Dose-dependent biphasic induction and transcriptional activity of nuclear factor kappa B (NF-(kappa)B) in EA.hy.926 endothelial cells after low-dose X-irradiation

Purpose: Low-dose radiotherapy is known to exert an anti-inflammatory effect, but the underlying radiobiological mechanisms are still elusive. It was recently reported that transforming growth factor (TGF) (beta)1 essentially contributes to the reduced adhesion of peripheral blood mononuclear cells to endothelial cells at low-dose X-irradiation. As the transcription factor nuclear factor kappa B (NF-(kappa)B) is crucially involved in mediating an inflammatory response by inducing the expression of cytokines and adhesion molecules, NF-(kappa)B DNA binding and transcriptional activity as well as its impact on the expression of TGF-(beta)1 in EA.hy.926 endothelial cells were analysed subsequently to low-dose radiotherapy. Materials and methods: Human EA.hy.926 endothelial cells were grown to subconfluence. Twenty hours after X-irradiation with single doses ranging from 0.3 to 3 Gy, the cells were activated with tumour necrosis factor-(alpha). Four hours later, the cells were harvested. NF-(kappa)B DNA-binding activity of nuclear extracts was analysed by electrophoretic mobility shift assay. The NF-(kappa)B subunits p50, p65/RelA, c-Rel and RelB of the NF-(kappa)B complexes were quantified by enzyme-linked immunoabsorbant assay. The transcriptional activity of NF-(kappa)B was measured using luciferase reporter gene assays in EA.hy.926 endothelial cells transiently transfected with the plasmid pB2xLuc. To correlate transcriptional activity to TGF-(beta)1 expression, NF-(kappa)B decoy oligonucleotides were used to inhibit NF-(kappa)B activity and TGF-(beta)1 secretion. Results: After low-dose radiotherapy, an increased NF-(kappa)B DNA-binding activity was observed in stimulated EA.hy.926 endothelial cells with a relative maximum (threefold induction) at 0.5 Gy. The NF-(kappa)B activation then decreased after X-irradiation at 0.6-0.8 Gy and subsequently increased again at doses of 1 and 3 Gy. This biphasic induction profile of NF-(kappa)B was confirmed by the analysis of the NF-(kappa)B-specific transcriptional activity. The latter showed a relative maximum at 0.5 Gy, a relative minimum between 0.5 and 1.0 Gy, and an increase at 3 Gy. Transfection of EA.hy.926 endothelial cells with NF-(kappa)B decoy oligonucleotides before irradiation resulted in a 50% reduction of TGF-(beta)1 secretion at 0.5 Gy compared with control oligonucleotides or untreated cells. Conclusions: Low-dose radiotherapy induces a biphasic activation of NF-(kappa)B with a relative maximum at 0.5 Gy. The induction by NF-(kappa)B of TGF-(beta)1 in endothelial cells might contribute to the anti-inflammatory properties of low-dose ionizing irradiation.