BETA-2 INTEGRIN-DEPENDENT PROTEIN-TYROSINE PHOSPHORYLATION AND ACTIVATION OF THE FGR PROTEIN-TYROSINE KINASE IN HUMAN NEUTROPHILS

Stimulation of adherent human neutrophils (PMN) with tumor necrosis fa ctor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigat ed the dependence of this response on beta 2 integrins by using PMN is olated from a leukocyte adhesion deficiency (LAD) patient, which do no t express beta 2 integrins, and by plating PMN on surface bound anti-b eta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF . Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti-Class I major h istocompatibility complex (MHC) antigens, antibodies triggered tyrosin e phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated wit h an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr prot ein-tyrosine kinase (p58(fgr)) activity, and its extent of phosphoryla tion in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58(fgr) in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activator s of beta 2 integrins such as phorbol-12-myristate 13-acetate (PMA), a nd formyl- methionyl-leucyl-phenylalanine (FMLP), induced activation o f p58(fgr) kinase activity. Activation of p58(fgr) kinase activity, an d phosphorylation in tyrosine, did not occur in PMN of a LAD patient i n response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigen s, antibodies inhibited activation of p58(fgr) kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findin gs demonstrate that, in PMN, beta 2 integrins are implicated in trigge ring of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fg r in cell signaling.