Endotoxin-induced macrophage gene expression depends on platelet-activating factor.
BACKGROUND
The development of multiple organ failure in septic patients is due to a systemic inflammation orchestrated by macrophages (Mphi). Elucidation and control of the mechanism involved in Mphi activation in sepsis is crucial to improving survival. An early event of Mphi activation involves the hydrolysis of membrane phospholipid by phospholipase A2 (PLA2) and subsequent generation of platelet-activating factor (PAF).


OBJECTIVE
We designed this study to test the hypothesis that Mphi gene expression depends on PAF.


METHODS
Rabbit alveolar Mphi were obtained by bronchoalveolar lavage and were stimulated with 10 ng/mL of Escherichia coli endotoxin lipopolysaccharide (LPS), PAF (1 micromol/L), LPS+/-CV3988 (10 micromol/L), a PAF receptor antagonist, or LPS+/-PLA2 inhibitors: AACOCF3 (50 micromol/L) or manoalide (10 micromol/L). After 4 hours of incubation, Mphi tumor necrosis factor (TNF) messenger RNA (mRNA) expression was assessed by Northern blot analyses. The TNF production in the Mphi supernatant was measured by L929 bioassays.


RESULTS
The LPS-stimulated Mphi expressed increased levels of TNF mRNA and produced an enormous amount of TNF. CV3988, a PAF antagonist, inhibited LPS-induced TNF mRNA. Furthermore, inhibiting PAF production with AACOCF3, or manoalide, also inhibited LPS-induced Mphi TNF mRNA expression. The effect of PAF depends on changes in intracellular calcium concentration. Inhibitors of calcium flux attenuated the PAF effects on LPS-stimulated Mphi.


CONCLUSIONS
Our data suggest that LPS-induced Mphi gene expression is mediated by PAF. It is likely that modulation of PAF production or activity may be beneficial in down-regulating the overactivity of Mphi in sepsis.
Lo
C J
CJ
Department of Surgery, University of California, Los Angeles 90095-6904, USA. clo@surgery.medsch.ucla.edu
Cryer
H G
HG
Fu
M
M
Kim
B
B
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D004731:Endotoxins; D004926:Escherichia coli; D015870:Gene Expression; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008297:Male; D010972:Platelet Activating Factor / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D004731:Endotoxins; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid