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


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.


CONCLUSIONS
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.
Hildebrandt
Guido
G
Department of Radiotherapy and Radiooncology, University of Leipzig, Germany. hilg@medizin.uni-leipzig.de
Loppnow
Gabriela
G
Jahns
Jutta
J
Hindemith
Marion
M
Anderegg
Ulf
U
Saalbach
Anja
A
Kamprad
Friedrich
F
D000704:Analysis of Variance; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003470:Culture Media; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D004307:Dose-Response Relationship, Radiation; D015870:Gene Expression; D006801:Humans; D016014:Linear Models; D008262:Macrophage Activation; D008962:Models, Theoretical; D009097:Multienzyme Complexes / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011829:Radiation Dosage; D013053:Spectrophotometry; D013997:Time Factors
D003470:Culture Media; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D009573:Nitrites; D012333:RNA, Messenger; D009569:Nitric Oxide; D012313:RNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex