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 or 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 moleuclar 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 petussis toxin, Several important conclusion can be drawn from these data. 1) GM‐CSF potentiates the rise in Ca 2+ , 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.