Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase.
We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.
O'Sullivan
M G
MG
Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157.
Chilton
F H
FH
Huggins
E M
EM
McCall
C E
CE
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D004790:Enzyme Induction; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D008070:Lipopolysaccharides / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000378:metabolism*; D011233:Precipitin Tests; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D011453:Prostaglandins / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011863:Radioimmunoassay; D013931:Thromboxanes / Q000378:metabolism
D015342:DNA Probes; D008070:Lipopolysaccharides; D011453:Prostaglandins; D012333:RNA, Messenger; D013931:Thromboxanes; D004247:DNA; D011451:Prostaglandin-Endoperoxide Synthases