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Published ahead of print on February 2, 2006, doi:10.1164/rccm.200506-947OC
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American Journal of Respiratory and Critical Care Medicine Vol 173. pp. 1016-1022, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200506-947OC

Original Article

A Novel I{kappa}B Kinase-beta Inhibitor Ameliorates Bleomycin-induced Pulmonary Fibrosis in Mice

Mami Inayama*, Yasuhiko Nishioka*, Momoyo Azuma, Susumu Muto, Yoshinori Aono, Hideki Makino, Kenji Tani, Hisanori Uehara, Keisuke Izumi, Akiko Itai and Saburo Sone

Departments of Internal Medicine and Molecular Therapeutics, and Molecular and Environmental Pathology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima; and Institute of Medicinal Molecular Design, Inc., Tokyo, Japan

Correspondence and requests for reprints should be addressed to Saburo Sone, M.D., Ph.D., Department of Internal Medicine and Molecular Therapeutics, Institute of Health Biosciences, the University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: ssone{at}clin.med.tokushima-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: I{kappa}B kinase-beta is a critical regulator in the activation of nuclear factor-{kappa}B (NF-{kappa}B), a transcription factor related to the expression and regulation of proinflammatory cytokines.

Objective: To evaluate if inhibition of I{kappa}B kinase-beta ameliorates pneumonitis and pulmonary fibrosis.

Methods: We examined whether a novel I{kappa}B kinase-beta inhibitor, IMD-0354, attenuates bleomycin-induced pulmonary fibrosis in mice.

Measurements and Main Results: Administration of IMD-0354 significantly improved the loss of body weight and survival of mice treated with bleomycin, whereas IMD-0354 alone did not cause any morphologic change in the lung. When mice were evaluated 28 d after bleomycin administration, IMD-0354 dose-dependently reduced the collagen content and fibrotic scores as shown by histologic examination. The findings in the bronchoalveolar lavage demonstrated that the proportions of neutrophils and lymphocytes were decreased in mice treated with IMD-0354 on Day 7 and 14, respectively. IMD-0354 treatment was confirmed to inhibit the activation of NF-{kappa}B, but not activator protein-1, in the lungs treated with bleomycin. The production of inflammatory cytokines tumor necrosis factor-{alpha} and interleukin-1beta was reduced in the lungs of mice treated with IMD-0354.

Conclusions: These results suggest that IMD-0354 might be useful to ameliorate the inflammation in the lungs induced by fibrotic injury and the subsequent fibrogenesis via inhibiting the expression of profibrotic cytokines related to the activation of NF-{kappa}B.

Key Words: interleukin-1beta • nuclear factor-{kappa}B, tumor necrosis factor-{alpha}

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the proliferation of fibroblasts and deposition of extracellular matrixes (1, 2). Recent reports advocated the novel hypothesis that repeated alveolar epithelial injury and the subsequent abnormal wound healing, but not chronic inflammation, are critical events in the pathogenesis of pulmonary fibrosis, especially in IPF. However, they also indicated that inflammatory responses are likely to modulate and accelerate tissue injury and aberrant wound healing in lung fibrogenesis (2, 3). Besides, in other types of pulmonary fibrosis, including acute interstitial pneumonia and nonspecific interstitial pneumonia, acute or chronic inflammation is more closely related to lung fibrogenesis (4–6). Because the relationship between inflammation and fibrogenesis is not entirely clear, the targeted inhibition of lung inflammation can still be a potential therapy for pulmonary fibrosis.

To date, corticosteroids have been used for treatment of patients with pulmonary fibrosis, although their clinical effects are limited (1, 7). Because corticosteroids have various biological effects, which in turn frequently lead to serious side effects (7), there is a need for more selective approaches to inhibit lung inflammation that might reduce the subsequent fibrosis without adverse effects. On the basis of these concepts, targeted inhibition of nuclear factor-{kappa}B (NF-{kappa}B), which is a transcription factor related to the expression and regulation of proinflammatory cytokines, including tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1beta, has been examined for its effects in reducing pulmonary fibrosis using the bleomycin model. Zhang and colleagues reported that administration of antisense oligonucleotides to NF-{kappa}B improved pulmonary fibrosis induced by bleomycin (8). However, Griesenbach and coworkers reported that intranasal inhalation of NF-{kappa}B decoy oligonucleotides failed to attenuate lung inflammation (9). These reports suggest that inhibition of NF-{kappa}B is a potential but not well established antifibrotic therapy.

The cytoplasmic binding of NF-{kappa}B to inhibitor of {kappa}B (I{kappa}B) prevents NF-{kappa}B activation. The serine phosphorylation of I{kappa}B allows its degradation by the 26S proteasome, and subsequently, NF-{kappa}B free of I{kappa}B translocates into the nucleus and activates gene transcription (10). There are two catalytic subunits of the I{kappa}B kinase (IKK) complex, IKK{alpha} and IKKbeta (11, 12). IKKbeta, but not IKK{alpha}, is required for TNF-{alpha}– and IL-1–mediated activation of NF-{kappa}B (13–15). Recently, it was reported that IKKbeta is a critical target to inhibit the NF-{kappa}B pathway in alveolar macrophages because inhibition of NF-{kappa}B–inducing kinase, which functions upstream of the IKK complex, does not affect the production of NF-{kappa}B–related proinflammatory cytokines (16).

We therefore examined whether targeted inhibition of IKKbeta reduced the pneumonitis/fibrosis induced by bleomycin in mice using a novel IKKbeta inhibitor, IMD-0354. This article reports the profound antifibrotic effects of IMD-0354 achieved via inhibition of the production of profibrotic cytokines. Some of these studies have been previously reported in the form of abstracts (17).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Detailed methods are described in the online supplement.

Mice and Materials
Eight-week-old C57BL/6 female mice were purchased from Charles River Japan, Inc. (Yokohama, Japan). Mice were maintained in the animal facility of the University of Tokushima under specific pathogen–free conditions according to the guidelines of our university (18). IMD-0354 (N-[3,5-bis-trifluoromethyl-phenyl]-5-chloro-2-hydroxy-benzamide; Figure 1) was kindly provided by the Institute of Medical Molecular Design (Tokyo, Japan). The specificity of IMD-0354 and the inhibitory activity for IKKbeta were shown in Table E1 of the online supplement and previous reports (19, 20). Bleomycin was purchased from Nippon Kayaku Co. (Tokyo, Japan).


Figure 1
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  		Figure 1. Chemical structure of IMD-0354.

 
Bleomycin Treatment
Osmotic minipumps (model 2001; Alza Pharmaceuticals, Palo Alto, CA) containing saline or bleomycin (125 or 150 mg/kg) were implanted subcutaneously (21, 22). Each experiment was performed in at least four mice per group.

Administration of IMD-0354
The IMD-0354 powder was dissolved in 0.5% carboxymethylcellulose (CMC; Sigma, Tokyo, Japan), and administered intraperitoneally into mice.

Bronchoalveolar Lavage
Bronchoalveolar lavage (BAL) was performed five times with saline (1 ml) using a soft cannula. After counting the cell number in the BAL fluid (BALF), cells were cytospun onto glass slides and stained with Diff-Quick (Baxter, Miami, FL) for cell classification.

Collagen Assay
The right lungs harvested on Day 28 were used for collagen assays. Total lung collagen was determined using the Sircol Collagen Assay kit (Biocolor Ltd., Belfast, Northern Ireland) according to the manufacturer's instructions (22).

Histopathology
The left lungs were fixed in 10% buffered formalin and embedded in paraffin. Sections (3–4 µm) were stained with hematoxylin and eosin. For the quantitative histologic analysis, a numeric fibrotic scale was used (Ashcroft score) (23). The mean score was considered the fibrotic score. Masson's trichrome staining was also performed.

Preparation of Nuclear Extracts
Preparation of nuclear extracts was performed using a CelLytic nuclear extraction kit (Sigma, Tokyo, Japan) according to the manufacturer's instructions (24). The nuclear extracts were kept at –80°C until use.

NF-{kappa}B and Activator Protein-1 ELISA
To evaluate the activation of NF-{kappa}B or activator protein-1 (AP-1), translocation of the p65 or c-Jun subunit into the nucleus was measured using a TransAM NF-{kappa}B p65 or c-Jun Transcription Factor Assay kit (Active Motif, Inc., Carlsbad, CA), respectively, according to the manufacturer's instructions (25). Results were determined by measuring the spectrophotometric absorbance at 655 nm and expressed as optical density (OD655).

Electrophoretic Mobility Shift Assay
To confirm the activation of NF-{kappa}B, the electrophoretic mobility shift assay was performed as previously described (26). Nuclear protein extracts (5 µg) were incubated with a 32P-labeled probe and loaded onto 5% polyacrylamide gels, and then electrophoresed and analyzed using an image analyzer (BAS 2000; Fuji Film Co., Tokyo, Japan).

ELISAs for TNF-{alpha} and IL-1beta
The concentrations of murine TNF-{alpha} and IL-1beta were quantified using ELISA kits (R&D Systems, Minneapolis, MN). The minimal detectable level of both TNF-{alpha} and IL-1beta was 5.1 pg/ml.

Statistical Analysis
Comparisons among multiple groups were analyzed using the one-way analysis of variance with Newman-Keuls post hoc correction (GraphPad Prism, version 3.0; GraphPad Software, San Diego, CA). Differences were considered statistically significant if p values were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IKKbeta Inhibitor IMD-0354 Improves the Survival and Weight Loss Induced by Bleomycin
We first examined the in vivo effects of a novel IKKbeta inhibitor, IMD-0354, on the survival and weight loss induced by bleomycin in mice. Mortality caused by bleomycin was dose dependent. Ten of 25 animals (40%) died from Day 16 to 24 after treatment with 150 mg/kg of bleomycin. The administration of 20 mg/kg/d of IMD-0354, however, significantly improved the survival rate of mice treated with bleomycin (Figure 2A). There were no deaths in the group of mice treated with both IMD-0354 and bleomycin.


Figure 2
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  		Figure 2. Effect of IMD-0354 on the survival rate and weight loss of mice treated with bleomycin. Mice were treated with 150 mg/kg of bleomycin (BLM). IMD-0354 (20 mg/kg) was given by intraperitoneal injection on a daily basis for the entire duration of the experiment (28 d). (A) The survival rates of mice treated with BLM alone (solid line, n = 16) or BLM + IMD-0354 (dotted line, n = 16) were examined. (B) The change in body weight of mice treated with saline alone (n = 10), IMD-0354 (n = 10), BLM alone (n = 16) or BLM + IMD-0354 (n = 16). Similar results were obtained in two separate experiments. *p < 0.001. CMC = carboxymethylcellulose. Diamonds = saline + CMC; squares = saline + IMD-0354; triangles = BLM + CMC; circles = BLM + IMD-0354.

 
The mice injected with bleomycin showed a significant loss of body weight from Day 14 as compared with those injected with saline, and weight loss did not recover until Day 28. In contrast, mice treated with both bleomycin and IMD-0354 (20 mg/kg/d) showed a significant weight loss on Day 14, but the weight partially recovered from Day 14 to 28 (Figure 2B). On Day 28, IMD-0354 significantly improved the weight loss caused by bleomycin (p < 0.001; Figure 2B), although mice in the group treated with both IMD-0354 and bleomycin still showed 15% weight loss on Day 28 as compared with the control group (saline + CMC). On the other hand, mice treated with saline + CMC or saline + IMD-0354 did not show any loss of body weight, indicating that 20 mg/kg/d of IMD-0354 was not toxic to mice.

Administration of IMD-0354 Ameliorates Bleomycin-induced Lung Fibrosis in Mice
To evaluate the antifibrotic effect of IMD-0354, mice were treated with 125 mg/kg of bleomycin and killed on Day 28. The fibrotic change in the lung was evaluated by histologic examination and measurement of total collagen content. As shown in Figure 3, administration of IMD-0354 alone did not generate any changes in lung morphology. However, when IMD-0354 was administered daily to bleomycin-treated mice, a significant reduction of fibrosis in the subpleural areas of lung was observed (Figure 3). These antifibrotic effects of IMD-0354 were also confirmed by histologic examination using the fibrotic score as described in METHODS (Figure 4B). The collagen assay demonstrated that treatment with IMD-0354 dose-dependently reduced the production of total collagen in bleomycin-treated lungs (Figure 4A). More than 5 mg/kg/d of IMD-0354 were effective in reducing the pulmonary fibrosis caused by bleomycin.


Figure 3
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  		Figure 3. Histologic examination of the antifibrotic effects of IMD-0354 on BLM-induced pulmonary fibrosis. Mice were treated with osmotic minipumps containing saline or BLM (125 mg/kg). IMD-0354 (20 mg/kg) was given by intraperitoneal injection on a daily basis for the entire duration of the experiment. On Day 28, mice were killed and histologic examination was performed by hematoxylin and eosin (H&E) staining (A–H) and Masson's trichrome staining (I–L; original magnification: A–D, x40; E–L, x100). A, E, I: saline alone; B, F, J: saline + IMD-0354; C, G, K: BLM alone; D, H, L: BLM + IMD-0354. Data are representative of three separate experiments. Bar = 200 µm.

 

Figure 4
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  		Figure 4. Quantitative examinations of the antifibrotic effects of IMD-0354 on BLM-induced pulmonary fibrosis. Mice were treated with osmotic minipumps containing saline or BLM. IMD-0354 (5 or 20 mg/kg) was given by intraperitoneal injection on a daily basis for the entire duration of the experiment. Mice were killed on Day 28. (A) Effects of IMD-0354 on collagen deposition after treatment with BLM. Collagen content in the right lung was measured using a Sircol collagen kit. Data are presented as mean ± SD of six mice. (B) Evaluation of fibrotic change in the lung using numeric fibrotic score. Histologic examination in the left lung was performed by H&E staining. The fibrotic score was determined by two pathologists as described in METHODS. Data are presented as mean ± SD of all fields examined in each group of six mice. Data are representative of two separate experiments.

 
Next, we examined the effect of the treatment schedule using IMD-0354 on the bleomycin-induced pulmonary fibrosis. Mice were separately treated with IMD-0354 from Day 0 to 14 (early treatment) or Day 14 to 28 (late treatment). As shown in Figures 5A and 5B, reduction of the antifibrotic effect of IMD-0354, as indicated by the fibrotic score and collagen content, was observed in both groups, but neither groups showed a significant difference from the bleomycin-alone group.


Figure 5
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  		Figure 5. Effects of treatment schedule of IMD-0354 on the antifibrotic effects in BLM-induced pulmonary fibrosis. Mice were treated with osmotic minipumps containing saline or BLM. IMD-0354 (20 mg/kg/d) was injected intraperitoneally from Day 0 to 14 (early treatment) or Day 14 to 28 (late treatment). Mice were killed on Day 28. (A) Effects of IMD-0354 on collagen deposition after treatment with BLM. Collagen content in the right lung was measured using a Sircol collagen kit. Data are presented as mean ± SD of eight mice. (B) Evaluation of fibrotic change in the lung using numeric fibrotic score. Histologic examination in the left lung was performed by H&E staining. The fibrotic score was determined by two pathologists as described in METHODS. Data are presented as mean ± SD of all fields examined in each group of eight mice. Data are representative of three separate experiments.

 
Cell Analysis of BALF
Next, we analyzed the cells in BALF to evaluate the effect of IMD-0354 on the inflammatory responses induced by bleomycin. Administration of bleomycin elevated the number of inflammatory cells, including macrophages, lymphocytes, and neutrophils, on Days 7, 14, and 28. Analysis of cell classification also showed that bleomycin treatment enhanced the percentage of lymphocytes and neutrophils on Days 7, 14, and 28. Administration of IMD-0354 significantly reduced the percentage of neutrophils and lymphocytes in BALF on Days 7 and 14, respectively (Table 1).


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  		TABLE 1. ANALYSIS OF BRONCHOALVEOLAR LAVAGE

 
Inhibition of Activation of NF-{kappa}B by IMD-0354
To confirm the inhibition of activation of NF-{kappa}B by IMD-0354, we used a p65 ELISA to examine the amount of p65 subunit of NF-{kappa}B in the nuclear fraction of lung tissues and BALF of bleomycin-treated mice with or without IMD-0354. Bleomycin induced the maximal nuclear translocation of p65 of NF-{kappa}B on Day 7 (data not shown). The treatment with IMD-0354 significantly inhibited the nuclear translocation of p65 subunit in both lung tissues and BALF (Figures 6A and 6B). On the other hand, the activation of AP-1, defined by the nuclear translocation of c-Jun subunit, was not inhibited by IMD-0354 (Figures 6C and 6D). Furthermore, an electrophoretic mobility shift assay showed that the DNA binding activity of NF-{kappa}B significantly decreased in nuclear extracts of the lungs in mice treated with both bleomycin and IMD-0354 as compared with bleomycin alone (Figure 6E), and that the positive bands disappeared by the addition of an excess of the unlabeled NF-{kappa}B oligonucleotides (Figure 6F).


Figure 6
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  		Figure 6. Inhibition of activation of nuclear factor (NF)-{kappa}B, but not activated protein (AP)-1, by IMD-0354 in the lungs treated with BLM. NF-{kappa}B activities in nuclear protein extracts of bronchoalveolar lavage fluid (BALF; A) and lung tissues (B) in mice treated with BLM with or without IMD-0354 on Day 7 were examined by measuring the level of the p65 subunit of NF-{kappa}B heterodimer using a trans AM kit (Active Motif, Inc., Carlsbad, CA). AP-1 activity was also examined in BALF (C) and lung tissues (D) by measuring the level of the c-Jun subunit of AP-1 heterodimer using a trans-AM kit. Data are presented as mean ± SD of four mice. The DNA binding activity of NF-{kappa}B in nuclear extracts of lung tissues was examined by an electrophoretic mobility shift assay (E). Competition experiments were performed by adding a 1,000-fold excess of unlabeled oligonucleotide before the labeled probes (F). Each lane in E and F shows the data from the nuclear extract of the lung of an individual mouse. Data are representative of two separate experiments.

 
IMD-0354 Inhibits the Expression of TNF-{alpha} and IL-1beta in Bleomycin-treated Lung
We examined whether IMD-0354 inhibited the expressions of TNF-{alpha} and IL-1beta, which is regulated by the activation of NF-{kappa}B, in homogenates and BALF of lungs treated with bleomycin. TNF-{alpha} was detected 3 to 7 d after the injection of bleomycin (data not shown). IMD-0354 reduced the level of TNF-{alpha} in both lung homogenates and BALF (Figures 7A and 7B). There was a significant difference in the level of TNF-{alpha} in BALF between the groups of bleomycin alone and bleomycin + IMD-0354 (8.3 ± 1.7 vs. 2.9 ± 1.5 pg/ml, p < 0.001) and in lung homogenates between these groups (44.2 ± 14.5 vs. 19.0 ± 5.8 pg/ml, p < 0.001). The level of IL-1beta in BALF was below the limit of sensitivity at all time points (data not shown). However, IL-1beta was detected in the lung tissue homogenates from mice treated with bleomycin. The level of IL-1beta in mice treated with bleomycin + IMD-0354 was significantly lower than that in mice treated with bleomycin alone (46.4 ± 22.7 vs. 90.6 ± 18.9 pg/ml, p < 0.05; Figure 7C).


Figure 7
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  		Figure 7. IMD-0354 inhibits the expression of tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1beta in BALF or lung homogenates of mice treated with BLM. The levels of TNF-{alpha} and IL-1beta in BALF and lung homogenates 7 d after treatment with BLM were measured by ELISA as described in METHODS. Data are shown as the mean ± SD of eight mice. Data are representative of two separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that a novel IKKbeta inhibitor, IMD-0354, effectively prevented bleomycin-induced pulmonary fibrosis via inhibition of the activation of NF-{kappa}B in the lungs. The analysis of BALF indicated that the antifibrotic effects of IMD-0354 were at least in part mediated by reducing the accumulation of neutrophils and lymphocytes in the lungs and the profibrotic cytokines TNF-{alpha} and IL-1beta.

The antiinflammatory effects of corticosteroids, which have been used for the therapy for pulmonary fibrosis, are mediated by various mechanisms that lead to several adverse events, including diabetes mellitus, osteoporosis, peptic ulcers, and so on (27, 28). Therefore, antifibrotic therapy targeting the activity of NF-{kappa}B might have the potential to reduce serious side effects as well as enhance the antiinflammatory and antifirotic effects. Recently, Zhang and coworkers showed the antifibrotic effects of antisense oligonucleotides to NF-{kappa}B, whereas the inhibition of NF-{kappa}B in lung homogenates was not achieved (8). Griesenbach and colleagues reported that NF-{kappa}B decoy oligonucleotides failed to inhibit lung inflammation induced by bleomycin because oligonucleotides administered intranasally did not reach the nuclei of lung tissue cells (9). These reports suggest that antifibrotic effects of oligonucleotides inhibiting the activity of NF-{kappa}B are still controversial.

In contrast to the previous findings, we clearly demonstrated here the successful inhibition of NF-{kappa}B, but not AP-1, in lung homogenates by systemic administration of IMD-0354. Furthermore, IMD-0354 effectively reduced the levels of TNF-{alpha} and IL-1beta in the lungs. These favorable results might be partly due to the use of a small compound with low molecular weight, not oligonucleotides. In fact, the molecular weight of IMD-0354 is low (383.7). It is likely that IMD-0354 is more easily delivered into lung tissue cells as compared with oligonucleotides, although we did not examine the concentration of IMD-0354 in the lung tissue.

On the other hand, the daily treatment of IMD-0354 throughout the experiments was required to exert the maximal antifibrotic effects because neither the early (Day 0 to 14) nor late (Day 15 to 28) treatment significantly prevented the pulmonary fibrosis induced by bleomycin. The reason for these results remains to be elucidated. Elevated activity of NF-{kappa}B was detected in the lung homogenates on Days 3 and 7 after bleomycin treatment in our system. However, the latent activity of NF-{kappa}B may have a role in the late phase of bleomycin-induced pulmonary fibrosis. Otherwise, the inhibitory activities for other kinases as well as biochemical actions of IMD-0354 are likely to be involved in the ability to attenuate bleomycin-induced pulmonary fibrosis. In particular, it is possible that IMD-0354 also has the antifibrotic, but not antiinflammatory, effects in the late phase of lung fibrogenesis. The direct effects of IMD-0354 on the growth and collagen production of lung fibroblasts are important targets of future experiments, since antiproliferative effects of IMD-0354 were recently reported (20). IMD-0354 may be a similar case to pirfenidone, which was originally developed as an antiinflammatory drug, but recently shown to have profound antifibrotic effects in vitro and in clinical trials for patients with IPF (29–31).

The analysis of BAL cells demonstrated that IMD-0354 reduced the number of accumulated neutrophils and lymphocytes on Day 7 and 14, respectively. The mechanism involved in inhibiting the accumulation of neutrophils by IMD-0354 is not clear. One possibility is that the inhibition of inflammatory cytokines in the lung, which are related to the activation of NF-{kappa}B, by IMD-0354 might contribute to reducing the accumulation of neutrophils and lymphocytes. Furthermore, we observed the reduction of macrophage inflammatory protein-2, which is known to be regulated by NF-{kappa}B and an important chemotactic factor for murine neutrophils, in the lungs treated with IMD-0354, although the difference did not reach a significant level (data not shown). Further studies regarding chemotactic factors to neutrophils and lymphocytes will be required.

A phase I study of IMD-0354 is now in progress. Recently, Onai and colleagues reported that IMD-0354 attenuated myocardial ischemia/reperfusion injury without general or cellular toxicity in rats (19). In our study, administration of IMD-0354 alone did not induce weight loss or any morphologic changes in the lungs. These results together with our data suggest that a novel IKKbeta inhibitor, IMD-0354, might be useful for the treatment of patients with pulmonary fibrosis, especially which is related to the activation of NF-{kappa}B.


   Acknowledgments
 
The authors thank Ms. Tomoko Oka for her technical assistance.


   FOOTNOTES
 
Supported by grants from the Ministry of Health and Welfare and the Ministry of Education, Science, Sports, and Culture of Japan.

* M.I. and Y.N. are joint first authors and contributed equally to this work. 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.200506-947OC on February 2, 2006

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

Received in original form June 20, 2005; accepted in final form February 2, 2006


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