The macrophage response to endotoxin requires platelet activating factor.
Platelet activating factor (PAF) is a key proinflammatory mediator of septic shock and is metabolized by PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of autodestructive excessive inflammatory responses such as post-injury multiple organ failure, and recombinant PAF-AH is being studied for the prevention of acute respiratory distress syndrome (ARDS). However, the potential role of PAF as an autocrine mediator of macrophage activation is unclear. We wanted to examine the role of PAF in the endotoxin- (LPS) induced macrophage response using PAF-AH. Rabbit alveolar macrophages were stimulated with LPS (10 ng/mL) with or without PAF-AH (0.1-10 microg/mL). Supernatants were collected to measure the production of tumor necrosis factor (TNF), interleukin 8 (Il-8), and prostaglandin E2 (PGE2). Cell monolayers were assessed for procoagulant activity (PCA). TNF mRNA production was determined by Northern blot and RNA stability was assessed. Evaluation of intracellular signaling pathways for LPS included western blots for phosphorylated p38 and ERK kinases and gel shift for nuclear factor-kappaB. There was a dose-response inhibition of TNF, PCA, Il-8, and PGE2 production following pretreatment with PAF-AH. Time course studies revealed effective inhibition of TNF production with administration of PAF-AH up to 2 h after LPS challenge. TNF mRNA production was inhibited, while mRNA stability was not affected. There was no effect on the phosphorylation of p38 or ERK 1 kinases; however, the nuclear translocation of NF-kappaB was inhibited. Macrophage cytokine production in response to endotoxin is PAF dependent. This effect involves the inhibition of TNF gene transcription and concomitant inhibition of NF-kappaB.
Bulger
Eileen M
EM
Department of Surgery, University of Washington, Seattle, USA.
Arbabi
Saman
S
Garcia
Iris
I
Maier
Ronald V
RV
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004731:Endotoxins; D066298:In Vitro Techniques; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010972:Platelet Activating Factor / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D004731:Endotoxins; D016328:NF-kappa B; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase