BOTH TNF RECEPTORS ARE REQUIRED FOR DIRECT TNF-MEDIATED CYTOTOXICITY IN MICROVASCULAR ENDOTHELIAL-CELLS

The conditions under which tumor necrosis factor-alpha (TNF) induces a poptosis in primary microvascular endothelial cells (MVEC) were invest igated. In the absence of sensitizing agents, TNF induced apoptosis af ter 3 days of incubation in confluent MVEC. In contrast, upon addition of the transcriptional inhibitor actinomycin D (Act. D), confluence w as no longer required and apoptosis occurred already after 16 h. To as sess the role of either TNF receptor (TNFR) type in apoptosis, MVEC is olated from mice genetically deficient in TNFR1 (Tnfr1 degrees mice) o r TNFR2 (Tnfr2 degrees mice) were incubated with TNF in the presence o r absence of Act. D. Under sensitized conditions, Tnfr2 degrees MVEC w ere lysed like controls, whereas Tnfr1 degrees MVEC were completely re sistant, indicating an exclusive role for TNFR1. In contrast, in the a bsence of Act. D, confluent monolayers of wild-type cells were lysed b y TNF, but both Tnfr1 degrees and Tnfr2 degrees 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 p rotection against the direct, but not the sensitized cytotoxicity of T NF. In conclusion, in pathophysiologically relevant conditions, both T NFR appear to be required for TNF-induced apoptosis in MVEC.