Induction of neutrophil chemotactic factor production by staurosporine in rat peritoneal neutrophils

1 Incubation of rat peritoneal neutrophils in medium containing various concentrations of staurosporine (6.4-64 nM) increased the neutrophil chemotactic activity in the conditioned medium in a time-and concentration-dependent manner. 2 Separation of the neutrophil chemotactic activity in the conditioned medium by isoelectric focusing revealed that staurosporine (64 nM) stimulated the production of basic (pH > 8) neutrophil chemotactic factors, while TPA (12-O-tetradecanoylphorbol 13-acetate, 49 nM) stimulated the production of both basic (pH>8) and acidic (pH 5) neutrophil chemotactic factors. 3 Determination by immunoassay of cytokine-induced neutrophil chemoattractant (CINC)-1, -2(alpha), -2(beta) and -3 in the conditioned medium at 4 h revealed that staurosporine (64 nM) and TPA (49 nM) strongly stimulated the production of CINC-3 (staurosporine, 133.0 +/- 3.8; TPA, 26.7 +/- 1.0; control, 0.32 +/- 0.01 ng ml(-1), means+/-s.e.mean from four samples) compared to CINC-1 (staurosporine, 55.0 +/- 1.2; TPA, 12.2 +/- 0.3; control, 0.56 +/- 0.01 ng ml(-1)) and CINC-2 (staurosporine, 1.09 +/- 0.03; TPA, 0.90 +/- 0.02; control, <0.10 ng ml(-1)). CINC-2(beta) was below the detectable amount (<0.078 ng ml(-1)). 4 The level of CINC-3 mRNA in the peritoneal neutrophils was determined by reverse transcription-polymerase chain reaction. Staurosporine (64 nM) and TPA (49 nM) enhanced the level of CINC-3 mRNA time-dependently, but had no effect on GAPDH mRNA levels. 5 Production of staurosporine-induced neutrophil chemotactic factor was inhibited by the protein kinase C inhibitors, H-7 (IC50, 12.3 mu M), calphostin C (IC50, 0.77 mu M) and Ro 31-8425 (24.3% inhibition at 10 mu M), and by the tyrosine kinase inhibitor, genistein (IC50, 68.5 mu M). Production of TPA-induced neutrophil chemotactic factor was also inhibited by both inhibitors. 6 Both the staurosporine-and the TPA-induced increase in CINC-3 mRNA levels were suppressed by H-7 and genistein.