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Published ahead of print on October 5, 2006, doi:10.1164/rccm.200606-862OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 1352-1360, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200606-862OC

Original Article

All-trans-Retinoic Acid Prevents Radiation- or Bleomycin-induced Pulmonary Fibrosis

Chiharu Tabata*, Yoshio Kadokawa*, Rie Tabata, Meiko Takahashi, Kae Okoshi, Yoshiharu Sakai, Michiaki Mishima and Hajime Kubo

Horizontal Medical Research Organization (HMRO), and Departments of Respiratory Medicine and Surgery, Graduate School of Medicine, Kyoto University, Kyoto; and Department of Internal Medicine, Hyogo Prefectural Tsukaguchi Hospital, Hyogo, Japan

Correspondence and requests for reprints should be addressed to Chiharu Tabata, M.D., HMRO, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan 606–8501. E-mail: ctabata{at}kuhp.kyoto-u.ac.jp


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Although radiotherapy is effective in treating lung cancers, resultant pulmonary injury is the main obstacle. Pulmonary fibrosis is characterized by progressive worsening in pulmonary function leading to high incidence of death. Currently, however, there has been little progress in effective preventive and therapeutic strategies.

Objectives: Previously, we reported that all-trans-retinoic acid (ATRA) reduced both irradiation-induced interleukin (IL)-6 production in lung fibroblasts and IL-6–dependent cell growth, and also directly inhibited the proliferation of lung fibroblasts after irradiation. In this study, we examined the preventive effect of ATRA on the progression of lung fibrosis both in irradiated and bleomycin-treated mice.

Measurements: We performed histologic examinations and quantitative measurements of IL-6, transforming growth factor (TGF)-beta1, and collagen type I{alpha}1 (COL1A1) in irradiated and bleomycin- treated mouse lung tissues with or without the administration of ATRA.

Results: Lethal irradiation effect was reduced by intraperitoneal administration of ATRA, and the overall survival rate at 16 wk was 30.0% without ATRA (n = 11), whereas it was 81.8% (n = 10) in the treatment group (p = 0.04). In vitro studies disclosed that the administration of ATRA reduced (1) irradiation-induced production of IL-6, TGF-beta1, and collagen from IMR90 cells, and (2) IL-6–dependent proliferation and TGF-beta1–dependent transdifferentiation of the cells, which could be the mechanism underlying the preventive effect of ATRA on lung fibrosis. Furthermore, ATRA ameliorated bleomycin-induced fibrosis in mouse lung tissues.

Conclusions: These data may provide a rationale to explore clinical use of ATRA for the prevention of radiation-induced lung fibrosis and other pathologic conditions involving pulmonary fibrosis.

Key Words: cytokines • interstitial lung disease • lung fibroblasts



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Pulmonary fibrosis caused by radiation therapy for lung cancer and of unknown etiology is characterized by progressive worsening in pulmonary function, leading to a high incidence of death. To date, there has been little progress toward effective preventive and therapeutic strategies.

What This Study Adds to the Field
The data provide a rationale to explore clinical use of all-trans-retinoic acid for the prevention of radiation-induced lung fibrosis and other pulmonary fibrosis.

 
Lung cancer is one of the most common solid tumors in many countries of the world, including the United States and Japan, and represents the primary cause of mortality among deaths from cancer. Although 30 to 40% of the patients with lung cancer can benefit from radiotherapy, approximately 20% of these patients develop radiation-induced pulmonary injury. Acute and subacute radiation pneumonitis, with late occurrence of fibrosis, are well-known risk factors for quality of life and survival of patients receiving radiotherapy to the thoracic region. However, there has been no previous report on effective treatment or prophylaxis.

According to clinical researchers investigating the circulating cytokines in relation to radiation-induced pulmonary injury, the levels of interleukin (IL)-6 and transforming growth factor (TGF)-beta1 may serve as a predictor for the occurrence of radiation pneumonitis (1–3). We previously reported that irradiation stimulated IL-6 production and accelerated transcription of its receptors, and increased cell proliferation via predictable IL-6/IL-6R autocrine/paracrine systems in human lung fibroblastic cell lines (4). All-trans-retinoic acid (ATRA), a physiologic metabolite of vitamin A, is known to affect cell differentiation, proliferation, and development, and has been widely used for differentiating therapy of acute promyelocytic leukemia, with the ability to overcome promyeolocytic leukemia (PML)/retinoic acid receptor (RAR) fusion protein. There have been several reports about the effects of ATRA on cytokine production (5–10). ATRA induced the growth inhibition of myeloma cells, which proliferated in IL-6 autocrine and paracrine mechanisms, with the reduction of both IL-6 production and its receptor expression (11–13). In the previous report, we demonstrated that ATRA reduced irradiation-induced production of both IL-6 and its receptors in the lung fibroblasts and IL-6–dependent cell growth, and also directly inhibited the proliferation of lung fibroblasts after irradiation as well as anti-human IL-6R antibodies (4).

Pulmonary fibrosis is frequently associated with collagen diseases, rheumatoid arthritis, and drugs, including anticancer agents. Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis, with a prevalence of 16 to 18 per 100,000 persons, and is characterized by progressive worsening in pulmonary function leading to high incidence of death (> 50% 5-yr mortality rate) due to ultimate respiratory failure (14). There have been a number of reports on cytokines being associated with lung fibrosis in animal models, including IL-6, TGF-beta1, and platelet-derived growth factor (PDGF), by overexpression of cytokine genes (15). Currently, however, there has been little progress in effective therapeutic and preventive strategies (16–18).

Here, we report the suggested inhibitory effect of ATRA on fibrotic changes both in irradiated and bleomycin-treated mouse lung tissues, resulting in the reduction of lethal effect caused by irradiation.


   METHODS
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 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
All cells (IMR-90, a cell derived from human fetal lung fibroblasts; Beas2B, a human bronchial epithelial cell line; HPAEC, a normal human pulmonary artery endothelial cell; and LEC, a normal human lymphatic endothelial cell) were cultured and irradiated with 8-Gy gamma rays as previously described (4). ATRA (Sigma Chemical Co., St. Louis, MO) was added to the growth medium to yield the final dimethyl sulfoxide solvent concentration of less than 0.2% (vol/vol). In some experiments, the cells were preincubated either with nuclear factor (NF)-{kappa}B inhibitor MG-132 (5 µM; Calbiochem, San Diego, CA) (19), Jun N-terminal kinase (JNK) inhibitor SP600125 (10 µM; Calbiochem) (20), p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580 (10 µM; Calbiochem) (21), or extracellular signal-regulated kinase (ERK)1/2 inhibitor PD98059 (25 µM; Calbiochem) (19) for 60 min before irradiation.

The concentrations of IL-6, TGF-beta1, tumor necrosis factor (TNF)-{alpha}, and IL-1beta were measured by ELISA kit (BioSource, Camarillo, CA) in the culture supernatants with or without ATRA (10–6 M) for 24 h.

Animal Studies
C57BL/6 female mice were purchased from Japan SLC (Shizuoka, Japan) and maintained in our specific pathogen–free animal facility. All animals were kept according to the Animal Protection Guidelines of Kyoto University. All protocols for animal use and euthanasia were reviewed and approved by the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Japan. After anesthesia with pentobarbital, gamma-ray radiation was administered as a single dose (20 Gy) to the entire thorax of 8-wk-old mice. Twelve-week-old mice were injected intraperitoneally with bleomycin sulfate (2 mg/mouse/d; Nippon Kayaku, Tokyo, Japan) on Days 1 and 8. In some experiments, mice were injected intraperitoneally with 0.5 mg of ATRA dissolved in 0.1 ml cottonseed oil or with 0.1 ml cottonseed oil alone (control mice). Injections were repeated three times weekly, as follows: (1) throughout the course, (2) for the first half period, or (3) for the later half period. The mice were killed at 16 and 22 wk after irradiation, or at 4 wk after bleomycin treatment, and histologic examination was performed by staining with hematoxylin and eosin or Azan. For a quantitative analysis of severity of fibrosis, the level of fibrosis was measured as a blue pixel number using Azan staining by the Analytical Digital Photomicroscopy technique using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).

Quantitative Real-Time Reverse Transcriptase–Polymerase Chain Reaction
RNA preparation and quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) were performed as previously described (4) using TaqMan gene expression products for human TGF-beta1, mouse IL-6, TGF-beta1, and collagen type I{alpha}1 (COL1A1). 18SrRNA served as an endogenous control (Applied Biosystems, Foster, CA).

Collagen Protein Measurement
IMR90 cells were 8-Gy irradiated and cultured in the presence or absence of ATRA, and after 7-d culture, cells were collected and examined for the amount of collagen using Sircol soluble collagen assay kit (Biocolor, Belfast, North Ireland).

Immunofluorescence Staining
Immunofluorescence staining was performed as previously described (4). The fixed cells were stained with mouse anti-human {alpha}-smooth muscle actin ({alpha}-SMA) monoclonal antibody (1:100; Sigma) followed by rhodamin-conjugated donkey anti-mouse antibody (1:1,000) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Hoechst 33258 fluorochrome (Sigma) was used for nuclear staining. The exposure time of fluorescence microscopy was fixed to 1.5 s.

Measurement of NF-{kappa}B p65 and p38MAPK
After treatment, nuclear and cytoplasmic extracts were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA), and nuclear NF-{kappa}B p65 and cytoplasmic phospho-p38MAPK (pThr180/pThr182) were detected by ELISA Kit (BioSource and Sigma, respectively).

Statistical Analysis
Results are given as the mean ± SD of values. Statistical analysis was performed using Bonferroni/Dunn multiple comparisons test. The estimate of the probability of survival was calculated by the Kaplan-Meier method and compared using the log-rank test.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Systemic Administration of ATRA on Survival Rate in Irradiated Mice
Eight-week-old mice received 20 Gy irradiation selectively to the thoracic field, with (n = 10) or without (n = 11) ATRA (0.5 mg/mouse) intraperitoneally administered three times per week during the time course. After irradiation, the survival rate at 20 d in mice that received ATRA was 73.3%, and 76.9% in mice that did not receive ATRA. The main causes of death in the early phase were probably acute lung injury or bone marrow failure, which were not related to the addition of ATRA. However, the mice without ATRA started to die after the 12th week, leading to a survival rate at the 16th week of 30%; however, this rate was 81.8% in ATRA-treated mice (p = 0.0412; Figure 1).


Figure 1
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  		Figure 1. Effect of all-trans-retinoic acid (ATRA) on the survival of mice after gamma-ray irradiation was administered as single dose (20 Gy) selectively to the entire thorax of 8-wk-old mice (n = 10 and 11, with and without ATRA, respectively). ATRA (0.5 mg/mouse) dissolved in 0.1 ml cottonseed oil or 0.1 ml cottonseed oil alone (control mice) was administered intraperitoneally three times per week during the time course.

 
Histologic Changes in Irradiated Mouse Lung with or without ATRA
The histologic changes at 16 wk are shown in Figure 2A. In the lung tissues from the irradiated mice without ATRA, pulmonary interalveolar septa became thickened and infiltrated by inflammatory cells, with collagen depositions in the interstitium disclosed by Azan staining that were far less remarkable than in those treated with ATRA. Mice treated by ATRA without irradiation showed no changes at all (data not shown).


Figure 2
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  		Figure 2. Effect of ATRA in irradiation-treated mouse lung. (A) Mice (n = 3 in each experiment) were killed at 16 wk after irradiation in the presence or absence of ATRA and histologic changes in the lung tissues were shown by hematoxylin and eosin and Azan staining (original magnification, x100). (B–D) Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to determine the changes in mRNA levels from the mouse lung tissues for (B) interleukin (IL)-6, (C) transforming growth factor (TGF)-beta1, and (D) collagen type I{alpha}1 (COL1A1), as described in METHODS. The levels of mRNA are represented as the ratio to 18SrRNA. The results are indicated as the mean ± SD of three separate experiments.

 
Levels of mRNA of IL-6, TGF-beta1, and COL1A1 in Mouse Lung Tissues
We previously reported that irradiation increased IL-6 production from cultured lung fibroblasts and lung tissues of mice, and that this could be reduced by the addition of ATRA (4). In this report, mRNA levels of IL-6 from lung tissues of mice 16 wk after 20 Gy irradiation were analyzed and shown to be approximately 15-fold elevated by real-time RT-PCR compared with control mice, which were significantly suppressed by the administration of ATRA (p < 0.0001) (Figure 2B).

TGF-beta1 is a well-known key cytokine in the process of human pulmonary fibrogenesis (22). We examined the effect of irradiation and ATRA on TGF-beta1 production in mouse lung tissues. The mRNA levels of TGF-beta1 (Figure 2C) and COL1A1 (Figure 2D), which reflect collagen synthesis, in the lung tissues of irradiated mice were highly elevated (approximately six- and twofold, respectively) compared with control mice without irradiation and ATRA. Notably, in irradiated mice treated with ATRA, the levels were markedly decreased (p < 0.0001 and p = 0.0001, respectively). Lung tissues from mice treated with ATRA without irradiation showed no changes in IL-6, TGF-beta1, and COL1A1 mRNA levels in comparison with control mice (data not shown).

Effect of ATRA on TGF-beta1 Production from Human Lung Fibroblasts
In vitro, we previously reported the inhibitory effect of ATRA on IL-6 production and proliferation of lung fibroblasts after irradiation. Thus, we performed experiments to examine the effect of ATRA on TGF-beta1 production of lung fibroblasts. IMR90 cells, human fetal lung fibroblasts, received 8 Gy irradiation with or without pretreatment with ATRA, and the concentration of TGF-beta1 in the culture supernatant and mRNA levels were measured 24 and 6 h later, respectively. The concentration of TGF-beta1 in the culture supernatant was increased (~ 1.3-fold) by irradiation, whereas this was decreased by the addition of ATRA (p = 0.0016; Figure 3A). The mRNA levels of TGF-beta1 were also increased (~ 2.5-fold) by irradiation, which was inhibited by ATRA (p < 0.0001; Figure 3B). On the other hand, neither irradiation nor ATRA had effect on the production of IL-6 or TGF-beta1 in Beas2B cells, HPAECs, and LECs (data not shown).


Figure 3
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  		Figure 3. Effect of ATRA on irradiation-induced TGF-beta1 production in lung fibroblasts. (A) TGF-beta1 concentration in the supernatants of confluent IMR90 cells, cultured in the presence or absence of 10–6 M ATRA for 24 h with or without 8 Gy irradiation, was measured by ELISA. At the time of harvest, cells were counted and the TGF-beta1 values were corrected as proportion to cell numbers. The results are indicated as the mean ± SD of three separate experiments. (B) IMR90 cells were cultured as in A for 6 h, and real-time RT-PCR was performed to determine the changes in mRNA levels. The levels of mRNA are represented as the ratio to 18SrRNA. The results are indicated as the mean ± SD of three separate experiments.

 
Effect of ATRA on Collagen Synthesis and Transdifferentiation of Lung Fibroblasts after Irradiation
TGF-beta1 is known to promote collagen synthesis by transdifferentiation of fibroblasts to myofibroblasts (23). We next examined the impact of irradiation on collagen synthesis, and transdifferentiation of IMR90 cells. As shown in Figure 4, in Sircol assay, increased synthesis (~ 1.4-fold) of collagen by irradiation was suppressed by ATRA (p < 0.0001). To study whether ATRA influenced this transdifferentiation process as well as cell proliferation, which we previously demonstrated (4), we examined the expression of {alpha}-SMA in cultured IMR90 cells. Although the cytoplasmic expression of {alpha}-SMA was increased by irradiation in a dose-dependent manner (Figure 5A), this was decreased by the addition of ATRA (p < 0.0001) (Figures 5B and 5C).


Figure 4
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  		Figure 4. Effect of irradiation and ATRA on collagen synthesis in lung fibroblasts. IMR90 cells were cultured in the presence or absence of 10–6 M ATRA for 7 d with or without 8 Gy irradiation, and cells were collected and examined for the amount of collagen using Sircol soluble collagen assay.

 

Figure 5
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  		Figure 5. Inhibitory effect of ATRA on irradiation-induced transdifferentiation of lung fibroblasts. (A) Dose-dependent stimulatory effect of irradiation on transdifferentiation of IMR90 cells. Cells were irradiated in the indicated doses, cultured for 7 d, and fixed. Immunofluorescence staining for expression of cytoplasmic {alpha}-smooth muscle actin ({alpha}-SMA) was performed as described in METHODS. (B) Inhibitory effect of ATRA on irradiaton-induced {alpha}-SMA expression in IMR90 cells. Cells received 8 Gy irradiation and were cultured in the presence or absence of 10–6 M ATRA for 7 d, and the cytoplasmic expression of {alpha}-SMA was demonstrated. (C) Representative immunofluorescence staining for B.

 
Involvement of NF-{kappa}B and p38MAPK in Irradiation-induced TGF-beta1 Production
To study the underlying mechanism of irradiation-induced stimulation of TGF-beta1 production, we asked whether any kinases are required for this process. The pretreatment with inhibitors for NF-{kappa}B or p38MAPK led to a decrease both in the concentrations of TGF-beta1 in the culture supernatant and in TGF-beta1 mRNA levels back to the control level (Figures 6A and 6B), whereas neither of the inhibitors for JNK or ERK1/2 affected the irradiation-induced TGF-beta1 production (data not shown). The level of nuclear NF-{kappa}B p65 was increased 1.4-fold by irradiation, which was suppressed by p38MAPK inhibitor as well as ATRA (p = 0.0022 and p = 0.0025, respectively; Figure 6C). The irradiation increased the level of phospho-p38MAPK (6.4-fold), which was decreased by ATRA (p < 0.0001). The addition of NF-{kappa}B inhibitor, on the other hand, had no effect on irradiation-induced increase of phospho-p38MAPK (Figure 6D). SB203580 inhibited the irradiation-induced p38MAPK activation (data not shown).


Figure 6
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  		Figure 6. Inhibitory effect of inhibitors for nuclear factor (NF)-{kappa}B or p38MAPK on irradiation-induced TGF-beta1 production. (A, B) IMR90 cells were cultured with or without 8 Gy irradiation in the presence or absence of 5 µM of MG-132 (inhibitor for NF-{kappa}B) or 10 µM of SB203580 (inhibitor for p38MAPK) for (A) 24 or (B) 6 h. ELISA and real-time RT-PCR were performed to determine the changes in (A) the concentration of TGF-beta1 in the culture supernatant and (B) TGF-beta1 mRNA levels after irradiation. (C) The activities of NF-{kappa}B were analyzed as described in METHODS. IMR90 cells were pretreated with or without ATRA (10–6 M) or SB203580 (10 µM), irradiated (8 Gy) cells were cultured for 2 h, and p65 amounts in nuclear protein extracts were analyzed. The results are indicated as the mean ± SD of three separate experiments. (D) The activities of p38MAPK were analyzed as described in METHODS. IMR90 cells were pretreated with or without ATRA (10–6 M) or MG-132 (5 µM), irradiated (8 Gy) cells were cultured for 2 h, and phospho-p38MAPK amounts in cytoplasmic protein extracts were analyzed. The results are indicated as the mean ± SD of three separate experiments.

 
Prevention of Bleomycin-induced Lung Fibrosis by ATRA
We next examined the effect of ATRA on bleomycin-induced lung fibrosis in mice as previously reported by Khalil and colleagues (24). As shown in Figure 7A, intraperitoneal administration of ATRA three times per week inhibited the collagen deposition in the bleomycin-treated mouse lung tissues. Elevated mRNA levels of IL-6 (Figure 7B), TGF-beta1 (Figure 7C), and COL1A1 (Figure 7D) (~ 1.5-, 1.5-, and 3-fold, respectively) in bleomycin-treated mouse lung tissues decreased after the treatment with ATRA (p = 0.0022, p = 0.0002, and p < 0.0001, respectively).


Figure 7
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  		Figure 7. Effect of ATRA on bleomycin-induced lung fibrosis. (A) 12-wk-old mice were injected intraperitoneally with bleomycin sulfate (Bleo; 2 mg/mouse/d) on Days 1 and 8. Administration of ATRA was performed in the same way as in irradiated mice. On Day 28, mice (n = 3 in each experiment) were killed and histologic changes were demonstrated by hematoxylin and eosin and Azan staining (original magnification, x100). (B–D) Real-time RT-PCR was performed to determine the changes in mRNA levels from lung tissues of mice with or without bleomycin and ATRA for (B) IL-6, (C) TGF-beta1, and (D) COL1A1. The levels of mRNA are represented as the ratio to 18SrRNA. The results are indicated as the mean ± SD of three separate experiments.

 
Early and Late Preventive Effects of ATRA on Irradiation- and Bleomycin-induced Lung Fibrosis
To study the "preventive" and "therapeutic" effects of ATRA on lung fibrosis, we examined the early (probably the inflammatory responses are mainly demonstrated) and late (probably the postinflammatory, fibrotic changes are mainly observed) effects of ATRA by transient administration to both irradiation- and bleomycin-treated mice. Mice received 20 Gy irradiation to the thoracic field, with or without ATRA intraperitoneally administered in three ways, as follows: (1) for the first 8 wk, (2) for the last 14 wk, and (3) throughout the course. Mice were killed at 22 wk after irradiation and the level of fibrosis was measured as the blue pixel number using Azan staining by the Analytical Digital Photomicroscopy technique. The results were the same in that the three administration courses led to the significant suppression of fibrosis in the lung (Figure 8A). In addition, mice were killed at 28 d after injection intraperitoneally with bleomycin in the absence or presence of ATRA, and the level of fibrosis was measured as above. ATRA was administrated in three ways: (1) for the first 14 d, (2) for the last 14 d, and (3) throughout the course, but again all administration courses resulted in the inhibition of lung fibrosis (Figure 8B). Taken together, these results demonstrate the "late," namely "therapeutic," effect of ATRA in both irradiation- and bleomycin-induced lung fibrosis models, in addition to the "early," namely "preventive," effect.


Figure 8
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  		Figure 8. Both early and late preventive effects of ATRA on irradiation- and bleomycin-induced lung fibrosis. (A) Mice (n = 3 in each experiment) were killed at 22 wk after irradiation in the presence or absence of ATRA and the level of fibrosis was measured as the blue pixel number in Azan staining by the Analytical Digital Photomicroscopy technique using Adobe Photoshop. ATRA was administrated in three ways: (1) for the first 8 wk, (2) for the last 14 wk, and (3) throughout the course. (B) Mice (n = 3 in each experiment) were killed at 28 d after injection intraperitoneally with bleomycin sulfate on Days 1 and 8 in the presence or absence of ATRA, and the level of fibrosis was measured as in A. ATRA was administrated in three ways: (1) for the first 14 d, (2) for the last 14 d, and (3) throughout the course.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed preventive and therapeutic effects of ATRA on the progression of lung fibrosis both in irradiated and in bleomycin-treated mice. Increased IL-6 and TGF-beta1 mRNA levels in the lung tissues of both models were inhibited by ATRA. ATRA also inhibited the irradiation-induced transdifferentiation process, in addition to the inhibitory effect on irradiation-induced cell proliferation as we previously reported (4). Taken together, ATRA appears to exert its effect through inhibition of both proliferation and differentiation of lung fibroblasts and prevents radiation- and bleomycin-induced pulmonary fibrosis in mice.

Several factors have been reported to be released from irradiated cells, including TGF-beta1, TNF-{alpha}, IL-1 and IL-6, PDGF, vascular endothelial growth factor, and fibroblast growth factor (25–31). Among these cytokines, the levels of IL-6 and TGF-beta1 appear to serve as a predictor for the occurrence of radiation pneumonitis (1–3). In fact, it has been shown that these levels in BAL fluid collected from irradiated areas increased progressively during lung irradiation, suggesting more direct local effects on the tissues (32). In this study, the IL-6 mRNA levels in the irradiated mouse lung tissues were markedly increased as compared with control, and the levels decreased by the addition of ATRA as shown in Figure 2B, which was compatible with our previous report (4). IL-6 stimulated proliferation of fibroblasts in a dose-dependent manner, and this effect was overcome by ATRA. On the other hand, it is well known that fibroblasts transdifferentiate to myofibroblasts, which express elevated levels of {alpha}-SMA, and consequently, display a markedly enhanced ability to contract extracellular matrix (33, 34). In this study, the expression of {alpha}-SMA was increased by irradiation in a dose-dependent manner, with a maximum effect at 8 Gy, which was the dose used throughout this study. As shown in Figure 5, ATRA decreased irradiation-induced {alpha}-SMA expression. Interestingly, IL-6 did not induce transdifferentiation of fibroblasts, whereas it was induced by TGF-beta1 (data not shown). In addition, irradiation increased the level of TGF-beta1 mRNA in mouse lung tissues, which was decreased by the addition of ATRA. There have been some reports about interaction between IL-6 and TGF-beta1 (35–37). However, IL-6 failed to induce TGF-beta1 production and vice versa in IMR90 cells (data not shown). Also, using ELISA assays, TNF-{alpha} and IL-1beta, both of which are cytokines reported to stimulate IL-6 (38, 39) and TGF-beta1 production (40–42), were undetected in irradiation-treated and untreated culture supernatants of IMR90 cells (data not shown). Because TGF-beta1 does not stimulate the proliferation of {alpha}-SMA–positive myofibroblasts, we propose a dual inhibitory effect of ATRA on IL-6–dependent proliferation and TGF-beta1–dependent transdifferentiation of fibroblasts, which may be the mechanism underlying the preventive and therapeutic effect of ATRA on lung fibrosis. Although there are some other cells that may contribute to the fibrogenesis in the lung, such as endothelial cells and epithelial cells, the concentration of IL-6 or TGF-beta1 in culture supernatants of Beas2B cells, HPAECs, and LECs was not affected by irradiation or addition of ATRA (data not shown).

To determine the cellular mechanism in the regulation of TGF-beta1 production by irradiation, we used some of the well-characterized pharmacologic inhibitors (40). It has been reported that exposure of cells to ionizing radiation induces simultaneous compensatory activation of NF-{kappa}B (43) and multiple MAPK pathways (44, 45). There are at least three distinct MAPK signal transduction pathways in mammalian cells that lead to activation of the ERK, JNK, and p38MAPK pathways. Here, we show the involvement of neither ERK nor JNK but the contribution of p38MAPK in the stimulation of TGF-beta1 production. Many signaling pathways can interact with each other through biochemical cross-talk, and the induction of most cytokine genes requires activation of NF-{kappa}B. However, such interactions between p38MAPK and NF-{kappa}B have not been fully investigated. In this study, we demonstrated that the inhibition of p38MAPK reduced irradiation-induced NF-{kappa}B p65 activation, whereas the inhibition of NF-{kappa}B had no effect on irradiation-induced p38MAPK activation (Figures 6C and 6D), which suggests that the activation of p38MAPK is necessary for NF-{kappa}B activation. These results suggest a possible mechanism where ATRA could reduce irradiation-induced TGF-beta1 production through a p38MAPK- and/or NF-{kappa}B–dependent pathway. These findings are summarized in Figure 9. As we previously reported, irradiation induced IL-6 production and cell proliferation of lung fibroblasts. ATRA reduced irradiation-induced IL-6 production through protein kinase C (PKC)-{delta}/NF-{kappa}B pathway. In the present study, we showed that irradiation stimulated TGF-beta1 production and accelerated transdifferentiation of fibroblasts. ATRA inhibited irradiation-induced TGF-beta1 production through the p38MAPK/NF-{kappa}B pathway, resulting in the inhibition of cell differentiation and collagen synthesis.


Figure 9
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  		Figure 9. Proposed mechanism for the inhibition of irradiation-induced fibrosis by ATRA. Irradiation stimulates IL-6 production through the protein kinase C (PKC)-{delta}/NF-{kappa}B pathway and IL-6–dependent proliferation of fibroblasts (demonstrated in our previous study). The production of TGF-beta1 is also increased by irradiation through p38MAPK and/or NF-{kappa}B pathway, and the fibroblasts are stimulated and differentiated to myofibroblasts by irradiation. ATRA activates PKC-{delta} and inhibits p38MAPK and/or NF-{kappa}B, resulting in the inhibition of both pathways and the decrease in collagen synthesis. Consequently, ATRA inhibits irradiation-induced lung fibrosis.

 
Pulmonary fibrosis is frequently associated with collagen diseases and rheumatoid arthritis, and with drugs including anticancer agents. We thus investigated another mouse model of lung fibrosis induced by bleomycin. We used a bleomycin model not as an "IPF model," which might be limited in the late postinflammatory phase of repair after injury, but as a model for drug-induced lung fibrosis, such as anticancer drugs. We treated mice with bleomycin by intraperitoneal administration instead of intratracheal administration as they are usually treated in many reports, because we wished to study the effect of the drug when added to the whole body since this administration better resembled the use of the drug in human cancer therapy. Pulmonary fibrosis is an inexorably progressive form of interstitial lung disease with various etiologies, including IPF. The median length of survival from the time of diagnosis of IPF varies between 2.5 and 3.5 yr, and there has been remarkably little progress in the development of effective therapeutic strategies (14, 16–18). Here, we report the preventive effect of ATRA on bleomycin-induced lung fibrosis in mice (Figure 7A). We also demonstrated that the treatment with bleomycin increased mRNA levels of both IL-6 and TGF-beta1. Although the involvement of TGF-beta1 in bleomycin-induced lung fibrosis is well known, we showed the effect of bleomycin on IL-6 production and the inhibitory effect of ATRA on the production of these causative cytokines. In addition, ATRA inhibited bleomycin-induced COL1A1 mRNA expression, which represents collagen synthesis. The precise cellular mechanism has not been fully investigated; however, we propose a possibility that IL-6 and TGF-beta1 play an important role in this mouse model and ATRA prevents lung fibrosis through the inhibition of these cytokines similarly to irradiation-induced lung fibrosis.

It has been previously reported that a PDGFR/cAbl/cKit kinase inhibitor, imatinib mesylate, was beneficial in the inhibition of lung fibrosis both by "antiinflammatory" and "antifibrotic" mechanisms in a rat bleomycin model (46). It is noteworthy that, in this report, we showed the late, namely therapeutic, effect of ATRA in both irradiation- and bleomycin-induced lung fibrosis models (Figure 8) in addition to the early, namely preventive, effect, because in clinical use, the therapeutic effect is often more important when the clinicians find that the fibrotic changes are already apparent in their patients after irradiation or that they are suffering from fibrosis of various etiologies.

ATRA is known to affect cell differentiation, proliferation, and development. There have been some reports demonstrating that ATRA induced the growth inhibition of IL-6–dependent myeloma cells (11–13) and small cell lung cancer cells (47, 48). Clinically, ATRA has been widely used in the differentiating therapy for acute promyelocytic leukemia (49). Furthermore, the oral administration of the drug results in good compliance. Our data may lead to the development of novel strategies incorporating ATRA in the prevention and treatment of various types of lung fibrosis.


   FOOTNOTES
 
Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology and the Special Coordination Funds for Promoting Science and Technology.

* These two authors contributed equally to this study. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200606-862OC on October 5, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 27, 2006; accepted in final form September 29, 2006


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anscher MS, Marks LB, Shafman TD, Clough R, Huang H, Tisch A, Munley M, Herndon IIJE, Garst J, Crawford J, et al. Using plasma transforming growth factor beta-1 during radiotherapy to select patients for dose escalation. J Clin Oncol 2001;19:3758–3765.[Abstract/Free Full Text]
  2. Chen Y, Rubin P, Williams J, Hernady E, Smudzin T, Okunieff P. Circulating IL-6 as a predictor of radiation pneumonitis. Int J Radiat Oncol Biol Phys 2001;49:641–648.[CrossRef][Medline]
  3. Chen Y, Williams J, Ding I, Hernady E, Liu W, Smudzin T, Finkelstein JN, Rubin P, Okunieff P. Radiation pneumonitis and early circulatory cytokine markers. Semin Radiat Oncol 2002;12:26–33.[CrossRef][Medline]
  4. Tabata C, Kubo H, Tabata R, Wada M, Sakuma K, Ichikawa M, Fujita S, Mio T, Mishima M. All-trans retinoic acid modulates radiation-induced proliferation of lung fibroblasts via IL-6/IL-6R system. Am J Physiol Lung Cell Mol Physiol 2006;290:597–606.
  5. Dubois C, Schlageter MH, de Gentile A, Balitrand N, Toubert ME, Krawice I, Fenaux P, Castaigne S, Najean Y, Degos L, et al. Modulation of IL-8, IL-1 beta, and G-CSF secretion by all-trans retinoic acid in acute promyelocytic leukemia. Leukemia 1994;8:1750–1757.[Medline]
  6. Hsu HC, Yang K, Kharbanda S, Clinton S, Datta R, Stone RM. All-trans retinoic acid induces monocyte growth factor receptor (c-fms) gene expression in HL-60 leukemia cells. Leukemia 1993;7:458–462.[Medline]
  7. Maeno T, Tanaka T, Sando Y, Suga T, Maeno Y, Nakagawa J, Hosono T, Sato M, Akiyama H, Kishi S, et al. Stimulation of vascular endothelial growth factor gene transcription by all trans retinoic acid through Sp1 and Sp3 sites in human bronchioloalveolar carcinoma cells. Am J Respir Cell Mol Biol 2002;26:246–253.[Abstract/Free Full Text]
  8. Matikainen S, Tapiovaara H, Vaheri A, Hurme M. Activation of interleukin-1 beta gene expression during retinoic acid-induced granulocytic differentiation of promyeloid leukemia cells. Cell Growth Differ 1994;5:975–982.[Abstract]
  9. Pelicano L, Li F, Schindler C, Chelbi-Alix MK. Retinoic acid enhances the expression of interferon-induced proteins: evidence for multiple mechanisms of action. Oncogene 1997;15:2349–2359.[CrossRef][Medline]
  10. Tkatch LS, Rubin KA, Ziegler SF, Tweardy DJ. Modulation of human G-CSF receptor mRNA and protein in normal and leukemic myeloid cells by G-CSF and retinoic acid. J Leukoc Biol 1995;57:964–971.[Abstract]
  11. Levy Y, Labaume S, Colombel M, Brouet JC. Retinoic acid modulates the in vivo and in vitro growth of IL-6 autocrine human myeloma cell lines via induction of apoptosis. Clin Exp Immunol 1996;104:167–172.[CrossRef][Medline]
  12. Ogata A, Nishimoto N, Shima Y, Yoshizaki K, Kishimoto T. Inhibitory effect of all-trans retinoic acid on the growth of freshly isolated myeloma cells via interference with interleukin-6 signal transduction. Blood 1994;84:3040–3046.[Abstract/Free Full Text]
  13. Sidell N, Taga T, Hirano T, Kishimoto T, Saxon A. Retinoic acid-induced growth inhibition of a human myeloma cell line via down-regulation of IL-6 receptors. J Immunol 1991;146:3809–3814.[Abstract]
  14. Coultas DB, Zumwalt RE, Black WC, Sobonya RE. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med 1994;150:967–972.[Abstract]
  15. Yoshida M, Sakuma J, Hayashi S, Abe K, Saito I, Harada S, Sakatani M, Yamamoto S, Matsumoto N, Kaneda Y, et al. A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta1, or platelet-derived growth factor B gene. Proc Natl Acad Sci USA 1995;92:9570–9574.[Abstract/Free Full Text]
  16. American Thoracic Society; European Respiratory Society. International multidisciplinary consensus classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2002;165:277–304.[Free Full Text]
  17. Gross TJ, Hunninghake GW. Idiopathic pulmonary fibrosis. N Engl J Med 2001;345:517–525.[Free Full Text]
  18. Mason RJ, Schwarz MI, Hunninghake GW, Musson RA. NHLBI workshop summary: pharmacological therapy for idiopathic pulmonary fibrosis: past, present, and future. Am J Respir Crit Care Med 1999;160:1771–1777.[Free Full Text]
  19. Jang BC. Jung TY, Paik JH, Kwon YK, Shin SW, Kim SP, Ha JS, Suh MH, Suh SI. Tetradecanoyl phorbol acetate induces expression of Toll-like receptor 2 in U937 cells: involvement of PKC, ERK, and NF-kappaB. Biochem Biophys Res Commun 2005;328:70–77.[CrossRef][Medline]
  20. Lee KY. Ito K, Hayashi R, Jazrawi EP, Barnes PJ, Adcock IM. NF-kappaB and activator protein 1 response elements and the role of histone modifications in IL-1beta-induced TGF-beta1 gene transcription. J Immunol 2006;176:603–615.[Abstract/Free Full Text]
  21. Fritz DK. Kerr C, Tong L, Smyth D, Richards CD. Oncostatin-M up-regulates VCAM-1 and synergizes with IL-4 in eotaxin expression: involvement of STAT6. J Immunol 2006;176:4352–4360.[Abstract/Free Full Text]
  22. Broekelmann TJ, Limper AH, Colby TV, McDonald JA. Transforming growth facter-beta is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 1991;88:6642–6646.[Abstract/Free Full Text]
  23. Grotendorst GR, Rahmanie H, Duncan MR. Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation. FASEB J 2004;18:469–479.[Abstract/Free Full Text]
  24. Khalil N, Whitman C, Zuo L, Danelpour D, Dreenberg A. Regulation of alveolar macrophage transforming growth factor-beta secretion by corticosteroids in bleomycin-induced pulmonary inflammation in rats. J Clin Invest 1993;92:1812–1818.[Medline]
  25. Hallahan DE, Spriggs DR, Beckett MA, Kufe DW, Weichselbaum RR. Increased tumor necrosis factor alpha mRNA after cellular exposure to ionizing radiation. Proc Natl Acad Sci USA 1989;86:10104–10107.[Abstract/Free Full Text]
  26. Rube CE, Uthe D, Schmid KW, Richter KD, Wessel J, Schuck A, Willich N, Rube C. Dose-dependent induction of transforming growth factor beta (TGF-beta) in the lung tissue of fibrosis-prone mice after thoracic irradiation. Int J Radiat Oncol Biol Phys 2000;47:1033–1042.[CrossRef][Medline]
  27. Sherman ML, Datta R, Hallahan DE, Weichselbaum RR, Kufe DW. Regulation of tumor necrosis factor gene expression by ionizing radiation in human myeloid leukemia cells and peripheral blood monocytes. J Clin Invest 1991;87:1794–1797.[Medline]
  28. Witte L, Fuks Z, Haimovitz-Friedman A, Vlodavsky I, Goodman DS, Eldor A. Effects of irradiation on the release of growth factors from cultured bovine, porcine, and human endothelial cells. Cancer Res 1989;49:5066–5072.[Abstract/Free Full Text]
  29. Woloschak GE, Chang-Liu CM, Jones PS, Jones CA. Modulation of gene expression in Syrian hamster embryo cells following ionizing radiation. Cancer Res 1990;50:339–344.[Abstract/Free Full Text]
  30. Abdollahi A, Li M, Ping G, Plathow C, Domhan S, Kiessling F, Lee LB, McMahon G, Grone HJ, Lipson KE, et al. Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis. J Exp Med 2005;201:925–935.[Abstract/Free Full Text]
  31. Kolb M. Margetts PJ, Anthony DC, Pitossi F, Gauldie J. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest 2001;107:1529–1536.[Medline]
  32. Barthelemy-Brichant N, Bosquée L, Cataldo D, Corhay JL, Gustin M, Seidel L, Thiry A, Ghaye B, Nizet M, Albert A, et al. Increased IL-6 and TGF-beta1 concentrations in bronchoalveolar lavage fluid associated with thoracic radiotherapy. Int J Radiat Oncol Biol Phys 2004;58:758–767.[CrossRef][Medline]
  33. Desmouliere A. Geinoz A, Gabbiani F, Gabbiani G. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993;122:103–111.[Abstract/Free Full Text]
  34. Sime PJ. Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 1997;100:768–776.[Medline]
  35. Gomes I. Mathur SK, Espenshade BM, Mori Y, Varga J, Ackerman SJ. Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis. J Allergy Clin Immunol 2005;116:796–804.[CrossRef][Medline]
  36. Elias JA. Lentz V, Cummings PJ. Transforming growth factor-beta regulation of IL-6 production by unstimulated and IL-1-stimulated human fibroblasts. J Immunol 1991;146:3437–3443.[Abstract]
  37. Zhang XL. Topley N, Ito T, Phillips A. Interleukin-6 regulation of transforming growth factor (TGF)-beta receptor compartmentalization and turnover enhances TGF-beta1 signaling. J Biol Chem 2005;280:12239–12245.[Abstract/Free Full Text]
  38. Brach MA, Gruss HJ, Kaisho T, Asano Y, Hirano T, Herrmann F. Ionizing radiation induces expression of interleukin 6 by human fibroblasts involving activation of nuclear factor-kappa B. J Biol Chem 1993;268:8466–8472.[Abstract/Free Full Text]
  39. Shibanuma M, Kuroki T, Nose K. Inhibition by N-acetyl-L-cysteine of interleukin-6 mRNA induction and activation of NF kappa B by tumor necrosis factor alpha in a mouse fibroblastic cell line, Balb/3T3. FEBS Lett 1994;353:62–66.[CrossRef][Medline]
  40. Sullivan DE. Ferris M, Pociask D, Brody AR. Tumor necrosis factor-alpha induces transforming growth factor-beta1 expression in lung fibroblasts through the extracellular signal-regulated kinase pathway. Am J Respir Cell Mol Biol 2005;32:342–349.[Abstract/Free Full Text]
  41. Sime PJ, Marr RA, Gauldie D, Xing Z. hewlwtt BR, Graham FL, Gauldie J. Transfer of tumor necrosis factor-{alpha} to rat lung induces severe pulmonary inflammation and patchy interstitial fibrogenesis with induction of transforming growth factor-beta1 and myofibloblasts. Am J Pathol 1998;153:825–832.[Abstract/Free Full Text]
  42. Phan SH, Gharaee-Kermani M, McGarry B, Kunkel S, Wolber FW. Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1beta and tumor necrosis factor-{alpha}. J Immunol 1992;149:103–106.[Abstract]
  43. Criswell T, Leskov K, Miyamoto S, Luo G, Boothman DA. Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation. Oncogene 2003;22:5813–5827.[CrossRef][Medline]
  44. Dent P, Yacoub A, Fisher PB, Hagan MP, Steven Grant S. MAPK pathways in radiation responses. Oncogene 2003;22:5885–5896.[CrossRef][Medline]
  45. Wang X. McGowan CH, Zhao M, He L, Downey JS, Fearns C, Wang Y, Huang S, Han J. Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest. Mol Cell Biol 2000;20:4543–4552.[Abstract/Free Full Text]
  46. Chaudhary NI, Schnapp A, Park JE. Pharmacologic differentiation of inflammation and fibrosis in the rat bleomycin model. Am J Respir Crit Care Med 2006;173:769–776.[Abstract/Free Full Text]
  47. Kalemkerian GP, Jasti RK, Celano P, Nelkin BD, Mabry M. All-trans-retinoic acid alters myc gene expression and inhibits in vitro progression in small cell lung cancer. Cell Growth Differ 1994;5:55–60.[Abstract]
  48. Weber E, Ravi RK, Knudsen ES, Williams JR, Dillehay LE, Nelkin BD, Kalemkerian GP, Feramisco JR, Mabry M. Retioic acid-mediated growth inhibition of small cell lung cancer cells is associated with reduced myc and increased p27Kip1 expression. Int J Cancer 1999;80:935–943.[CrossRef][Medline]
  49. Huang M, Ye Y, Chen S, Chai J, Lu J, Zhoa L, Gu L, Wang Z. Retioic acid-mediated growth inhibition of small cell lung cancer cells is associated with reduced myc and increased p27Kip1 expression. Blood 1988;72:567–572.[Abstract/Free Full Text]




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