Related Experiment Videos
Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular
G Camussi1, F Bussolino, G Salvidio
1Department of Pathology, School of Medicine, State University of New York at Buffalo 14214.
Abstract:
Murine tumor necrosis factor (mTNF) stimulates production of platelet-activating factor (PAF) by cultured rat peritoneal macrophages in amounts comparable to those formed during treatment with the calcium ionophore A23187 or phagocytosis of zymosan. The cell-associated PAF that was released into the medium was identical to synthetic PAF, as determined with physicochemical, chromatographic, and enzymatic assays. Furthermore, de novo synthesis of PAF by macrophages was demonstrated by the incorporation of radioactive precursors such as [3H]acetyl-coenzyme A or [3H]2-lyso-PAF. Macrophages incubated with mTNF for 4 h synthesized PAF only during the first h of treatment. At this time, the amount of cell-associated PAF was approximately equal to that released into the medium. The cell-associated PAF decreased afterwards, whereas that in the medium did not correspondingly increase, suggesting that some PAF was being degraded. The response of rat macrophages to different doses of mTNF and human TNF (hTNF) was examined. Maximal synthesis of PAF was obtained with 10 ng/ml of mTNF and 50 ng/ml of hTNF. This finding may be explained by a lower affinity of hTNF for TNF receptors of rat cells. The hTNF stimulated production of PAF by human vascular endothelial cells cultured from the umbilical cord vein. The time course of PAF synthesis was slower than that observed with macrophages, with maximal production between 4 and 6 h of treatment. Optimal synthesis of PAF was obtained with 10 ng/ml of hTNF. Only 20-30% of the PAF synthesized by endothelial cells was released into the medium, even after several hours of incubation. Synthesis of PAF in response to TNF was also detected in rat polymorphonuclear neutrophils, but not in human tumor cells and dermal fibroblasts. Therefore, production of PAF is a specialized response that is transient in macrophages continuously treated with TNF, and that appears to be controlled by unidentified regulatory mechanisms.
Insights
Murine tumor necrosis factor (mTNF) stimulates platelet-activating factor (PAF) production in rat macrophages. This PAF synthesis is transient and regulated by unknown mechanisms, with varying responses across different cell types.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Tumor necrosis factor (TNF) is a key inflammatory cytokine.
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammation.
- Understanding the interplay between TNF and PAF is crucial for inflammatory disease research.
Purpose of the Study:
- To investigate the role of murine tumor necrosis factor (mTNF) in stimulating platelet-activating factor (PAF) production by macrophages.
- To characterize the kinetics and regulation of TNF-induced PAF synthesis.
- To compare TNF-induced PAF production across different cell types.
Main Methods:
- Cultured rat peritoneal macrophages and human umbilical cord vein endothelial cells were treated with mTNF and human TNF (hTNF).
- PAF production was quantified using physicochemical, chromatographic, and enzymatic assays.
- De novo synthesis was confirmed via radioactive precursor incorporation ([3H]acetyl-coenzyme A, [3H]2-lyso-PAF).
Main Results:
- mTNF stimulated PAF production in rat macrophages comparable to ionophore or zymosan treatment.
- PAF synthesis in macrophages was transient, occurring mainly in the first hour of mTNF exposure.
- hTNF also induced PAF production in human endothelial cells, but with slower kinetics and less release into the medium compared to macrophages.
- PAF synthesis in response to TNF was observed in rat neutrophils but not in human tumor cells or fibroblasts.
Conclusions:
- TNF is a potent stimulator of PAF production in specific cell types, notably macrophages and endothelial cells.
- The transient nature of PAF synthesis in macrophages suggests tight regulatory control.
- Differences in TNF affinity and cell-specific responses highlight the complexity of TNF-mediated inflammatory signaling.