Radiation-induced lung injury (RILI) is one of the most common complications for patients receiving thoracic radiation. When it occurred, the dose of radiation has to be reduced. So it is one of the most important factors of reducing local control rate. Even with the most advanced three-dimensional conformal radiotherapy, there are still a considerable number of patients with varying degrees of lung injury (radiation pneumonitis and pulmonary fibrosis) [
In recent years, in-depth studies on the molecular mechanisms of RILI suggest that multicytokine-mediated cell-cell interactions play an important role in radiation pneumonitis and pulmonary fibrosis [
A clinical study showed that dexamethasone reduces IL-17A expression in the lungs of asthmatic patients and the severity of lung inflammation [
Inbred male C57BL/6J mice (
The study protocols were approved by the institutional ethics committees of Liaocheng People’s Hospital. Mice were kept in a group of four per cage in pathogen-free rooms and were supplied with standard laboratory diet and water. A total of 104 mice were divided into three groups randomly: (1) Sham group (
Unanesthetized mice were fixed in a plastic jig designed by ourselves which contained 12 mice at the same time. The whole thorax was irradiated by ELEKTA precise linear accelerator at a single dose of 15 Gy only once. Radiation was performed according to the characteristics as follows: beam energy: 6 MV-photons; dose rate: 3.0 Gy/min [
Mice were sacrificed at a predetermined time of 1, 4, 8, and 16 weeks after irradiation (
The lung tissues were fixed with 10% formalin solution, paraffin-embedded, and sectioned at an average thickness of 3 mm with a microtome. The slices obtained were stained with haematoxylin and eosin (H&E) to evaluate the inflammation and with Masson staining to identify the fibrosis in the lung. Extent of the alveolitis and pulmonary fibrosis was graded on a scale of 0 (normal) to 3 (severe) according to Szapiel’s method [
Sections obtained from paraffin-embedded tissues were placed in an antigen retrieval solution (DAKO, Denmark), followed by peroxide and protein blocking. Sections were incubated with primary antibodies specific to IL-17A (1 : 200) and then stained using a sensitive avidin-streptavidin-DAB peroxidase kit (BioGenex) according to the manufacturer’s instructions. Five fields (200x) per lung specimen were examined randomly. Pictures obtained by microscope camera system were analyzed by Image-Pro Plus 6.0 image analysis system (Media Cybernetics Corporation), and integrated optical densities (IOD values) were obtained. The average IOD value of all the photos of each group represents the IOD value of the group.
The contents of IL-17A, TGF-
As a biochemical index of parenchymal collagen content, hydroxyproline was determined by alkaline hydrolysis assay. Briefly, hydroxyproline was released from lung tissue homogenates by acid hydrolysis. The hydroxyproline content of the hydrolyzation products was assessed calorimetrically at 550 nm. The results were represented as micrograms per gram lung.
All statistical analyses were carried out using SPSS 16.0 statistical software. Data are represented as mean ± SD. Measurement data were analyzed by one-factor ANOVA and
The expression of IL-17A located in the cytoplasm by immunohistochemistry. We observed a stronger expression of IL-17A by alveolar macrophages, lymphocyte, type II alveolar cells, and bronchiolar epithelium cells after radiotherapy. There are only slight expressions in lung epithelial cells for control group. The intensity of IL-17A OD values increased from 1 week to 4 weeks, then began to weaken, and sustained a higher level at 16 weeks than basal line (Figure
The average optical density of IL-17A analysis and comparison in each group (
Group | 1 w | 4 w | 8 w | 16 w |
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Sham |
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RT# |
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RT + DXM## |
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IL-17A expression in lung tissue detected by immunohistochemical method. It can be seen that cytoplasmic staining of RT group gradually deepened from the 1st week, peaked in the 4th week, declined in the 8th week, and reached a lower level in the 16th week but slightly higher than Sham group (Table
The contents of IL-17A in the BALF were analyzed by ELISA kits. Radiation stimulated an increase in the levels of IL-17A in RT group, started at 1 week, peaked at 4 weeks, and dropped at 8 weeks. The results are correspondent with IL-17A immunohistochemical analysis. And dexamethasone reduced the level of IL-17A in BALF of irradiated mice (Figure
Expression of IL-17A in BALF at the indicated time points. The mice were sacrificed at the time of 1, 4, 8, and 16 weeks after irradiation, and BALF was collected for analysis of IL-17A contents. The contents of IL-17A in the BALF were analyzed by ELISA kits. Radiation stimulated an increase in the levels of IL-17A, but dexamethasone attenuated the IL-17A level in BALF. #
Alveolitis is the main lesion before 8 weeks after irradiation, and since then the degree of alveolitis began to gradually be mild, lung parenchyma structural damage, disorder, and focal fibrosis began to appear, and it gradually evolved into fibrosis. We examined hydroxyproline content of mice lung tissue at 16 weeks and found that of RT group significantly higher than the Sham group (
Comparison of the pathology grade of alveolitis and pulmonary fibrosis.
Lung injury | Group |
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Grade | |||
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0 | I | II | III | |||
Alveolitis | Sham | 24 | 21 | 3 | 0 | 0 |
RT | 24 | 0 | 3 | 10 | 11 | |
RT + DXM | 24 | 0 | 17* | 3* | 4* | |
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Lung fibrosis | Sham | 8 | 8 | 0 | 0 | 0 |
RT | 10 | 0 | 1 | 7 | 2 | |
RT + DXM | 12 | 0 | 8# | 3# | 0 |
Alveolitis:
Lung fibrosis:
Representative samples of pathological changes in Sham group (a), (d), RT group (b), (e), and RT + DXM group (c), (f). Dexamethasone attenuates radiation-induced pneumonitis and pulmonary fibrosis. Mice were administered dexamethasone (0.42 mg/kg/day) intraperitoneally on days 1 to 30 after irradiation. Mice were sacrificed on specific time point, and lung tissue was fixed and excised into tissue sections for the detection of pulmonary inflammation and collagen deposition by H&E or Masson staining. In (a), (b), and (c), dexamethasone promoted the resolution of the radiation-induced pulmonary inflammation in the 4th week as indicated by H&E staining of the lung sections. (a) Sham group. (b) RT group, significant radiation pneumonitis and inflammatory cell infiltration (black arrows position). (c) RT + DXM group, inflammatory cell infiltration was significantly reduced without significant leakage. In (d), (e), and (f), dexamethasone promoted the resolution of the radiation-induced pulmonary fibrosis in the 16th week as indicated by Masson staining of the lung sections. (d) Sham group. (e) RT group, fibrosis stage, a large number of collagen depositions (black arrow position). (f) RT + DXM group, collagen deposition was significantly reduced compared with RT group; the alveolar structure is relatively intact.
Content of lung hydroxyproline was determined by alkaline hydrolysis assay. Hydroxyproline content was significantly higher at 16 weeks than Sham group. Dexamethasone reduced lung hydroxyproline content which can reflect the content of collagen. *
The level of TNF-
The content of TGF-
Group | TGF- |
IL-6 (pg/mL) | TNF- |
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RT + DXM |
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Mice began to die on the 85th day after irradiation; dexamethasone applications delayed the time of death in mice and improved the survival rate of mice receiving total thoracic irradiation. In the radiation group, 13 mice (65.0%) died within 180 days following the radiation. Only 6 mice (30.0%) of dexamethasone treatment group died on days 121, 139, 146, 158, 165, and 178, respectively. The survival rate in the treatment group was higher than in the radiation group (
Effect of dexamethasone on the survival of mice after irradiation. C57BL/6J mice were treated with a single dose (15 Gy) to the entire thorax. Dexamethasone treatment increased the survival rates of irradiated mice. The survival rates at 180 days in Sham (
Previous studies proved that IL-17A was significantly increased in a variety of chronic inflammatory disease models, and blocking IL-17A signals can relieve inflammation and fibrosis, thus confirming that IL-17A plays an important role in inflammatory processes [
In this study, the model of RILI was established through C57BL/6 mice irradiated with 15 Gy in the chest. Immunohistochemistry and ELISA methods were used to detect IL-17A in the lung tissue and BALF. The results showed that expression of IL-17A was elevated gradually in lung tissue after irradiation, peaked in the fourth week, and began to decline in the eighth week. This trend is consistent with IL-17A content in BALF. The result implies that IL-17A may be another important cytokine involved in RILI in addition to IL-6, TGF-
Recent studies suggest that the incidence of radiation-induced pulmonary fibrosis is not simply the result of chronic outcome of radiation pneumonitis. In fact, in the early radiation injury, along with the radiation pneumonitis, fibrosis has been launched [
Biological effects of IL-17A have not been reported in RILI, and the exact mechanism is unclear. However, IL-17A contribution to idiopathic or drug-induced pulmonary fibrosis can be summarized as TGF-
Basic pharmacological studies confirmed that dexamethasone can regulate gene transcription and expression of a variety of inflammatory cytokines [
In summary, IL-17A may play an important role in the process of radiation-induced lung injury, especially in the early phase. Dexamethasone applications attenuate the severity of radiation pneumonitis and pulmonary fibrosis, by reducing inflammatory and fibrogenic cytokine expression, thereby enhancing the survival time of irradiated mice. Indeed, the further experimental studies should be carried out by using IL-17A knockout mice or IL-17A antagonist to study radiation pneumonitis and fibrosis, thus establishing the direct evidence of IL-17A in radiation-induced lung injury.
The authors report no conflict of interests.
This work was supported by a Grant from Liaocheng Municipal Science Research and Development Program (no. 2012NS14). The authors thank Dr. Jun-long Xu (Pathologist from the Department of Pathology, Liaocheng People’s Hospital, Liaocheng, China) for his expert suggestions and technical assistance.