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Endotoxin-induced macrophage gene expression depends on platelet-activating factor
1Department of Surgery, University of California, Los Angeles 90095-6904, USA. clo@surgery.medsch.ucla.edu
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.
Design:
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.
Insights
Platelet-activating factor (PAF) drives macrophage gene expression in sepsis. Inhibiting PAF or its receptor reduces inflammatory responses, suggesting PAF modulation as a sepsis treatment.
Area of Science:
- Immunology
- Molecular Biology
- Sepsis Pathophysiology
Background:
- Sepsis-induced multiple organ failure stems from systemic inflammation orchestrated by macrophages.
- Macrophage activation in sepsis is a critical factor for patient survival.
- Early macrophage activation involves phospholipase A2 (PLA2) and platelet-activating factor (PAF) generation.
Purpose of the Study:
- To investigate the hypothesis that macrophage gene expression is dependent on PAF.
- To elucidate the role of PAF in lipopolysaccharide (LPS)-induced macrophage activation.
Main Methods:
- Rabbit alveolar macrophages were stimulated with LPS, PAF, PAF receptor antagonist (CV3988), or PLA2 inhibitors (AACOCF3, manoalide).
- Tumor necrosis factor (TNF) mRNA expression was assessed via Northern blot.
- TNF production was quantified using L929 bioassays.
Main Results:
- LPS stimulation significantly increased TNF mRNA and TNF production in macrophages.
- PAF receptor antagonist CV3988 inhibited LPS-induced TNF mRNA expression.
- PLA2 inhibitors (AACOCF3, manoalide) also inhibited LPS-induced TNF mRNA, indicating PAF's role in its production.
- PAF's effects were linked to intracellular calcium concentration changes, as calcium flux inhibitors attenuated LPS-induced responses.
Conclusions:
- LPS-induced macrophage gene expression is mediated by PAF.
- Modulating PAF production or activity could be a therapeutic strategy to reduce macrophage overactivity in sepsis.