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Published ahead of print on June 1, 2006, doi:10.1164/rccm.200509-1534OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 550-556, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200509-1534OC

Original Article

Gefitinib Prevents Bleomycin-induced Lung Fibrosis in Mice

Yoshiki Ishii, Sakae Fujimoto and Takeshi Fukuda

Department of Pulmonary Medicine and Clinical Immunology, Dokkyo Medical University School of Medicine, Mibu, Tochigi, Japan

Correspondence and requests for reprints should be addressed to Yoshiki Ishii, M.D., Ph.D., Department of Pulmonary Medicine and Clinical Immunology, Dokkyo Medical University School of Medicine, 800 Kitakobayashi, Mibu, Tochigi 321-0293, Japan. E-mail: ishiiysk{at}dokkyomed.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Transforming growth factor-{alpha} and epidermal growth factor (EGF), the ligands for EGF receptor (EGFR), stimulate fibroblast proliferation and play an important role in the pathogenesis of pulmonary fibrosis. Therefore, inhibition of the EGFR signal by an EGFR tyrosine kinase inhibitor (EGFR-TKI) may prevent pulmonary fibrosis. However, there is a possibility that blocking the EGFR signal may inhibit epithelial cell repair, thereby exaggerating lung fibrosis.

Objective: To investigate the effect of EGFR-TK inhibition on lung fibrosis.

Methods: We looked at the effects of the EGFR-TKIs gefitinib (20, 90, 200 mg/kg) and AG1478 (12 mg/kg) on a bleomycin-induced lung fibrosis model in mice.

Measurements and Main Results: Gefitinib prevented lung fibrosis at all three doses. Furthermore, in those mice that did not receive bleomycin treatment, gefitinib at 200 mg/kg did not induce lung fibrosis. Immunohistochemistry revealed that phosphorylation of EGFR in lung mesenchymal cells induced by bleomycin was inhibited by gefitinib. AG1478 also attenuated the lung fibrosis. In vitro studies further demonstrated that the addition of gefitinib or AG1478 suppressed the EGFR ligand–induced proliferation of lung fibroblasts.

Conclusions: These findings suggest that, in the preclinical setting, EGFR-TKIs may have a protective effect on lung fibrosis induced by bleomycin. Because these molecular targeted drugs may have differing effects depending on species and individuals, a cautious interpretation is warranted.

Key Words: epidermal growth factor • EGF receptor tyrosine kinase inhibitor • fibroblasts • interstitial lung disease • molecular targeted drug

Molecular targeted drugs have been attracting a great deal of attention as novel cancer therapies, with the goal of inhibition of cancer cell proliferation by the suppression of growth signals through growth factor receptor tyrosine kinase inhibition (1, 2). Growth factor receptors are located not only in cancer cells but also in normal cells, playing a role in cell proliferation. The main pathologic feature of idiopathic pulmonary fibrosis is proliferation of fibroblasts stimulated with various growth factors. Therefore, inhibition of growth factor receptor signaling in fibroblasts may be useful in the treatment of fibrosis. Growth factors in fibroblasts include transforming growth factor-{alpha} (TGF-{alpha}) and epidermal growth factor (EGF) as well as TGF-beta, platelet-derived growth factor, and insulin-like growth factor-1. There are many reports indicating that TGF-{alpha} and EGF, ligands for EGF receptor (EGFR), play an important role in the pathogenesis of pulmonary fibrosis. TGF-{alpha} was increased in the bronchoalveolar lavage of patients with idiopathic pulmonary fibrosis and was immunolocalized to type II epithelial cells, fibroblasts, and the vascular endothelium (3). Expression of TGF-{alpha} and EGFR mRNA was also increased in fibrotic lung tissue after bleomycin-induced lung injury in rats (4). Furthermore, conditional expression of TGF-{alpha} caused pulmonary fibrosis in transgenic mice (5), whereas TGF-{alpha} deficiency reduced pulmonary fibrosis in TGF-{alpha} knockout mice (6). An in vitro study also showed that the ligands stimulated fibroblast proliferation (7). Therefore, blocking EGFR-mediated signaling by EGFR tyrosine kinase inhibitors (EGFR-TKIs) could be useful in the treatment of pulmonary fibrosis. In fact, the EGFR-TKI AG1478 reduced the pulmonary fibrosis induced by vanodium pentoxide in rats (8).

However, interstitial pneumonia and acute lung injury have been reported in approximately 5.8% of Japanese patients (with a mortality rate of 2.3%) treated with another EGFR-TKI, gefitinib (9–12), which is already used clinically for lung cancer therapy (2, 13). It is unclear whether the injury is caused by the inhibition of EGFR signaling or by another mechanism possibly not related to gefitinib. In addition, interstitial lung disease (ILD) is a condition that may be associated with lung cancer itself (14). In a study using a murine model of bleomycin-induced pulmonary fibrosis, gefitinib at a dose of 200 mg/kg (a dose close to the maximum tolerated dose and the highest dose used in xenograft models [15]) augmented the lung fibrosis (16). Therefore, in this study, we looked at the inhibitory effect of three doses of gefitinib on bleomycin-induced lung fibrosis; considering that the minimum inhibitory concentration for transplanted tumors in nude mice is 12.5 mg/kg (15), we chose 20 mg/kg as a probable effective dose for EGFR-TK inhibition, 200 mg/kg as a dose 10 times that of the effective dose, and 90 mg/kg as an intermediate dose. The effect of gefitinib was also compared with AG1478 in the same experimental system.

Some of the results of these studies have been previously reported in the form of an abstract (17).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Additional details are provided in the online supplement.

Fibroblast Proliferation Assay
Proliferation of human fetal lung fibroblasts (HFL-1) in response to TGF-{alpha} and EGF was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation using a cell proliferation ELISA kit (Roche Diagnostics Corporation, Indianapolis, IN). Gefitinib (10–6 M; AstraZeneca, Osaka, Japan), AG1478 (10–6M; Calbiochem, La Jolla, CA), or vehicle was added to the cultures 30 min before addition of the growth factors.

Animal Treatment
Bleomycin (3 mg/kg; Nippon Kayaku Co., Tokyo, Japan) was intratracheally administered in 60 µl saline to the male C57BL/6 mice (8–10 wk old; Japan Clea, Tokyo, Japan). On Days 3, 7, and 14 after bleomycin treatment, the animals were killed and the lungs were removed en bloc. Animals were allocated to seven groups, as follows: (1) intratracheal saline + vehicle givenally, (2) intratracheal saline + 200 mg/kg of oral gefitinib, (3) intratracheal bleomycin + oral vehicle, (4) intratracheal bleomycin + 20 mg/kg of oral gefitinib, (5) intratracheal bleomycin + 90 mg/kg of oral gefitinib, (6) intratracheal bleomycin + 200 mg/kg of oral gefitinib, (7) intratracheal bleomycin + 12 mg/kg of intraperitoneal AG1478. Gefitinib suspension in 1% Tween 80 (0.2 ml) was given daily by gavage from Day 1 to Day 13; AG1478 was given intraperitoneally at a daily dose of 12 mg/kg in dimethyl sulfoxide solution from Day 1 to Day 13. For the saline and the bleomycin control groups (groups 1 and 3), a daily dose of vehicle (1% Tween 80 solution) was given orally. All experiments were performed in accordance with National Institutes of Health guidelines and protocols approved by the Dokkyo Medical University School of Medicine Subcommittee on Research Animal Care.

Histologic Evaluation
The right lung was fixed in 10% buffered formalin, and stained with hematoxylin and eosin and Masson's trichrome. Histologic grading of fibrosis was performed by three experienced histopathologists using a blinded semiquantitative scoring system for extent and severity of fibrosis in lung parenchyma based on previous studies (18, 19) with modifications. Severity of fibrosis was scored according to the method of Ashcroft and colleagues (20), with minor modifications as follows: The area of the fibrosis field for each grade and the ratio to the entire field of the section were calculated using a film scanner and the NIH Image software (National Institutes of Health, Bethesda, MD). The sum of the product of ratio multiplied by the grade was used as the score for each section. The mean score of the four sections was considered as the fibrosis score for the animal.

Collagen Assay
The left lung was homogenized and the collagen content determined using the Sircol Collagen Assay kit (Biocolor Ltd., Belfast, Northern Ireland) (21).

Immunohistochemistry
Lung tissues were prepared according to the Amex method (22). Sections taken from paraffin-embedded samples were immunostained for EGFR and phosphorylated EGFR by the labeled streptavidin-biotin (LSAB) method using a Dako LSAB+/HRP kit (Dako-Cytomation, Glostrup, Denmark) (23). To evaluate fibroblast proliferation and expression of EGFR on fibroblasts, lungs were double-immunostained for fibroblast-specific marker S100A4 (24) and EGFR. For the representative samples, immunofluorescent double-staining for S100A4 and EGFR was also performed. For a semiquantitative analysis of receptor expression, more than 500 cells per immunostained section were observed to count positive cells. The labeling index was calculated as follows: labeling index (%) = positive cells/all counted cells x 100.

Statistical Analysis
Data are expressed as means ± SEM. Statistical significance was determined by one-way analysis of variance or t test. p values less than 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Cell Proliferation Assay
We examined the effect of gefitinib and AG1478 on EGF ligand–induced HFL-1 cell proliferation in vitro. TGF-{alpha} and EGF stimulated proliferation of the cells. The addition of gefitinib or AG1478 significantly inhibited the growth of the cells induced by TGF-{alpha} or EGF in a dose-dependent manner (Figures 1 and 2).


Figure 1
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  		Figure 1. Gefitinib and AG1478 inhibited in vitro transforming growth factor (TGF)-{alpha}–stimulated fibroblast proliferation. Cell proliferation was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. BrdU absorbance (OD) was measured at 450 nm with 690 nm as reference wavelength. (n = 6 in each group; *p < 0.01 vs. TGF-{alpha}, 0 ng/ml; #p < 0.01 vs. vehicle.)

 

Figure 2
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  		Figure 2. Gefitinib and AG1478 inhibited in vitro epidermal growth factor (EGF)–stimulated fibroblast proliferation. Cell proliferation was determined by BrdU incorporation. (n = 6 in each group; *p < 0.01 vs. EGF, 0 ng/ml; #p < 0.01 vs. vehicle.)

 
Bleomycin-induced Pulmonary Fibrosis
Histologic examination of mouse lung revealed that bleomycin induced marked inflammatory cell infiltration with fibrosis in the lungs (Figure 3). The fibrosis score for mice given gefitinib 200 mg/kg without bleomycin treatment showed no significant change. In bleomycin-treated mice, gefitinib at doses of 20, 90, and 200 mg/kg significantly prevented the bleomycin-induced lung fibrosis (Figures 3 and 4). There was no significant difference in the inhibitory effect of the inhibiters at any dose. AG1478 also attenuated the lung fibrosis (Figures 3 and 4). The two EGFR-TKIs also significantly reduced the bleomycin-induced lung collagen accumulation (Figure 5).


Figure 3
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  		Figure 3. Representative pathologic findings of lung tissue by Masson's trichrome stain obtained 14 d after bleomycin instillation. (A) Vehicle given orally with saline instillation. (B) Vehicle given orally with bleomycin instillation. (C) Gefitnib 200 mg/kg given orally with bleomycin instillation. (D) Intraperitoneal AG1478 with bleomycin instillation. Mice were given orally the vehicle alone or gefitinib (200 mg/kg) 1 h before and on Days 1–13 after an intratracheal injection of bleomycin (3 mg/kg). AG1478 (12 mg/kg) was injected intraperitoneally 1 h before bleomycin instillation and on Days 1–13. Original magnification, x100.

 

Figure 4
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  		Figure 4. Fibrosis scoring based on severity and area of fibrosis. The fibrosis score for gefitinib 200 mg/kg without bleomycin showed no significant change. Gefitinib at doses of 20, 90, and 200 mg/kg significantly prevented the bleomycin-induced lung fibrosis. AG1478 also prevented the fibrosis. (n = 6–8 in each group; *p < 0.001 vs. saline + vehicle, #p < 0.001 vs. bleomycin + vehicle.) AG = AG1478 12.5 mg/kg; BLM = bleomycin; G20 = gefitinib 20 mg/kg; G90 = gefitinib 90 mg/kg; G200 = gefitinib 200 mg/kg; Veh = vehicle.

 

Figure 5
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  		Figure 5. Collagen content in lung tissues. Bleomycin augmented the lung collagen content. Gefitinib at doses of 20, 90, and 200 mg/kg significantly prevented the increased collagen content. AG1478 also prevented the collagen content. (n = 6–8 in each group; *p < 0.001 vs. saline + vehicle, #p < 0.001 vs. bleomycin + vehicle.) AG = AG1478 12.5 mg/kg; BLM = bleomycin; G20 = gefitinib 20 mg/kg; G90 = gefitinib 90 mg/kg; G200 = gefitinib 200 mg/kg; Veh = vehicle.

 
Gefitinib Inhibited Bleomycin-induced Phosphorylation of EGFR
Immunohistologic examination was conducted to confirm changes of EGFR and phosphorylated EGFR expression in the lung during the bleomycin-induced fibrosis process and to confirm that phosphorylation of EGFR could be inhibited by gefitinib. EGFR expression in the lung of the control group was positive in about 15% of cells, mainly epithelial cells and interstitial cells. The expression was not augmented by bleomycin, and a slight decrease in labeling index was observed after 14 d (Figure 6). On the other hand, phosphorylated EGFR expression was significantly increased 3 d after bleomycin treatment in the epithelial cells of fibrotic lung tissue and in interstitial fibroblast-like cells, compared with the control. Gefitinib significantly reduced this expression (Figures 7 and 8). There was a decreasing tendency of phosphorylated EGFR expression with time; however, even on Day 7, gefitinib significantly reduced the phosphorylation compared with the bleomycin control group (Figure 8).


Figure 6
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  		Figure 6. Immunohistochemical labeling index of EGFR expression on lung tissues after the intratracheal instillation of bleomycin. (n = 3–6; *p < 0.05, ** p < 0.01 vs. control.) BLM = bleomycin; G20 = gefitinib 20 mg/kg; G200 = gefitinib 200 mg/kg.

 

Figure 7
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  		Figure 7. Immunohistochemical labeling index of phosphorylated EGFR expression on lung tissues after the intratracheal instillation of bleomycin. (n = 3–6; *p < 0.05 vs. control, #p < 0.05 vs. bleomycin, ##p < 0.02 vs. bleomycin.) BLM = bleomycin; G20 = gefitinib 20 mg/kg; G200 = gefitinib 200 mg/kg.

 

Figure 8
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  		Figure 8. Representative findings on immunohistochemical staining for phosphorylated EGFR. Lung tissues were obtained from mice 7 d after intratracheal instillation of bleomycin. Control mice were given a saline instillation (A). Mice were treated with orally given vehicle (B) or gefitinib (200 mg/kg; C) after bleomycin instillation. There was no specific staining in preparations incubated with control IgG (D).

 
Fibroblast Proliferation and EGFR Expression on Fibroblasts In Vivo
To determine whether the bleomycin treatment induces fibroblast proliferation and whether EGFR is expressed in fibroblasts cells in vivo, lungs were double-immunostained for fibroblast-specific marker S100A4 and EGFR. The number of S100A4-positive cells was significantly increased with time at Days 3, 7, and 14 in the bleomycin group as compared with the control group (Figure 9). Gefitinib treatment at doses of 20 and 200 mg/kg significantly attenuated the bleomycin-induced increase in S100A4-positive cell number (Figure 9). Double-inmmunostaining clearly revealed that EGFR is expressed on the S100A4-positive fibroblasts (Figures 10 and 11). The percentage of EGFR-positive cells in S100A4-positive cells (EGFR labeling index) was about 10% in the control untreated lungs (Figure 12). Bleomycin treatment slightly increased this index up to 16% on Day 3, but the index was not altered on Days 7 and 14 (Figure 12).


Figure 9
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  		Figure 9. Number of S100A4-positive cells in the lung tissues after the intratracheal instillation of bleomycin. Quantification was performed by counting the numbers of positive cells/mm2 in a minimum of three randomly chosen microscopic fields. (n = 3–6; *p < 0.01 vs. control, #p < 0.01 vs. bleomycin). BLM = bleomycin; G20 = gefitinib 20 mg/kg; G200 = gefitinib 200 mg/kg.

 

Figure 10
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  		Figure 10. Representative findings on double immunohistochemical staining for fibroblast-specific marker S100A4 (gray/black) and EGFR (pink/red). Lung tissues were obtained from mice 14 d after intratracheal instillation of bleomycin. Control mice were given a saline instillation (A). Mice were treated with orally given vehicle (B) or gefitinib (200 mg/kg; C) after bleomycin instillation. There was no specific staining in preparations incubated with control IgG (D).

 

Figure 11
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  		Figure 11. Representative findings on double immunofluorescence staining for fibroblast-specific marker S100A4 and EGFR. Lung tissues were obtained from mice 14 d after intratracheal instillation of bleomycin. (A) Samples were stained with anti-S100A4 antibody (green). (B) Samples were stained with anti-EGFR antibody (red). (C) Double-immunofluorescence staining of S100A4 and EGFR (yellow). Arrows indicate positive-staining cells.

 

Figure 12
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  		Figure 12. EGFR labeling index in S100A4-positive cells on lung tissues after the intratracheal instillation of bleomycin. (n = 3–6, *p < 0.05 vs. control.) BLM = bleomycin; G20 = gefitinib 20 mg/kg; G200 = gefitinib 200 mg/kg.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In those mice that had not received bleomycin treatment to induce lung fibrosis, no changes to the lung were observed after the administration of gefitinib, even at the high dose of 200 mg/kg, which was close to the maximum tolerated dose and the highest dose used in xenograft models (15). Gefitinib at each of the dose levels—20, 90 and 200 mg/kg—significantly prevented the lung fibrosis induced by intratracheal bleomycin. The fibrosis was effectively reduced by gefitinib 20 mg/kg, which was about twice the growth-inhibitory dose (12.5 mg/kg) used on transplanted tumors in nude mice (15). A similar suppression effect was observed at a dose of 200 mg/kg, 10 times the low dose and close to the maximum tolerated dose, without aggravation of fibrosis. AG1478 also significantly attenuated the fibrosis, indicating that EGFR-TK inhibition itself had a protective effect on lung fibrosis in a mouse bleomycin-induced pulmonary fibrosis model.

Immunohistochemical staining revealed that gefitinib treatment significantly decreased fibroblast proliferation induced by bleomycin in vivo and that EGFR was expressed on fibroblasts almost constantly. Meanwhile, EGFR phosphorylation of the lungs was induced by bleomycin early on Day 3, which, however, was suppressed by gefitinib. This evidence was considered to support the in vitro observation that the EGFR-TKIs prevented the fibroblast proliferation induced by EGF ligands, as well as the in vivo observation that the EGFR-TKIs inhibited the bleomycin-induced lung fibrosis.

EGFR is also expressed and phosphorylated in alveolar or airway epithelial cells, and therefore, gefitinib is considered to have had effects on those cells. It has been suggested that epithelial injury and delay of its repair could play an important role in the fibrogenesis process (25, 26). Therefore, inhibition of epithelial regeneration by an EGFR-TKI might promote fibrogenesis. Moreover, blocking of EGFR could induce epithelial apoptosis (27). In the present study, although we did not directly examine whether or not retardation of epithelial repair or promotion of apoptosis was induced, fibrogenesis was clearly reduced as a result, indicating that inhibition of fibroblast migration and proliferation could result in prevention of fibrosis even in a retarded condition of epithelial regeneration.

EGFR is expressed by many cell types in the lung, including the epithelium, smooth muscle cells, endothelium, and fibroblasts (28). Ligands for the EGFR found in the lung include TGF-{alpha}, EGF, amphiregulin, and heparin-binding EGF. Some studies demonstrated that the ligands were abundantly localized in the lung due to elevated production in pathologic lung fibrosis (3, 4). Expression of EGFR was also increased (4). Pulmonary fibrosis by bleomycin in TGF-{alpha}–knockout mice was reduced compared with wild-type mice (6), whereas the fibrosis was induced in TGF-{alpha} transgenic mice (5). Those findings suggested that EGFR and its ligands were significantly involved in pathology of lung fibrosis, and therefore blocking of both signal transductions could be a useful treatment for pulmonary fibrosis. In fact, the EGFR-TKI AG1478 reduced the pulmonary fibrosis induced by vanadium pentoxide in rats (8). The results of our study are thus consistent with those of a sequence of studies.

Suzuki and colleagues reported that gefitinib augmented bleomycin-induced pulmonary fibrosis in mice (16). Their study used 200 mg/kg (a subtoxic dose) of gefitinib; therefore, in this study, three doses were chosen: 200 mg/kg as a high dose, 20 mg/kg as enough to inhibit EGFR, and 90 mg/kg as an intermediate dose. Fibrosis was significantly reduced in all three dose groups without any aggravation due to the drug. There were no significant differences between the experimental methods, except for the difference in strains used: ICR and C57BL/6 mice, in which exposure for gefitinib showed little difference between the two strains based on the observed maximum serum concentration (Cmax), the area under the serum concentration—time curve from 0 to 24 h (AUC0–24), and the terminal half-life of gefitinib (AstraZeneca, unpublished data). The reason for the discrepancy between the results is not known.

ILD is a condition considered to be associated with lung cancer (14). Since gefitinib was clinically introduced as an anticancer drug for lung tumors, interstitial pneumonia or acute lung injury have been seen in patients treated with gefitinib (9–12). The incidence of interstitial pneumonia is about 5% in Japanese patients, which is higher than the incidence reported globally of 0.8% (29). There are no large differences in the incidence of interstitial pneumonia between gefitinib and new-generation cytotoxic anticancer agents such as paclitaxel, docetaxel, irinotecan, vinorelbine, gemcitabine, and amrubicin, according to the reported incidences of interstitial pneumonia (0.1–6.2%) in the late phase 2 trials in Japan (data from the package inserts of the agents). As for possible mechanisms of pathogenesis, pharmacology of EGFR-TKIs, drug toxicity independent of pharmacology, and immunoreaction have been postulated. The results of this study reveal that neither universal effects induced by the drug itself at a high dose nor repair inhibition triggered by inflammation in the host could have contributed to the development of ILD.

In clinical studies with gefitinib in patients with lung cancer, a greater antitumor effect was observed in adenocarcinomas, females, and nonsmokers (11, 12), and an ethnic difference was evident, with a greater antitumor effect seen in Japanese populations than in non-Japanese populations (30). Also, mutation of the EGFR gene has been known to be involved as a determinant of response to gefitinib therapy (31–33): gefitinib was more effective in patients with mutations on exon 18, 19, and 21 of the EGFR gene. There was a higher rate of mutation-positive patients in the groups that exhibited a greater clinical effect with gefitinib, such as nonsmokers or Japanese patients (32, 34). On the contrary, reports of lung injury in association with gefitinib therapy were also higher in Japanese populations than in non-Japanese populations. This suggested the potential involvement of genetic factors, although this has not been demonstrated (10). Smoking and underlying pulmonary fibrosis have been identified as risk factors for interstitial pneumonia and acute lung injury (11). Although it is possible that gefitinib could induce unbalanced repair after lung injury and thereby could result in lung fibrosis, our results have shown that no lung fibrosis was observed with gefitinib itself, even at a high dose, and that gefitinib reduced rather than increased the bleomycin-induced lung fibrosis.

The present study demonstrated that EGFR-TKIs, such as gefitinib and AG1478, attenuated bleomycin-induced lung fibrosis in mice, possibly by inhibiting growth signaling in fibroblasts. This suggests that EGFR-TKIs might be useful for the treatment of pulmonary fibrosis, although many issues remain to be resolved. Moreover, imatinib, which is a platelet-derived growth factor (a growth factor in fibroblasts) receptor TKI, has been shown to suppress bleomycin-induced lung fibrosis (35–37), and its clinical use has been discussed. However, because interstitial pneumonia has been also reported as a complication of imatinib (38), the mechanism of pathogenesis should be clarified.

Although the mechanism of induction of clinical interstitial pneumonia and lung injury could not be explained in this study, one hypothesis could be that blocking EGFR could alter the balance between repair and fibrosis after lung injury in a negative direction by genetic factors in particular individuals, leading to the induction of fibrosis. Pathologic elucidation using genetic analysis in lung fibrosis cases is necessary to improve understanding.


   Acknowledgments
 
The authors thank Kaori Nagashima and Hirata Hisato for excellent technical assistance.


   FOOTNOTES
 
Supported by grants from the Japanese Ministry of Health and Welfare.

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.200509-1534OC on June 1, 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 September 30, 2005; accepted in final form June 1, 2006


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