Effects of lipopolysaccharide on the response of C57BL/6J mice to whole thorax irradiation

Abstract

Background and purpose: Inflammatory and fibrogenic processes play a crucial role in the radiation-induced injury in the lung. The aim of the present study was to examine whether additive LPS exposure in the lung (to simulate respiratory infection) would affect pneumonitis or fibrosis associated with lung irradiation.

Material and methods: Wildtype C57Bl/6J (WT-C57) and TNFα, TNFR1 and TNFR2 knockout ((-/-)) mice, in C57Bl/6J background, were given whole thorax irradiation (10 Gy) with or without post-irradiation intratracheal administration of LPS (50μg/mice). Functional deficit was examined by measuring breathing rate at various times after treatment. Real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry were used to analyze the protein expression and m-RNA of Interleukin-1 alpha (IL-1α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumour Necrosis Factor alpha (TNFα) and Transforming Growth Factor beta (TGFβ) in the lung at various times after treatment. Inflammatory cells were detected by Mac-3 (macrophages) and Toluidine Blue (mast cells) staining. Collagen content was estimated by hydroxyproline (total collagen) and Sircol assay (soluble collagen). Levels of oxidative damage were assessed by 8-hydroxy-2-deoxyguanosine (8-OHdG) staining.

Results: LPS exposure significantly attenuated the breathing rate increases following irradiation of WT-C57, TNFR1(-/-) and TNFR2(-/-)mice and to a lesser extent in TNFα(-/-) mice. Collagen content was significantly reduced after LPS treatment in WT-C57, TNFR1(-/-) and TNFα(-/-) mice and there was a trend in TNFR2(-/-) mice. Similarly there were lower levels of inflammatory cells and cytokines in the LPS treated mice.

Conclusions: This study reveals a mitigating effect of early exposure to LPS on injury caused by irradiation on lungs of C57Bl mice. The results suggest that immediate infection post irradiation may not impact lung response negatively in radiation-accident victims, however, further studies are required in different animal models, and with specific infectious agents, to confirm and extend our findings.

Figure 1

The mean breathing rate (±SEM) for groups of mice (a) C57WT, (b) TNFR1 −/−, (c) TNFR2−/−, (d) TNFα−/−, as a function of time after being given either 0 or 10 Gy (+/− LPS) to the whole lung at time zero. Dashed black line indicates the control mice, gray line indicates radiation treated mice and black line indicates radiation + LPS treated mice. A preliminary version of some of these results has been published previously (10).

Figure 2

Quantitative analysis of IHC staining for cytokines IL-1α (a), IL-1β (b), IL-6 (c), TNFα (d) and TGFβ (e), at 12 and 24 weeks following lung irradiation (10Gy) with/without post-irradiation (within 1h) intratracheal administration of LPS. Percent positivity is the ratio of positive pixels/total number of positive and negative pixels in the tissue section (air spaces excluded). Each bar represents the mean (±SEM for 6 mice).

Figure 3

(a) Mac-3 staining for activated macrophages in the mouse lungs at 12 and 24 weeks following lung irradiation (10Gy) with/without post-irradiation (within 1h) intratracheal administration of LPS. (b) Toluidine Blue staining for mast cells in the mouse lungs at 12 and 24 weeks following lung irradiation (10Gy) with/without post-irradiation (within 1h) intratracheal administration of LPS. (c) 8-OHdG staining (oxidative stress/DNA damage) in the mouse lungs at 12 and 24 weeks following lung irradiation (10Gy) with/without post-irradiation (within 1h) intratracheal administration of LPS. Percent positivity is the ratio of positive pixels/total number of positive and negative pixels in the tissue section (air spaces excluded). Each bar represents the mean (±SEM for 6 mice).

Figure 4

(a) Hydroxyproline content (μg of hydroxyproline/100 mg of wet lung tissue) at 12 and 24 weeks following lung irradiation (10Gy) with/without post-irradiation (within 1h) intratracheal administration of LPS. (b) Sircol assay (mg collagen/65mg wet lung tissue) showed a small increase in recently-synthesized (RS) collagen after irradiation in the mouse lungs at 12 and 24 weeks following lung irradiation (10Gy) with/without post-irradiation (within 1h) intratracheal administration of LPS. Each bar represents the mean (±SEM for 6 mice).