB 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was not significantly different from that in normal neutrophils (< 10% adherence with normal, MoAb 60.3-treated, and patients neutrophils). In medium containing 10% autologous or heterologous human plasma the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelium monolayers was significantly reduced (35% .+-. 7% of normal neutrophils in 7 experiments). Although phorbol myristate acetate (PMA) (10 ng/ml) and calcium ionophore A23187 [calcimycin] (10-5 mol/l) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum-free medium (40-85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAB 60.3 (< 5% adherence). The adherence of PMA-activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (< 5% adherence). When intact normal neutrophils were added to the patient''s neutrophils, the patient''s neutrophils adhered normally in the presence of PMA (70-80% adherence). The migration of neutrophils from patient 1 and normal neutrophils treated with MoAB 60.3 across endothelial monolayers on polycarbonate filters in response to formylemethionyl-leucyl-phenylalanine (FMLP) (10-7 mol/l) was also markedly decreased compared to normal neutrophils (< 30% of control neutrophils at 90 min). GP-150 was not detected in the postsecretory medium of PMA-activated normal neutrophils on analysis by SDS-PAGE. Activation of normal neutrophils with PMA increased binding of MoAb 60.3 and the inhibition of PMA-induced neutrophil adherence by MoAB 60.3 required continued presence of the MoAb during the incubation. GP-150 is not a secreted protein but remains membrane-associated and the new antigen expressed during activation is critical to increased adhesiveness. The neutrophil membrane glycoprotein GP-150 is required for augmented neutrophil adherence to and chemotaxis across endothelial monolayers in vitro. The inability of the patients'' neutrophils to augment adherence to endothelium when activated may account for the failure of the patients'' neutrophils to migrate to sites of inflammation in vivo.