Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: role of protein kinase and G-proteins.
Granulocyte-macrophage colony-stimulating factor, GM-CSF, potentiates superoxide generation produced by human neutrophils stimulated with fMet-Leu-Phe and platelet-activating factor, PAF, but not by phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan. The potentiation is greatest in fMet-Leu-Phe-stimulated cells. This indicates that the actions of only certain receptors are potentiated by GM-CSF. Incubation of the cells with the protein kinase inhibitor H-7 or with the protein synthesis inhibitor cyclohexamide before the addition of GM-CSF does not affect the observed potentiation. The rationales behind these studies are to examine the roles of protein kinase C and protein synthesis in the action of GM-CSF. The data suggest that neither protein kinase C nor protein synthesis is necessary for GM-CSF action. On the other hand, no potentiation can be seen in the presence of cytochalasin B. Unlike intact cells, GM-CSF does not enhance superoxide production by cytoplasts stimulated with fMet-Leu-Phe. The rationale behind the use of cytoplasts is to examine the role of granules and/or nucleus in GM-CSF action, and the data indicate that one or more of these two components is necessary for the priming effect of GM-CSF. The amount of actin associated with the cytoskeleton under control of fMet-Leu-Phe-stimulated condition is the same in normal and GM-CSF-treated human neutrophils. Botulinum D toxin ADP-ribosylates a protein with a molecular weight of 22 kDa. This ribosylation is reduced in homogenates obtained from cells pretreated with botulinum D toxin or GM-CSF. Botulinum D toxin does not affect the basal or the fMet-Leu-Phe-induced rise in the intracellular concentration of free calcium in human neutrophils. GM-CSF also increases the rise in intracellular concentration of free calcium in human neutrophils stimulated with PAF or fMet-Leu-Phe. The increases are inhibited by pertussis toxin. Several important conclusion can be drawn from these data. 1) GM-CSF potentiates the rise in Ca2+i produced by PAF and fMet-Leu-Phe, and these potentiations are inhibited in pertussis-toxin-treated cells. 2) GM-CSF does not prime cytoplasts to stimulation by fMet-Leu-Phe. This suggests that the granules and/or nucleus are necessary for the priming action. 3) The priming by GM-CSF is not mediated by the H-7-sensitive protein kinase C, botulinum D-sensitive G-protein, or protein synthesis.
Mege
J L
JL
Department of Physiology, University of Connecticut Health Center, Farmington 06032.
Gomez-Cambronero
J
J
Molski
T F
TF
Becker
E L
EL
Sha'afi
R I
RI
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000199:Actins / Q000032:analysis / Q000502:physiology; D001905:Botulinum Toxins / Q000494:pharmacology; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D003115:Colony-Stimulating Factors; D003598:Cytoskeletal Proteins; D006133:Growth Substances; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D010566:Virulence Factors, Bordetella; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D037342:Pertussis Toxin; D011494:Protein Kinases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate