Cross-linking of CD18 in human neutrophils induces an increase of intracellular free Ca²⁺, exocytosis of azurophilic granules, quantitative up-regulation of CD18, shedding of L-selectin, and actin polymerization

Polymorphonuclear leukocytes (PMNs) exert most of their physiological functions while adherent to surfaces rather than in suspension. PMN adhesion is largely dependent on the function of the β₂ integrins, CD11a,b,c/CD18. We mimicked engagement of β₂ integrins by antibody cross-linking of CD18 on isolated human PMNs using both intact monoclonal antibody and F(ab')₂ fragments. Within seconds of CD18 cross-linking, we observed a significant, transient rise of intracellular free Ca²⁺ concentration by 200-300 nM, which was largely due to Ca²⁺ mobilization from intracellular stores. The Ca²⁺ signal was blocked after pretreatment with phorbol myristate acetate, an activator of protein kinase C, but not with herbimycin A, a potent inhibitor of tyrosine kinases. In addition to the rise of intracellular free Ca²⁺ concentration, CD18 cross-linking induced exocytosis of azurophilic granules (release of 26% of total PMN elastase), which was significantly inhibited by herbimycin A. Moreover, 2.2-fold up-regulation of CD18 antigen and significant down-regulation of surface expression of the granulocyte adhesion molecule L-selectin were induced. Granulocyte F-actin content as measured by nitrobenzoxadiazole-phallacidin increased significantly 1 min after CD18 cross-linking. By contrast, CD18 crosslinking by soluble antibodies did not induce superoxide production, but PMNs bound to immobilized monoclonal antibodies against CD18 released significant amounts of superoxide. Initial signaling through β₂ integrins does not appear to be mediated by a phospholipase C isoform activated through tyrosine phosphorylation, because the Ca²⁺ signal was not altered by herbimycin A. However, more complex cellular responses including exocytosis were found to require tyrosine phosphorylation. We show that engagement of β₂ integrins provides an important stimulatory signal to PMNs inducing degranulation, modulation of L-selectin, and cytoskeletal changes.