Radiation-induced lung injury and inflammation in mice: role of inducible nitric oxide synthase and surfactant protein D

Abstract

Reactive nitrogen species (RNS) generated after exposure to radiation have been implicated in lung injury. Surfactant protein D (SP-D) is a pulmonary collectin that suppresses inducible nitric oxide synthase (iNOS)-mediated RNS production. Herein, we analyzed the role of iNOS and SP-D in radiation-induced lung injury. Exposure of wild-type (WT) mice to γ-radiation (8 Gy) caused acute lung injury and inflammation, as measured by increases in bronchoalveolar lavage (BAL) protein and cell content at 24 h. Radiation also caused alterations in SP-D structure at 24 h and 4 weeks post exposure. These responses were blunted in iNOS(-/-) mice. Conversely, loss of iNOS had no effect on radiation-induced expression of phospho-H2A.X or tumor necrosis factor (TNF)-α. Additionally, at 24 h post radiation, cyclooxygenase expression and BAL lipocalin-2 levels were increased in iNOS(-/-) mice, and heme oxygenase (HO)-1(+) and Ym1(+) macrophages were evident. Loss of SP-D resulted in increased numbers of enlarged HO-1(+) macrophages in the lung following radiation, along with upregulation of TNF-α, CCL2, and CXCL2, whereas expression of phospho-H2A.X was diminished. To determine if RNS play a role in the altered sensitivity of SP-D(-/-) mice to radiation, iNOS(-/-)/SP-D(-/-) mice were used. Radiation-induced injury, oxidative stress, and tissue repair were generally similar in iNOS(-/-)/SP-D(-/-) and SP-D(-/-) mice. In contrast, TNF-α, CCL2, and CXCL2 expression was attenuated. These data indicate that although iNOS is involved in radiation-induced injury and altered SP-D structure, in the absence of SP-D, it functions to promote proinflammatory signaling. Thus, multiple inflammatory pathways contribute to the pathogenic response to radiation.

Keywords: iNOS; lung injury; radiation; reactive nitrogen species; surfactant protein D.

FIG. 1.

Effects of radiation exposure on BAL protein and cell content. BAL was collected from untreated mice or 24 h and 4 weeks (wk) after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation, and analyzed for protein (upper panel) or cell content (lower panel). Each bar is the mean ± SE (n = 3–6 mice/treatment group). ^aSignificantly (P ≤ 0.05) different from respective untreated mice; ^bSignificantly (P ≤ 0.05) different from WT mice at the same post exposure time point; ^cSignificantly (P ≤ 0.05) different from iNOS^−/− mice at the same post exposure time point; ^dSignificantly (P ≤ 0.05) different from SP-D^−/− mice at the same post exposure time point.

FIG. 2.

Effects of radiation exposure on BAL SP-D structure. SP-D protein was analyzed in BAL collected from untreated (U) mice or 24 h and 4 weeks (wk) after exposure of WT and iNOS^−/− mice to radiation. Upper panel, SP-D protein analyzed in native gels; Lower panel, SP-D protein analyzed in denatured gels.

FIG. 3.

Effects of radiation exposure on lung HO-1 expression. Lung sections, prepared from untreated mice or 24 h and 4 weeks (wk) after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation, were stained with antibody to HO-1. Binding was visualized using a Vectastain kit. Original magnification, 600×. Representative sections from 3 mice/treatment group are shown.

FIG. 4.

Effects of radiation exposure on lung cell DNA damage. Lung sections, prepared from untreated mice or 6 h and 24 h after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation, were stained with antibody to phospho-H2A.X. Binding was visualized using a Vectastain kit. Original magnification, 600×. Representative sections from 3 mice/treatment group are shown.

FIG. 5.

Effects of radiation exposure on lung lipocalin-2. BAL and lungs were collected from untreated mice or 24 h and 4 weeks (wk) after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation. Upper panel, Total RNA was isolated from lung tissue, reverse-transcribed, and mRNA analyzed by real-time PCR as described in the Materials and Methods section. Data are presented as fold change from untreated WT control. Lower panel, BAL protein was analyzed by western blotting for expression of lipocalin-2. Protein band intensity was quantified using ImageJ software. Bars are the mean ± SE (n = 3 mice/treatment group). ^aSignificantly (P ≤ 0.05) different from respective untreated group; ^bSignificantly (P ≤ 0.05) different from WT mice at the same post exposure time point.

FIG. 6.

Effects of radiation exposure on lung mRNA expression of proinflammatory mediators. Lungs were collected from untreated mice or 24 h and 4 weeks (wk) after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation. Total RNA was isolated, reverse-transcribed, and mRNA analyzed by real-time PCR as described in the Materials and Methods section. Data are presented as fold change from untreated WT control. Each bar represents the mean ± SE (n = 3–5 mice/treatment group). ^aSignificantly (P ≤ 0.05) different from respective untreated group; ^bSignificantly (P ≤ 0.05) different from WT mice at the same post exposure time point; ^cSignificantly (P ≤ 0.05) different from iNOS^−/− mice at the same post exposure time point; ^dSignificantly (P ≤ 0.05) different from iNOS^−/−/SP-D^−/− mice at the same post exposure time point.

FIG. 7.

Effects of radiation exposure on lung COX-2 expression. Lung sections, prepared from untreated mice or 24 h and 4 weeks (wk) after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation, were stained with antibody to COX-2. Binding was visualized using a Vectastain kit. Original magnification, 600×. Representative sections from 3 mice/treatment group are shown.

FIG. 8.

Effects of radiation exposure on lung Ym1 expression. Lung sections, prepared from untreated mice or 24 h and 4 weeks (wk) after exposure of WT, iNOS^−/−, SP-D^−/−, and iNOS^−/−/SP-D^−/− mice to radiation, were stained with antibody to the alternatively activated/profibrotic macrophage marker, Ym1. Binding was visualized using a Vectastain kit. Original magnification, 600×. Representative sections from 3 mice/treatment group are shown.