Neutrophil priming by granulocyte colony stimulating factor and its modulation by protein kinase inhibitors.
Upon stimulation by various ligands, freshly isolated human peripheral neutrophils (PMN) respond in a variety of ways, such as superoxide (O2-.) generation, phagocytosis enzyme release, migration etc. Chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and opsonized zymosan activate neutrophils by a receptor-mediated mechanism, while phorbol myristate acetate and dioctanoylglycerol activate the cells by a mechanism involving Ca(2+)-and phospholipid-dependent protein kinase (PKC). Receptor-mediated but not PKC-mediated O2-. generation in PMN was enhanced by the priming of recombinant human granulocyte colony stimulating factor (G-CSF). FMLP-dependent luminol chemiluminescence was also enhanced by G-CSF. However, no appreciable enhancement was observed in FMLP-induced intracellular calcium ion concentration ([Ca2+]i). Enhancement of FMLP-induced generation of O2-. by G-CSF was inhibited by genistein or alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was stimulated by staurosporine and 1-(5-isoquinolinesulfonyl)-3-methyl-piperazine (H-7), inhibitors of PKC. The ED50 values of genistein and ST 638 for the inhibition of the FMLP-induced O2-. generation from G-CSF were 0.5 and 5 microM, respectively. In contrast, O2-. generation by PKC activation without G-CSF priming was inhibited by stauroporine and H-7, but was stimulated by genistein and ST 638. These results suggested that the enhancing effect of G-CSF on receptor-mediated generation of the O2-. might be regulated by protein kinases, such as TK and PKC, and that the TK inhibitor selectively inhibited the G-CSF-primed receptor-mediated O2-. generation of neutrophils.
Tanimura
M
M
Department of Urology, Kochi Medical School, Japan.
Kobuchi
H
H
Utsumi
T
T
Yoshioka
T
T
Kataoka
S
S
Fujita
Y
Y
Utsumi
K
K
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D002934:Cinnamates / Q000494:pharmacology; D019833:Genistein; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008165:Luminol; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D019311:Staurosporine; D013440:Sulfides / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism
D000470:Alkaloids; D002934:Cinnamates; D007529:Isoflavones; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D013440:Sulfides; C060747:ST 638; D013481:Superoxides; D016179:Granulocyte Colony-Stimulating Factor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008165:Luminol; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D019311:Staurosporine; D002118:Calcium