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

B. Walzog, R. Seifert, A. Zakrzewicz, P. Gaehtgens, K. Ley

Research output: Contribution to journalArticlepeer-review

71 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)625-635
Number of pages11
JournalJournal of Leukocyte Biology
Volume56
Issue number5
DOIs
StatePublished - 1994
Externally publishedYes

Keywords

  • Actin
  • CD18
  • Calcium
  • Cytoskeleton
  • Degranulation
  • Integrin
  • Selectin

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Cell Biology

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