Beta2 integrins mediate protein tyrosine phosphorylation in human neutrophils.
Tyrosine kinases play a prominent role in various intracellular signal transduction pathways. In this study, beta2 integrin (CD11/CD18)-mediated adhesive interactions of human neutrophils (PMN) were mimicked by antibody cross-linking of CD11/CD18. Cross-linking of CD18 induced tyrosine phosphorylation of several proteins with apparent molecular masses of approximately 120, 78, 72, 65, and 56 kDa, respectively, as shown by anti-phosphotyrosine immunoblotting of whole cell lysates. Cross-linking of the alpha-subunits of the beta2 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 beta2 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.
Walzog
B
B
Department of Physiology, Freie Universität Berlin, Germany.
Offermanns
S
S
Zakrzewicz
A
A
Gaehtgens
P
P
Ley
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018821:CD18 Antigens / Q000502:physiology*; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D011506:Proteins; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine