Cellular and molecular aspects of the anti-inflammatory effects of low-dose radiation therapy

For decades an anti-inflammatory and analgesic effect of low-dose X-irradiation (LD-RT) has clinically been well established in the treatment of a plethora of benign diseases and chronic degenerative disorders with empirically identified single doses < 1 Gy to be most effective. Although considerable progress has been achieved in the understanding of immune modulatory effects of ionising radiation, especially in the low-dose range, the underlying molecular mechanisms are currently not fully resolved. Nevertheless, a modulation of endothelial cell (EC) activity has already been proven to comprise a key element in the therapeutic effects of LD-RT. In line with that, a putative interrelationship between DNA damage repair and a discontinuous dose-response relationship following low-dose irradiation was recently suggested. Moreover, a mechanistic involvement of reactive oxygen species (ROS) production and the cellular antioxidative response to give rise or contribute to these phenomena in endothelial cells remain elusive. Thus, in the present study, radiation effects with a particular focus on low-dose irradiation of ECs were investigated. To analyse DNA repair capacity, phospho-histone H2AX foci were assayed at 1 h, 4 h and 24 h after irradiation. ROS production, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and transcription factor nuclear factor E2-related factor 2 (Nrf2) expression and activity were analysed by western immunoblotting, fluorometric 2',7'-dichlorodihydrofluorescein-diacetate (H2DCFDA), colorimetric assays, flow-cytometry and real-time PCR, respectively. A functional impact of ROS on γH2AX foci numbers and on peripheral blood mononuclear cell (PBMC) adhesion to ECs was assayed in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and the Nrf2 activator AI-1. Irrespective of inflammatory stimulation by tumour necrosis factor-α (TNF-α), immortalised EA.hy926 ECs cells revealed a linear dose-response characteristic of γH2AX foci levels at 1 h and 4 h after irradiation. By contrast, at 24 h after irradiation a discontinuity in residual γH2AX foci levels with locally elevated values following a 0.5 Gy exposure were observed. This effect was unlikely caused by modulation of DNA damage repair, as proven by small molecule inhibitors targeting either the repair pathways homologous recombination (HR) or non-homologous end joining (NHEJ). However, the discontinuity in γH2AX foci levels was abolished by treatment with N-acetyl-L-cysteine (NAC), indicating an involvement of ROS. In line with that, in EA.hy926 ECs a discontinuous expression and enzymatic activity of SOD, CAT and GPx concomitant with a lowered expression and DNA-binding activity of the redox sensitive transcription factor Nrf2 most pronounced after a dose of 0.5 Gy was observed. Finally, scavenging of ROS by NAC or activation of Nrf2 by AI-1 significantly diminished a lowered adhesion of PBMC to EC, typically detectable following irradiation with a dose of 0.5 Gy In conclusion, these results indicate a non-linear regulation of ROS production, major compounds of the antioxidative system including SOD, CAT, GPx and Nrf2 expression and activity in EA.hy926 EC following irradiation with doses < 1 Gy. This functionally contributes to a discontinuous level of residual γH2AX foci and a hampered leukocyte/EC adhesion. These data may thus contribute a further component to the plethora of mechanisms implicated in the anti-inflammatory effects of low-dose X-ray exposure