Abstract
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.
Highlights
From the SDiuision of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, South Australia andthe §Department of Physiology, Uniuersity of Melbourne, Melbourne, Victoria 3052, Australia
Despite these and other biological effects GM-CSF was qualitative andnot the result of of GM-CSF invitro and its incipient clinical use, little is larger amounts of platelet-activating factor (PAF) being synthesized since similar known about effects of GM-CSF which may shed light on the amounts were generated in response to the calcium mechanism of action of this cytokine. ionophore A23187 but no released PAF could be de- Investigations of signals stimulated by GM-CSF have tected
We show that human GM-CSF stimulates the production of PAF from human neutrophils in a time- and concentration-dependent manner and through a pertussis toxin-sensitive G protein
Summary
PAF Synthesis and Extraction-Purified neutrophils at concentrations which ranged from lo to 5 X 107/ml were incubated in assay medium at 37 "C with appropriate stimulus or medium for defined periods of time (see "Results"), after which the cells were rapidly cooled on ice and aliquots dispensed and centrifuged at 4 "C for 5 min a t 450 X g. The mixtures were incubated a t 37 "C for various periods of time up to 60 min (see "Results"). Under these conditions, >98% neutrophils could be recovered indicating that little or no adherence to the surface of the tubes had occurred.
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