Inhibition of the iNOS pathway in inflammatory macrophages by low-dose X-irradiation in vitro. Is there a time dependence?

Abstract

Background: Low radiation doses (< or = 1.25 Gy), if applied 6 h before or after stimulation, are known to inhibit the inducible nitric oxide synthase (iNOS) pathway in inflammatory macrophages in vitro. We therefore investigated the time dependence and the underlying molecular mechanism of this effect, since it may be involved in the clinically observed anti-inflammatory and analgesic efficacy of low-dose radiotherapy.

Material and methods: Metabolic activity, nitric oxide (NO) production, iNOS- and hemoxygenase 1-(HO-1-)protein and -mRNA expression by macrophages in vitro after stimulation with LPS/IFN-gamma (0.1 microg ml(-1)/100 U ml(-1)) were investigated. Irradiation was performed at 6, 4, 2 h before and 0, 2, 4, 6 h after stimulation with doses ranging from 0.3 to 10 Gy. For each group, three independent experiments were performed over a period of 30 h with sampling intervals of 3 h.

Results: In stimulated macrophages, metabolic activity was not affected by radiation doses up to 10 Gy. A dose-dependent modulation of the cumulative NO production was observed with significant inhibition by low radiation doses < or = 1.25 Gy) and return to control level and even higher concentrations by higher doses (< or = 5 Gy). The degree of inhibition did not show any significant time dependence within the experimental time window used. The iNOS-mRNA expression 3-18 h following stimulation and subsequent irradiation was not affected by doses < or = 1.25 Gy. The iNOS-protein expression 6-24 h following stimulation and subsequent irradiation was reduced by doses < or = 1.25 Gy. By contrast, neither HO-1-protein nor HO-1-mRNA expression at the same time points was influenced by these low doses.

Conclusion: The inhibitory interference of low radiation doses with the iNOS pathway in inflammatory macrophages appears to be based on radiation effects on the translational and posttranslational control mechanisms of iNOS activity. However, contrary to our working hypothesis this is not related to radiation-induced induction of HO-1 expression and thereby increased degradation of heme which is essential for iNOS activity. Thus, other posttranslational modifications such as the proteasome degradation pathway might be involved.