Background: The mechanism by which tumor necrosis factor (TNF) activates endothelial cells in not well understood. Endothelial cells express both the 55 kd (TNF-R1) and 75 kd (TNF-R2) TNF receptors, and it has been shown that TNF-R2 is expressed predominantly on the cell surface whereas TNF-R1 is localized largely in an intracellular compartment. In order to identify structural features responsible for these distributions and to further elucidate transduction of the TNF signal, we have overexpressed epitope-tagged (FLAG) TNF-R1 and TNF-R2 in human umbilical vein endothelial cells (HUVEC).

Methods: HUVEC were transfected with expression vector constructs of the full length cDNA for either TNF-R1 or TNF-R2. The receptors were epitope-tagged with FLAG on the amino terminus. Green fluorescent protein(GFP) was co-transfected as an independent marker fo transfected EC. The transfections were done using DEAE/dextran. After harvesting, the cells were divided and half were permeabilized. They then underwent immunofluorescent staining and FACS analysis.

Results: FACS analysis using GFP as a reporter showed the transfection efficiency to be about 10%. Analysis of the nonpermeabilized cells showed staining for TNF-R2 but not for TNF-R1. The permeabilized cells showed staining for both TNF-R1 and TNF-R2. This pattern of staining of the epitope-tagged receptors is consistent with the localization observed for the endogenous receptors.

Conclusion: In this experiment we demonstrated that transfected TNF receptors are expressed in endothelial cells. Epitope tagging allowed detection of the transfected receptors as distinct from the endogenous receptors. Furthermore, this experiment showed that the epitope tag did not disrupt the localization pattern of TNF-R1 and TNF-R2. Using these techniques it will be possible to further study the relationship between the two receptors and their role in TNF activation of endothelial cells.