Related Experiment Videos
Both TNF receptors are required for direct TNF-mediated cytotoxicity in microvascular endothelial cells
R Lucas1, I Garcia, Y R Donati
1Department of Anaesthesiology, Pharmacology and Surgical Intensive Care, University Medical Center, University of Geneva, Switzerland. lucas@cmu.unige.ch
Abstract:
The conditions under which tumor necrosis factor-alpha (TNF) induces apoptosis in primary microvascular endothelial cells (MVEC) were investigated. In the absence of sensitizing agents, TNF induced apoptosis after 3 days of incubation in confluent MVEC. In contrast, upon addition of the transcriptional inhibitor actinomycin D (Act. D), confluence was no longer required and apoptosis occurred already after 16 h. To assess the role of either TNF receptor (TNFR) type in apoptosis, MVEC isolated from mice genetically deficient in TNFR1 (Tnfr1o mice) or TNFR2 (Tnfr2o mice) were incubated with TNF in the presence or absence of Act. D. Under sensitized conditions, Tnfr2o MVEC were lysed like controls, whereas Tnfr1o MVEC were completely resistant, indicating an exclusive role for TNFR1. In contrast, in the absence of Act. D, confluent monolayers of wild-type cells were lysed by TNF, but both Tnfr1o and Tnfr2o MVEC were resistant to TNF-mediated toxicity, indicating a requirement for both TNFR types. Overexpression of the anti-apoptotic protein bcl-xL in MVEC led to a protection against the direct, but not the sensitized cytotoxicity of TNF. In conclusion, in pathophysiologically relevant conditions, both TNFR appear to be required for TNF-induced apoptosis in MVEC.
Insights
Tumor necrosis factor-alpha (TNF) triggers apoptosis in microvascular endothelial cells (MVEC) differently depending on conditions. Both TNF receptors (TNFR) are crucial for TNF-induced apoptosis in MVEC under pathophysiologically relevant conditions.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Tumor necrosis factor-alpha (TNF) is a key cytokine involved in inflammation and cell death.
- Microvascular endothelial cells (MVEC) play critical roles in vascular homeostasis and disease.
- Understanding TNF-induced apoptosis in MVEC is vital for various pathological conditions.
Purpose of the Study:
- To investigate the conditions governing TNF-induced apoptosis in primary MVEC.
- To elucidate the specific roles of TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) in this process.
Main Methods:
- Primary microvascular endothelial cells (MVEC) were treated with TNF alone or with actinomycin D (Act. D).
- MVEC from TNFR1-deficient (Tnfr1o) and TNFR2-deficient (Tnfr2o) mice were used to assess receptor roles.
- Overexpression of bcl-xL was employed to study anti-apoptotic mechanisms.
Main Results:
- TNF induced apoptosis in confluent MVEC after 3 days without Act. D.
- Confluence was not required for apoptosis when Act. D was present, with rapid cell death observed.
- TNFR1 was exclusively required for sensitized apoptosis, while both TNFR1 and TNFR2 were necessary for direct apoptosis in confluent cells.
- Overexpression of bcl-xL protected against direct TNF cytotoxicity but not sensitized apoptosis.
Conclusions:
- TNF-induced apoptosis in MVEC is highly dependent on cellular conditions and the presence of transcriptional inhibitors.
- TNFR1 plays a critical role in sensitized apoptosis, whereas both TNFR1 and TNFR2 are essential for direct apoptosis in confluent MVEC.
- These findings highlight the complex interplay of TNFR signaling in endothelial cell apoptosis relevant to pathological settings.