Mechanisms of platelet-activating factor-induced lipid body formation: requisite roles for 5-lipoxygenase and de novo protein synthesis in the compartmentalization of neutrophil lipids.
Lipid bodies, lipid rich cytoplasmic inclusions, are characteristically abundant in vivo in leukocytes associated with inflammation. Because lipid bodies are potential reservoirs of esterified arachidonate and sites at which eicosanoid-forming enzymes may localize, we evaluated mechanisms of lipid body formation in neutrophils (PMN). Among receptor-mediated agonists, platelet activating factor (PAF), but not C5a, formyl-methyl-phenylalanine, interleukin 8, or leukotriene (LT) B4, induced the rapid formation of lipid bodies in PMN. This action of PAF was receptor mediated, as it was dose dependently inhibited by the PAF receptor antagonist WEB 2086 and blocked by pertussis toxin. Lipid body induction by PAF required 5-lipoxygenase (LO) activity and was inhibited by the 5-lipoxygenase-activating protein antagonist MK 886 and the 5-LO inhibitor zileuton, but not by cyclooxygenase inhibitors. Corroborating the dependency of PAF-induced lipid body formation on 5-LO, PMN and macrophages from wild-type mice, but not from 5-LO genetically deficient mice, formed lipid bodies on exposure to PAF both in vitro and in vivo within the pleural cavity. The 5-LO product inducing lipid body formation was not LTB4 but was 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE], which was active at 10-fold lower concentrations than PAF and was also inhibited by pertussis toxin but not by zileuton or WEB 2086. Furthermore, 5-HETE was equally effective in inducing lipid body formation in both wild-type and 5-LO genetically deficient mice. Both PAF- and 5(S)-HETE-induced lipid body formation were inhibited by protein kinase C (PKC) inhibitors staurosporine and chelerythrine, the phospholipase C (PLC) inhibitors D609 and U-73122, and by actinomycin D and cycloheximide. Prior stimulation of human PMN with PAF to form lipid bodies enhanced eicosanoid production in response to submaximal stimulation with the calcium ionophore A23187; and the levels of both prostaglandin (PG) E2 and LTB4 correlated with the number of lipid bodies. Furthermore, pretreatment of cells with actinomycin D or cycloheximide inhibited not only the induction of lipid body formation by PAF, but also the PAF-induced "priming" for enhanced PGE2 and LTB4 in PMN. Thus, the compartmentalization of lipids to form lipid bodies in PMN is dependent on specific cellular responses that can be PAF receptor mediated, involves signaling through 5-LO to form 5-HETE and then through PKC and PLC, and requires new protein synthesis. Since increases in lipid body numbers correlated with priming for enhanced PGE2 and LTB4 production in PMN, the induction of lipid bodies may have a role in the formation of eicosanoid mediators by leukocytes involved in inflammation.
Bozza
P T
PT
Harvard Thorndike Laboratory, Beth Israel Hospital, Boston, Massachusetts 02215, USA.
Payne
J L
JL
Goulet
J L
JL
Weller
P F
PF
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000187:drug effects*; D050356:Lipid Metabolism; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D016859:Lipoxygenase Inhibitors; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011500:Protein Synthesis Inhibitors; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D001094:Arachidonate 5-Lipoxygenase; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins