β 2 integrins mediate protein tyrosine phosphorylation in human neutrophils

Tyrosine kinases play a prominent role in various intracellular signal transduction pathways. In this study, β 2 integrin (CD11/CD18)‐mediated adhesive interactions of human neutrophils (PMN) were mimicked by antibody cross‐linking of GD11/CD18. Cross‐linking of CD18 induced tyrosine phosphorylation of several proteins with apparent molecular masses of ~120, 78, 72, 65, and 56 kDa, respectively, as shown by anti‐phosphotyrosine immunoblotting of whole cell lysates. Cross‐linking of the α‐subunits of the β 2 integrins demonstrated that only cross‐linking of Mac‐1 but not LFA‐1 or gp150/95 triggered tyrosine signaling. The tyrosine phosphorylations showed a rapid and transient time course. Comparison of the CD18‐mediated tyrosine phosphorylation patterns with those induced by chemoattractants gave similar results. The observed tyrosine phosphorylation was specific, since binding and non‐binding irrelevant primary antibodies had no effect. Furthermore, F(ab') 2 fragments of the anti‐CD18 antibody IB4 and addition of F(ab') 2 fragments of secondary antibody were sufficient to induce tyrosine phosphorylation. Inhibition of tyrosine kinases by herbimycin A resulted in partial inhibition of the CD18‐mediated tyrosine phosphorylation of the 120‐ and 65‐kDa proteins and in complete inhibition of tyrosine phosphorylation of the 78‐ and 56‐kDa proteins. In unstimulated PMN, the tyrosine phosphatase inhibitor sodium orthovanadate had no effect on tyrosine phosphorylation of the 120‐kDa protein, but induced phosphorylation of the 78‐, 72‐, 65‐, and 56‐kDa proteins. These results indicate that the β 2 integrin‐mediated intracellular signaling cascade involves different tyrosine phosphorylation (and dephosphorylation) events, which may play a role in the regulation of PMN functions during inflammation.