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-2_α (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.

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