C5a as a model for chemotactic factor-stimulated tyrosine phosphorylation in the human neutrophil

Abstract

Human neutrophils were exposed to the chemotactic factors C5a, FMLP, IL-8, leukotriene B4, and PAF, for 30 s, and subsequently analyzed for protein tyrosine phosphorylation by immunoblotting whole cell lysates with a polyclonal antiphosphotyrosine Ab. All chemotactic factors caused the rapid de novo tyrosine phosphorylation of a broad band of approximately 120 kDa and increased the phosphotyrosine content of several other proteins, including two with molecular masses of 60 and 56 kDa that were present in the unstimulated neutrophil. Tyrosine phosphorylation was evident as early as 10 s after stimulation and was maintained for 1 to 3 min before dephosphorylation occurred. The extent of tyrosine phosphorylation was dependent on the concentration of chemotactic factor, with stimulation observed at concentrations as low as 10 to 100 nM. To investigate the pathway used by chemotactic factors to transduce this signal, neutrophils were treated with PMA. PMA also stimulated tyrosine phosphorylation in the neutrophil but with a slower response time and a different pattern of affected proteins. Additional experiments suggested that tyrosine phosphorylation is involved in the regulation of the neutrophil respiratory burst because the tyrosine kinase inhibitor, herbimycin A, inhibited C5a-induced protein tyrosine phosphorylation and also prevented C5a- and FMLP-induced superoxide anion production. Herbimycin A also inhibited PMA-induced tyrosine phosphorylation and superoxide anion production. To confirm that the ability to stimulate tyrosine phosphorylation was intrinsic to the C5a receptor, tyrosine phosphorylation was examined in both undifferentiated U937 cells (C5a receptor negative) and cAMP differentiated U937 cells (C5a receptor positive). C5a induced tyrosine phosphorylation only in differentiated U937 cells. Analysis of the C5a receptor mRNA using the PCR confirmed its presence in differentiated and its absence in undifferentiated U937 cells. Therefore, C5a stimulates tyrosine phosphorylation via a receptor-mediated mechanism and U937 cells provide a system in which G-coupled receptor-mediated tyrosine phosphorylation can be investigated.