Effect of Granulocyte-Macrophage Colony-Stimulating Factor on Superoxide Production in Cytoplasts and Intact Human Neutrophils: Role of Protein Kinase and G-Proteins

Abstract

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.