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

Abstract

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(α), -2(β), 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-2α, (staurosporine, 1.09 ± 0.03; TPA, 0.90 ± 0.02; control, < 0.10 ng ml-1). CINC-2(β) 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 μM), calphostin C (IC50, 0.77 μM) and Ro 31-8425 (24.3% inhibition at 10 μM), and by the tyrosine kinase inhibitor, genistein (IC50, 68.5 μM) 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.