title: 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.
abstract: 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 beta 2 integrins, CD11a,b,c/CD18. We mimicked engagement of beta 2 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 cross-linking 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 beta 2 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 beta 2 integrins provides an important stimulatory signal to PMNs inducing degranulation, modulation of L-selectin, and cytoskeletal changes.
authors.affiliation: Department of Physiology, Freie Universität Berlin, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Walzog
authors.forename: R
authors.initials: R
authors.lastname: Seifert
authors.forename: A
authors.initials: A
authors.lastname: Zakrzewicz
authors.forename: P
authors.initials: P
authors.lastname: Gaehtgens
authors.forename: K
authors.initials: K
authors.lastname: Ley
mesh_terms: D000199:Actins / Q000378:metabolism; D016227:Benzoquinones; D018845:CD11 Antigens / Q000378:metabolism / Q000502:physiology*; D018821:CD18 Antigens / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D015550:Cell Degranulation / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D019041:L-Selectin; D047029:Lactams, Macrocyclic; D008562:Membrane Glycoproteins / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism / Q000502:physiology*; D011809:Quinones / Q000494:pharmacology; D016028:Receptors, Leukocyte-Adhesion / Q000502:physiology*; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation
chemical_list: D000199:Actins; D016227:Benzoquinones; D018845:CD11 Antigens; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D047029:Lactams, Macrocyclic; D008562:Membrane Glycoproteins; D011809:Quinones; D016028:Receptors, Leukocyte-Adhesion; D013481:Superoxides; D019041:L-Selectin; D017828:Rifabutin; C020754:herbimycin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium