Background and purpose: Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the knowledge of the underlying molecular mechanisms is still scarce. The authors have recently reported that transforming growth factor beta 1 (TGF-beta(1)) essentially contributes to a reduced endothelial adhesion of mononuclear cells (PBMC) following LD-RT. Furthermore, TGF-beta(1) secretion was associated with the induction of the transcription factor nuclear factor kappa B (NF-kappaB). However, the time course of adhesion, TGF-beta(1) expression, and NF-kappaB activity following LD-RT has not been thoroughly investigated yet.
Material and methods: The human EA.hy.926 endothelial cell line (EA.hy.926 EC) was grown to 95% confluence. Immediately after stimulation with the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), EA.hy.926 EC were irradiated with single doses ranging from 0.3 up to 3 Gy. Adhesion assays were performed 4, 12, and 24 h after irradiation. Nuclear extracts and culture supernatants were harvested after 4, 8, 12, 20, 24, 30, and 40 h. NF-kappaB DNA-binding activity was analyzed by electrophoretic mobility shift assays (EMSA) and TGF-beta(1) secretion by enzyme-linked immunosorbent assay (ELISA). The functional impact of TGF-beta(1) on the course of leukocyte/EC adhesion was analyzed by neutralizing TGF-beta(1) in parallel with stimulation/irradiation of the EA.hy.926 EC.
Results: 4 and 24 h after irradiation of EA.hy.926 EC in the dose range between 0.3 and 0.7 Gy, a reduced adhesion of PBMC compared to nontreated controls could be observed. However, 12 h after irradiation a relative maximum of adhesion (up to 30% increase) was seen at a dose of 0.3 Gy. TGF-beta(1) secretion and NF-kappaB DNA-binding activity displayed a similar biphasic kinetics of induction with a relative minimum 12 h after irradiation. Neutralization of TGF-beta(1) activity restored adhesion at 4 and 24 h after LD-RT of EA.hy.926 EC, but it did not influence leukocyte adhesion 12 h after irradiation.
Conclusion: LD-RT of stimulated human EA.hy.926 EC is followed by a biphasic time course of NF-kappaB activity and an increased secretion of TGF-beta(1). The kinetics shows peak levels at 4-8 h and 24-30 h after LD-RT and results in a biphasic leukocyte/EC adhesion profile.