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Mechanisms of reprogrammed macrophage endotoxin signal transduction after lipopolysaccharide pretreatment
M A West1, S C Seatter, J Bellingham
1Department of Surgery, Hennepin County Medical Center, University of Minnesota, Minneapolis 55415, USA.
Background:
Dysregulation of macrophage tumor necrosis factor (TNF) and interleukin-(IL-1) release results from repetitive lipopolysacharride (LPS) stimulation. In this study we investigated the mechanisms of LPS pretreatment (LPSp) signal transduction producing altered LPS-activated (LPSa) cytokine release.
Methods:
Murine macrophages were treated with medium alone, actinomycin D, cycloheximide, a protein kinase C inhibitor (H7), or the nitric oxide synthase inhibitor L-NMA. Macrophages were then pretreated with 100 ng/ml LPSp and cultured in medium alone, a nitric oxide donor (sodium nitroprusside), or a cyclic adenosine monophosphate donor (8 bromoadenosine) for 20 hours. Cultures were then washed, and fresh medium containing 1 microgram/ml LPSa was added. TNF and IL-1 release in 24-hour supernatant was measured by bioassays.
Results:
LPSp inhibited TNF and enhanced IL-1 release. The results with actinomycin D and cycloheximide suggested that LPSp effects did not require transcription, but IL-1 enhancement required protein synthesis. Addition of 8-bromo-cyclic adenosine monophosphate, H7, or nitroprusside prevented LPSp-induced augmentation of IL-1 but had no effect on inhibition of TNF release. Inhibition of LPSp-induced nitric oxide production with L-NMA had no effect on TNF or IL-1.
Conclusions:
Complex, independent, but incompletely understood signal transduction pathways for LPSp-induced alterations in LPSa-stimulated macrophage TNF and IL-1 release were shown. Understanding altered signal transduction from prior LPS stimulation may suggest new therapies to control dysregulated macrophage cytokine release in sepsis.
Insights
Repetitive lipopolysaccharide (LPS) stimulation alters macrophage cytokine release. LPS pretreatment (LPSp) inhibits TNF but enhances IL-1, involving complex pathways that may offer new sepsis therapies.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Repetitive lipopolysaccharide (LPS) stimulation dysregulates macrophage release of tumor necrosis factor (TNF) and interleukin-1 (IL-1).
- Investigating the mechanisms of LPS pretreatment (LPSp) signal transduction is crucial for understanding altered LPS-activated (LPSa) cytokine release.
Purpose of the Study:
- To elucidate the signal transduction pathways involved in LPSp-induced alterations of LPSa-stimulated macrophage cytokine release.
- To determine the roles of protein synthesis, nitric oxide, and cyclic adenosine monophosphate in these altered pathways.
Main Methods:
- Murine macrophages were subjected to various treatments including actinomycin D, cycloheximide, protein kinase C inhibitor (H7), and nitric oxide synthase inhibitor (L-NMA).
- Macrophages were pretreated with LPSp and then exposed to nitric oxide or cyclic adenosine monophosphate donors before LPS activation.
- TNF and IL-1 release were quantified using bioassays in the supernatant of treated macrophage cultures.
Main Results:
- LPS pretreatment (LPSp) inhibited TNF release while enhancing IL-1 release.
- LPSp effects on TNF did not require transcription, but IL-1 enhancement necessitated protein synthesis.
- Cyclic adenosine monophosphate, protein kinase C inhibition, and nitric oxide donors modulated IL-1 release but not TNF inhibition; nitric oxide production inhibition had no effect.
Conclusions:
- LPSp-induced alterations in LPSa-stimulated macrophage TNF and IL-1 release involve complex, independent, and not fully understood signal transduction pathways.
- Understanding these altered pathways may lead to novel therapeutic strategies for controlling dysregulated macrophage cytokine release in conditions like sepsis.