Biology contribution
Compartmental responses after thoracic irradiation of mice: Strain differences

https://doi.org/10.1016/j.ijrobp.2005.02.037Get rights and content

Purpose: To examine and compare the molecular and cellular processes leading to radiation fibrosis and pneumonitis in C57BL/6J and C3H/HeN mice.

Methods and Materials: At indicated times after various doses of thoracic irradiation, the cell populations obtained by bronchoalveolar lavage of C57BL/6J mice were differentially analyzed by cytology and assessed by RNase protection (RPA) assay for levels of cytokines and related genes. The molecular responses in bronchial alveolar lavage (BAL) populations were compared with those in whole lung of C57BL/6J mice and with those of C3H/HeN mice. The former strain develops late radiation fibrosis, whereas the latter develop subacute radiation pneumonitis.

Results: In C57BL/6J mice, a decrease in the total number of BAL cells was found 1 week after 6, 12, or 20 Gy thoracic irradiation with a subsequent dose-dependent increase up to 6 months. After 12 and 20 Gy, large, foamy macrophages and multinucleated cells became evident in BAL at 3 weeks, only to disappear at 4 months and reappear at 6 months. This biphasic response was mirrored by changes expression of mRNA for proinflammatory cytokines and the Mac-1 macrophage-associated antigen. As with BAL, whole lung tissue also showed biphasic cytokine and Mac-1 mRNA responses, but there were striking temporal differences between the two compartments, with changes in whole lung tissue correlating better than BAL with the onset of fibrosis in this strain. The radiation-induced proinflammatory mRNA responses had strain-dependent and strain-independent components. Thoracic irradiation of C3H/HeN induced similar increases in tumor necrosis factor (TNF)-α, interleukin (IL)-1α/β, and interferon (IFN)-γ mRNA expression in lung as it did in C57BL/6J mice during the “presymptom” phase at 1–2 months. However, immediately preceding and during the pneumonitic time period at 3–4 months, TNF-α and IL-1α/β mRNAs were highly upregulated in C3H/HeN mice, which develop pneumonitis, but not in C57BL/6J mice, which do not. At the onset of radiation fibrosis in C57BL/6J mice (5–6 months), irradiated lungs had increased levels of IL-1α/β and IFN-γ mRNA expression, but the TNF-α response was, notably, still muted.

Conclusions: The major molecular and cellular events in lungs of C57BL/6J and C3H/HeN mice, which develop late fibrosis and subacute pneumonitis after thoracic irradiation respectively, take place within the interstitium and are not reflected within BAL populations. The initial proinflammatory responses are similar in the two strains, but later responses reflect the latent time to lesion development. TNF-α expression at 3–4 months may be important in radiation-induced pneumonitis, and its downregulation is important in avoiding this radiation-induced complication.

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.

References (44)

  • N.J. Gross

    Pulmonary effects of radiation therapy

    Ann Intern Med

    (1977)
  • N.J. Gross

    The pathogenesis of radiation-induced lung damage

    Lung

    (1981)
  • E.L. Travis et al.

    Late functional and biochemical changes in mouse lung after irradiationDifferential effects of WR-2721

    Radiat Res

    (1985)
  • B.E. Lehnert et al.

    Temporal sequence of early alterations in rat lung following thoracic X-irradiation

    Int J Radiat Oncol Biol Phys

    (1991)
  • W. Westermann et al.

    Th2 cells as effectors in postirradiation pulmonary damage preceding fibrosis in the rat

    Int J Radiat Oncol Biol Phys

    (1999)
  • C.J. Johnston et al.

    Inflammatory cell recruitment following thoracic irradiation

    Exp Lung Res

    (2004)
  • M.W. Epperly et al.

    Correlation of ionizing irradiation-induced late pulmonary fibrosis with long-term bone marrow culture fibroblast progenitor cell biology in mice homozygous deletion recombinant negative for endothelial cell adhesion molecules

    In Vivo

    (2004)
  • M.W. Epperly et al.

    Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis

    Am J Respir Cell Mol Biol

    (2003)
  • C.E. Rube et al.

    Irradiation induces a biphasic expression of pro-inflammatory cytokines in the lung

    Strahlenther Onkol

    (2004)
  • J.H. Hong et al.

    Bronchoalveolar lavage and interstitial cells have different roles in radiation-induced lung injury

    Int J Radiat Oncol Biol Phys

    (2003)
  • C.J. Johnston et al.

    Early and persistent alterations in the expression of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor alpha mRNA levels in fibrosis-resistant and sensitive mice after thoracic irradiation

    Radiat Res

    (1996)
  • J.H. Hong et al.

    Rapid induction of cytokine gene expression in lung after single and fractionated doses of radiation

    Int J Radiat Oncol Biol Phys

    (1999)
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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.

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