Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils.
Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX-2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM-CSF, TNF-alpha, and fMLP, induced COX-2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX-2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX-2 protein expression was initiated. In LPS-treated neutrophils, efficient induction of COX-2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX-2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX-2, raising the possibility that phospholipase D activation might take part in the process of COX-2 induction. Major COX-2-derived prostanoids synthesized by inflammatory neutrophils were identified by liquid-chromatography and tandem mass-spectrometry as TXA2 and PGE2. The agonist-induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX-2 inhibitor NS-398. These results show that COX-2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX-2 isoform is preeminent over COX-1 for the stimulated-production of prostanoids, and also suggest that neutrophil COX-2 displays a distinct profile of expression among circulatory cells.
Pouliot
M
M
Centre de Recherche en Rhumatologie et Immunologie, and Laval University, Québec, Canada. pouliot@zeus.bwh.harvard.edu
Gilbert
C
C
Borgeat
P
P
Poubelle
P E
PE
Bourgoin
S
S
Créminon
C
C
Maclouf
J
J
McColl
S R
SR
Naccache
P H
PH
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D051546:Cyclooxygenase 2; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010587:Phagocytosis; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000097:blood / Q000235:genetics*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D000906:Antibodies; D000939:Epitopes; D007527:Isoenzymes; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015054:Zymosan; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate