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Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells
G Camussi1, L Biancone, E L Iorio
1Dipartimento di Biochimica e Biofisica, I Facoltá di Medicina e Chirurgia, Universitá di Napoli, Italy.
Abstract:
Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Gram-negative bacteria porins and lipopolysaccharide (LPS) stimulate platelet activating factor (PAF) synthesis in human kidney cells. This occurs rapidly via the phospholipase A2 (PLA2) pathway, independent of TNF, unlike LPS-induced PAF synthesis.
Area of Science:
- Cell Biology
- Immunology
- Microbiology
Background:
- Porins are proteins in gram-negative bacteria outer membranes.
- Platelet activating factor (PAF) is a mediator of inflammation and endotoxic shock.
- Human glomerular mesangial cells (MC) play a role in kidney inflammation.
Purpose of the Study:
- To investigate the role of bacterial porins and lipopolysaccharide (LPS) in stimulating Platelet Activating Factor (PAF) synthesis in human glomerular mesangial cells (MC).
- To elucidate the signaling pathways involved in PAF production induced by porins and LPS.
Main Methods:
- Cultured human glomerular mesangial cells (MC) were stimulated with purified porins and LPS.
- Platelet activating factor (PAF) synthesis was measured over time.
- The role of protein synthesis, tumor necrosis factor (TNF), and phospholipase A2 (PLA2) activity was assessed using inhibitors (cycloheximide, anti-TNF antibodies, p-bromodiphenacylbromide - PBDB) and labeled precursors.
- PAF synthesis pathway via remodeling involving 2-lyso-PAF was investigated.
Main Results:
- Porins rapidly stimulated PAF synthesis in MC (peak at 20 min), independent of LPS and TNF.
- LPS also stimulated PAF synthesis, but with a biphasic pattern (peaks at 10 min and 3-6 hours), with the later peak dependent on protein synthesis and TNF.
- Both porins and LPS induced PAF synthesis via the remodeling pathway, involving PLA2-dependent mobilization of arachidonic acid and 2-lyso-PAF.
- Inhibition of PLA2 by PBDB blocked PAF synthesis.
Conclusions:
- Bacterial porins are potent stimulators of rapid PAF synthesis in MC through a PLA2-dependent pathway.
- LPS-induced PAF synthesis is more complex, involving both rapid and delayed phases, with the latter dependent on TNF.
- Understanding these pathways is crucial for addressing inflammation and endotoxic shock in gram-negative bacterial infections.