International Journal of Radiation Oncology*Biology*Physics
Biology contributionCompartmental responses after thoracic irradiation of mice: Strain differences
Introduction
Radiation-induced pulmonary damage is a frequent complication in patients receiving irradiation for lung or breast cancers. Patients might develop subacute pneumonitis or late fibrosis months to years after radiation exposure. Inbred mouse strains develop similar responses, showing genetic differences in their propensity to develop pneumonitis or fibrosis after irradiation (1). Radiation pneumonitis and fibrosis apparently involve distinct and separate target cells (2, 3, 4, 5, 6, 7) and have distinct, highly reproducible, latent periods before their onset. Their pathophysiologies are complex and unclear, although it is believed that infiltration of inflammatory cells plays a significant role (8, 9, 10, 11, 12, 13).
Many studies have shown time-dependent, recurring molecular responses after thoracic irradiation that may remain subclinical or correlate with the expression of functional damage (14, 15). Expression of cytokines (14, 16, 17, 18), chemokines (19), and cell adhesion molecules (20, 21) has been reported and implicated as being actively involved in pathogenesis. It has been hypothesized that macrophages/monocytes that infiltrate the lung after irradiation mediate these responses (8, 22, 23). The type of cytokines released during this latent period might critically determine the outcome. It has been shown that, after a single dose of 12.5 Gy irradiation, levels of transforming growth factor (TGF)-β1 mRNA were increased at 8 and 26 weeks after irradiation in the C57BL/6J mouse strain, whereas the C3H/HeJ strain remained at control levels (24). The same group found strain-dependent variation in radiation-induced interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α mRNA levels and suggested roles for these cytokines in the development of radiation-induced fibrosis (16). In contrast, work by Franko et al. showed that elevated levels of TGF-β1 in C57L/J mice were not essential to the initiation of fibrosis and concluded that they were likely more related to its progression (25). They also found only a minor difference in TNF-α production between C57L/J and C3HeB/FeJ strains and concluded that it did not appear to be biologically meaningful (25). These contradictory reports might result from slight genetic differences in the mouse strains, the methods used, and the time points examined. Our early studies showed that early proinflammatory cytokine expression in response to irradiation is not related to the lesion that develops (18), but that late changes are associated with lesion onset (15).
This study was designed to systematically explore and compare the molecular and cellular responses in different strains of mice in response to radiation-induced lung injury. We had previously demonstrated a compartmentalized response in bronchial alveolar lavage (BAL) and interstitial populations during the development of radiation-induced pneumonitis in C3H/HeN mice (15). We extended this approach further to investigate the molecular and cellular responses during the progression of radiation-induced pulmonary fibrosis in C57BL/6J mice. The C57BL/6J mouse is the most common model for studying the pathophysiology of radiation-induced fibrosis (26). Studies have suggested that activated neutrophils (27), lymphocytes (28), and alveolar macrophages (10, 22) are involved. This concept is further supported by the observed elevation of inflammatory cytokine mRNAs within the lung and of proteins in the peripheral circulation during the progression of radiation-induced fibrosis (16, 24, 29, 30). Our study reasserts the distinct temporal and spatial changes in proinflammatory cytokine gene expression in different cellular compartments of the irradiated lung. The comparison of the temporal expression of individual cytokine mRNA during the development of radiation-induced pneumonitis and fibrosis demonstrates that early alterations during the presymptom phase is strain-independent, whereas late changes are strain-dependent. Control of the late cytokine response may critically determine the outcome in terms of pneumonitis or fibrosis.
Section snippets
Mice and radiation treatments
C57BL/6J mice were purchased from the National Laboratory Animal Center, Taiwan, and housed in Chung Gung University Laboratory Animal Center, Taiwan. Seven 8-week-old male mice were used for experiments. For lung irradiation, unanesthetized mice were restrained in Perspex tubes (Becton Dickinson, Franklin Lakes, NJ) (18). The whole thorax was irradiated by 6 MV X-rays from a linear accelerator, with a dose rate of 2–3 Gy/min and a 1.5-cm bolus on the surface. The field was 2.5 cm in length in
Development of lethality after thoracic irradiation
We first examined the responses of these mice to a range of single doses of radiation. In agreement with the reports in the literature (4, 33), C57BL/6J mice resisted death from pneumonitis after radiation doses not in excess of 20 Gy and died mainly of fibrosis (Figs. 1a−d) between 5 and 8 months (see Fig. 2a). The LD50/360 was 12.50 ± 0.01 Gy (see Fig. 2b). Fibrosis began at 4 months and progressively increased in degree with time (Figs. 1a-d). The extent of fibrosis at 6 months was
Discussion
By comparing the cellular and molecular responses between BAL and interstitial tissues, we had previously demonstrated a compartmentalized response of proinflammatory cells and molecules during the development of radiation-induced pneumonitis in C3H/HeN mice (15). We extended this approach further to investigate the molecular and cellular responses during the progression of radiation-induced pulmonary fibrosis in a C57BL/6J mouse model. In agreement with previous findings in C3H/HeN mice (15)
Acknowledgments
The authors thank Mr. Ching-Jung Lo, Mr. Yi-Chen Liu, and Miss Chin-Yi Lee for their help in dosimetry verification, mouse irradiation, and the counting of bronchial alveolar cells.
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Supported by NHRI-EX92-9030SL, NSC 93-2314-B-182-082, and CMRP836 to J.-H.H.. C.-S.C. is supported by NSC 92-2320-B-007-009 and NHRI-EX92-9121BI grants.