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Platelet-activating factor biosynthesis by cultured mesangial cells is modulated by proteinase inhibitors
L Biancone1, C Tetta, E Turello
1Laboratorio di Immunopatologia, Università di Torino, Italy.
Abstract:
Rat mesangial cells stimulated with calcium ionophore A23187 and phagocytosis were shown to produce platelet-activating factor (PAF), a mediator of inflammation and endotoxic shock. In the study presented here, the cultured human mesangial but not epithelial cells synthetized PAF not only in response to calcium ionophore A23187 and phagocytosis of immunoglobulin G-coated latex beads, but also after stimulation with cytokines such as tumor necrosis factor-alpha and interleukin-1 beta. PAF synthetized after stimulation with A23187 and to a lesser extent with phagocytosis was partially released. In contrast, PAF synthesized by stimulation with tumor necrosis factor-alpha and interleukin-1 beta remained cell associated. Experiments with labeled precursors demonstrated that PAF was synthetized via the remodeling pathway that involves the activation of phospholipase A2 and of an acetyl-coenzymeA:2-lyso-PAF acetyltransferase. Synthetic inhibitors of serine proteases as well as plasma alpha 1-proteinase inhibitor inhibited the activation of phospholipase A2 detected as release of (14C) arachidonic acid and the activation of acetyl-CoA:2-lyso-PAF acetyltransferase at concentrations 100-fold lower than those present in plasma. This raises the question about the ability of mesangial cells to synthetize PAF in vivo. However, the inhibitory effect of plasma alpha 1-proteinase inhibitor may be abrogated by oxidative inactivation due to a concomitant stimulation of mesangial cell respiratory burst or in zones of close contact among cells or matrix, which have been shown to exclude antiproteinases.
Insights
Human mesangial cells produce platelet-activating factor (PAF) in response to various stimuli, including cytokines. Plasma alpha 1-proteinase inhibitor can block this production, but its effectiveness may be limited in vivo.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Platelet-activating factor (PAF) is a mediator of inflammation and endotoxic shock.
- Rat mesangial cells produce PAF when stimulated by calcium ionophore A23187 and phagocytosis.
Purpose of the Study:
- To investigate PAF synthesis in cultured human mesangial cells.
- To determine the stimuli that induce PAF production in these cells.
- To elucidate the mechanism of PAF synthesis and its regulation.
Main Methods:
- Stimulation of human mesangial cells with calcium ionophore A23187, phagocytosis of IgG-coated latex beads, tumor necrosis factor-alpha, and interleukin-1 beta.
- Measurement of PAF synthesis and release.
- Use of labeled precursors to identify the PAF synthesis pathway (remodeling pathway).
- Assessment of the effects of synthetic serine protease inhibitors and plasma alpha 1-proteinase inhibitor on PAF synthesis.
Main Results:
- Human mesangial cells, but not epithelial cells, synthesized PAF in response to A23187, phagocytosis, tumor necrosis factor-alpha, and interleukin-1 beta.
- PAF release varied depending on the stimulus: partially released after A23187/phagocytosis, cell-associated after cytokine stimulation.
- PAF synthesis occurred via the remodeling pathway involving phospholipase A2 and acetyl-CoA:2-lyso-PAF acetyltransferase.
- Synthetic inhibitors and plasma alpha 1-proteinase inhibitor inhibited key enzymes in PAF synthesis at low concentrations.
Conclusions:
- Human mesangial cells are capable of synthesizing PAF in response to diverse stimuli.
- The remodeling pathway is the primary route for PAF synthesis in these cells.
- The in vivo relevance of mesangial cell PAF synthesis is questioned due to potential inhibition by plasma alpha 1-proteinase inhibitor, although this inhibition may be overcome under certain conditions.