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Published ahead of print on December 20, 2007, doi:10.1165/rcmb.2007-0299OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 566-571, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0299OC

Role of Interleukin-6 in Bleomycin-Induced Lung Inflammatory Changes in Mice

Fumitake Saito1, Sadatomo Tasaka1, Ken-ichiro Inoue2, Keisuke Miyamoto1, Yasushi Nakano1, Yuko Ogawa1, Wakako Yamada1, Yoshiki Shiraishi1, Naoki Hasegawa1, Seitaro Fujishima3, Hirohisa Takano2 and Akitoshi Ishizaka1

1 Division of Pulmonary Medicine, and 3 Department of Emergency and Critical Care Medicine, Keio University School of Medicine, Tokyo, Japan; and 2 Inhalation Toxicology Research Team, National Institute for Environmental Studies, Tsukuba, Japan

Correspondence and requests for reprints should be addressed to Sadatomo Tasaka, M.D., Division of Pulmonary Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: tasaka{at}cpnet.med.keio.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Interleukin-6 (IL-6) is known to be involved in the pathogenesis of various inflammatory diseases, but its role in bleomycin (BLM)-induced lung injury and subsequent fibrotic changes remains to be determined. We evaluated the role of IL-6 in the lung inflammatory changes induced by BLM using wild-type (WT) and IL-6–deficient (IL-6–/–) mice. The mice were treated intratracheally with 1 mg/kg BLM and killed 2, 7, or 21 days later. Lung Inflammation in the acute phase (Days 2 and 7) was assessed by differential cell counts in bronchoalveolar lavage (BAL) fluid and cytokine levels in the lung. Lung fibrotic changes were evaluated on Day 21 by histopathology and collagen assay. On Day 2, BLM administration induced significant increases in the numbers of total cells, macrophages, and neutrophils in BAL fluid, which were attenuated in IL-6–/– mice (P < 0.05). Lung pathology also showed inflammatory cell accumulation, which was attenuated in the IL-6–/– mice compared with WT mice. In WT mice, elevated levels of TGF-β1 and CCL3 were observed 2 and 7 days after BLM challenge, respectively. On Day 7, BLM-induced inflammatory cell accumulation did not differ between the genotypes. Lung pathology 21 days after BLM challenge revealed significant fibrotic changes with increased collagen content, which was attenuated in IL-6–/– mice. Although the TGF-β1 level in the lung did not differ between the genotypes on Day 21, CCL3 was significantly lower in IL-6–/– mice. These results indicate that IL-6 may play an important role in the pathogenesis of BLM-induced lung injury and subsequent fibrotic changes.

Key Words: IL-6 • bleomycin • fibrosis • TGF-β • CCL3



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This study enhances our knowledge of the pathogenesis of lung injury and subsequent fibrotic change. IL-6 plays an important role in the development of bleomycin-induced lung inflammation and fibrosis, possibly through the up-regulation of TGF-β1 and CCL3.

 
Pulmonary fibrosis is characterized morphologically by thickening of the alveolar septa with collagen deposition and myofibroblast proliferation, and by a diffuse inflammatory infiltrate (1). Much of the information regarding the development of pulmonary fibrosis has been acquired with a well-characterized animal model in which lung fibrosis is induced by intratracheal administration of the antineoplastic agent bleomycin (BLM). In rodents, BLM administration induces acute inflammatory response, which resembles acute respiratory distress syndrome (ARDS), followed by fibroblast proliferation and increased collagen content in the lung (2). ARDS is the most severe form of lung injury induced by diverse causes such as sepsis, aspiration of gastric contents, severe pneumonia, and multiple trauma (3). In addition, when persistent inflammation of ARDS fails to resolve, fibroproliferation occurs with collagen deposition and progressive lung fibrosis (4, 5). Various mediators are involved in the pathogenesis of pulmonary fibrosis, including those subsequent to ARDS, although the detailed mechanisms are still not well understood (6, 7).

IL-6 is known to mediate many inflammatory processes in the lung, and its dysregulated release has been implicated in the pathogenesis of a variety of respiratory disorders (7, 8). Several genetic studies in both animals and humans have shown a possible association between IL-6 and the development of fibrosis (9, 10). In addition, the IL-6 concentration in bronchoalveolar lavage (BAL) fluid was 100-fold higher in patients with ARDS than in normal subjects, and remained above the normal range for up to 21 days when the disease was persistent (7). Although IL-6 is secreted by BLM-challenged endothelial cells and macrophages in rats (11, 12), little is known about the role of IL-6 and the interaction between IL-6 and other mediators during development of BLM-induced lung injury. We hypothesized that IL-6 might mediate persistent inflammation and subsequent fibrotic changes in the lung.

In the present study, we evaluated the inflammatory response to intratracheal BLM and subsequent fibrotic changes, comparing wild-type (WT) and IL-6–deficient (IL-6–/–) mice. To determine the effect of IL-6 deficiency on the acute phase of BLM-induced lung inflammation, we collected BAL fluid or lung samples for pathology 2, 7, or 21 days after the BLM challenge. We examined the accumulation of inflammatory cells in BAL fluid as well as changes in lung pathology. To evaluate the chronic phase that is characterized by lung fibrosis, we examined lung pathology and collagen content using the lung samples obtained 21 days after BLM administration. The levels of transforming growth factor-β1 (TGF-β1) and macrophage inflammatory protein-1{alpha}/CC chemokine ligand 3 (MIP-1{alpha}/CCL3), both of which are known to be involved in the pathogenesis of lung fibrosis (13, 14), were also measured in the lungs.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
IL-6–/– mice (B6.129S2-Il6tm1Kopf/J) and their WT controls were purchased from the Jackson Laboratory (Bar Harbor, ME) and routinely bred in the vivarium of National Institute for Environmental Studies (NIES), Tsukuba, Japan. All experiments were performed with mice at the age of 8 to 12 weeks. Animals received humane care according to the NIES guidelines for animal welfare, conforming to NIH guidelines.

Model of Intratracheal Bleomycin Injury
Mice (20–25 g) were anesthetized with intraperitoneal ketamine (120 mg/kg) and xylazine (12 mg/kg). Intratracheal instillation of BLM (1 mg/kg) or vehicle (sterile isotonic saline solution) in a volume of 2 ml/kg was performed via a Microsprayer (PennCentury, Philadelphia, PA). All mice were killed by deep anesthesia 2, 7, or 21 days after the BLM instillation.

Histopathologic Determination of Lung Inflammation and Fibrosis
The lungs were fixed by intratracheal instillation of 10% neutral phosphate-buffered formalin and paraffin embedded. The tissues were cut into 3-µm sections and stained with hematoxylin-eosin (H-E) or Masson's trichrome for morphologic analysis.

To determine the severity of lung inflammation on Days 2 and 7, pulmonary neutrophils were quantified by morphometric analysis in histologic sections that were stained with H-E (15). Neutrophil emigration was quantitated by counting the number of neutrophils in 200 randomly selected alveoli and was expressed as the number of neutrophils per alveolus.

Fibrotic change on Day 21 was evaluated with Ashcroft score, a numerical fibrotic scoring scale, in histologic sections that were stained with Masson's trichrome (16). A score of 0 to 1 was grouped as no fibrosis, 2 to 3 as minimal, 4 to 5 as moderate, and 6 to 8 as severe fibrosis. Grading was performed by a single investigator in a blinded fashion.

Preparation and Analysis of Bronchoalveolar Lavage
To evaluate inflammatory cell accumulation in alveolar space, BAL fluid was collected by cannulating the trachea and lavaging the lung with three separate 1.0-ml volumes of sterile saline, each volume being instilled and withdrawn three times. The average volume retrieved was 90%, and the recovery rates did not differ by treatment or genotype. The fluid collections were combined and cooled to 4°C. The lavage fluid was centrifuged at 300 x g for 10 minutes, and the total cell count was determined on a fresh fluid specimen using a hemocytometer. Differential cell counts were assessed on cytologic preparation. Slides were prepared using a Cytospin (Tomy Seiko, Tokyo, Japan) and were stained with Diff-Quik (International Reagents Co., Kobe, Japan).

Measurement of Cytokine Levels in the Lung
For cytokine measurement, the lungs were subsequently homogenized with 10 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA (Sigma, St. Louis, MO), 0.1 mM phenylmethanesulfonyl fluoride (Nacalai Tesque, Kyoto, Japan), 1 µM pepstatin A (Peptide Institute, Osaka, Japan), and 2 µM leupeptin (Peptide Institute). The homogenates were then centrifuged at 10,500 x g for 1 hour, and the supernatants were stored at –80°C. Enzyme-linked immunosorbent assays (ELISAs) for TGF-β1 and CCL3 (R&D Systems, Minneapolis, MN) were performed following the manufacturer's instructions.

Analysis of Collagen Content in the Lung
Total lung collagen content was determined using the Sircol collagen assay (Biocolor Ltd., Belfast, UK) according to the manufacturer's instructions. Briefly, frozen caudal lobes from the right lung of each animal were homogenized in 500 µl radio-immunoprecipitation assay buffer containing protease inhibitors. Homogenates were centrifuged at 13,000 x g for 10 minutes at 4°C. Sirius red reagent (50 µl) was added to each lung homogenate (50 µl) and mixed for 30 minutes. The collagen–dye complex was precipitated by centrifugation at 16,000 x g for 5 minutes, washed with ethanol, and dissolved in 0.5 M NaOH. Finally, absorbance was measured at 540 nm and compared with a calibration curve generated using the collagen standard provided by the manufacturer.

Statistical Analysis
Data are reported as mean ± SEM. Differences among groups were determined using ANOVA followed by post hoc analysis with the Bonferroni's test for multiple comparisons. A P value less than 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Profiles of Inflammatory Cells in the Airspace after Bleomycin Administration
To examine the effect of IL-6 deficiency on BLM-induced neutrophil emigration, lung histology on Day 2 (i.e., earlier phase of acute lung inflammation) and Day 7 (i.e., later phase of acute lung inflammation) was evaluated. The number of neutrophils per alveolus is shown in Figure 1A. On Day 2, intratracheal BLM induced significant neutrophil emigration within the lungs, which was significantly greater in WT mice than in IL-6–/– mice (P < 0.05). On Day 7, both WT and IL-6–/– mice that received BLM challenge showed further neutrophil accumulation in the lungs compared with the control animals with saline instillation (P < 0.01). There was no significant difference in the BLM-induced neutrophil infiltration between WT and IL-6–/– mice on Day 7 (Figure 1A). Representative microscopic findings are shown in Figure 1B. By 7 days after administration, the lungs of BLM-treated animals exhibited marked hemorrhages and congestion with infiltration of inflammatory cells predominantly consisting of neutrophils.


Figure 1
Figure 1
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  		Figure 1. IL-6 deficiency attenuates acute neutrophil infiltration after bleomycin (BLM) administration. (A) Neutrophil emigration was quantitated by counting the number of neutrophils in 200 randomly selected alveoli and was expressed as the number of neutrophils per alveolus. IL-6–deficient mice (solid bars) revealed decreased neutrophil emigration 2 days after the BLM instillation compared with wild-type (WT) mice (open bars), whereas neutrophil emigration 7 days after the BLM challenge did not differ between the genotypes. All values are expressed as the mean + SE (n = 6 in each group). *P < 0.05 were considered to be significantly different from the corresponding value of the control mice with saline instillation. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the WT mice. (B) Representative examples of lung pathology 2 or 7 days after the instillation of saline or BLM. Original magnification: x200.

 
Since histopathologic changes were reflected by commensurate increases in total BAL cells, we examined total and differential cell counts in BAL fluid obtained on Days 2, 7, and 21. On Day 2, intratracheal BLM significantly increased the total cell count in BAL fluid in WT mice, which was significantly suppressed in IL-6–/– mice (Figure 2A). Macrophage, neutrophil, and lymphocyte counts in BAL fluid were also increased 2 days after the BLM challenge compared with the mice administered saline. There were significant differences in macrophage and neutrophil counts in BAL fluid between the two genotypes examined. On Day 7, the counts of total cells, macrophages, neutrophils, and lymphocytes in BAL fluid were further increased in the mice that received BLM instillation (Figure 2B). There was no significant difference in the counts of total cells, macrophages, neutrophils, and lymphocytes in BAL fluid between the two genotypes examined. Twenty-one days after BLM treatment, WT mice revealed significant increases in the counts of total cells, macrophages, and lymphocytes in BAL fluid, compared with the control animals (Figure 2C). In IL-6–/– mice, BLM-induced increases in the counts of total cells and macrophages were significantly reduced.


Figure 2
Figure 2
Figure 2
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  		Figure 2. Total and differential cell counts in bronchoalveolar lavage (BAL) fluid collected 2, 7, or 21 days after intratracheal instillation. (A) Two days after BLM challenge, WT mice showed significant increases in the counts of total cells, macrophages, neutrophils, and lymphocytes in BAL fluid compared with the control mice that received saline intratracheally. In IL-6–deficient mice, BLM-induced increases in total cells, macrophages, and neutrophils were significantly attenuated compared with WT mice. (B) On Day 7, the counts of total cells, macrophages, neutrophils, and lymphocytes were significantly greater in BLM-treated mice than in control animals that received saline intratracheally. Neither total nor differential cell counts differed between the mouse genotypes. (C) Twenty-one days after BLM challenge, WT mice showed significant increases in the counts of total cells, macrophages, and lymphocytes in BAL fluid compared with the control mice that received saline intratracheally. In IL-6–deficient mice, BLM-induced increases in total cells and macrophages were significantly attenuated compared with WT mice. All values are expressed as the mean ± SE (n = 6). *P < 0.05 was considered to be significantly different from the corresponding value of the control mice with saline instillation. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the WT mice.

 
Histopathological Determination of Lung Fibrotic Changes
To examine the effect of IL-6 deficiency on BLM-induced fibrotic changes, lung histology on Day 21 were evaluated. Representative microscopic findings after H-E and Masson's trichrome staining are shown in Figure 3. Lung pathology after H-E staining demonstrated marked thickening of the alveolar septa and infiltration predominated by mononuclear cells (Figure 3A). Masson's trichrome staining for collagen expression revealed that intratracheal BLM induced significant fibrotic change in the lung, which was mitigated in IL-6–/– mice (Figure 3B).


Figure 3
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  		Figure 3. Representative examples of lung pathology 21 days after the instillation of saline or BLM. (A) Hematoxylin-eosin stain. (B) Masson's trichrome stain. Original magnification: x200.

 
The Ashcroft sores for more quantitative assessment of the lung fibrotic changes were shown in Figure 4. In WT mice, the score was significantly higher in those treated with BLM than in those with saline (P < 0.05). The IL-6 deficiency significantly decreased the increase in the score after BLM challenge, compared with the WT mice (P < 0.05).


Figure 4
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  		Figure 4. The Ashcroft sores for quantitative assessment of the lung fibrotic changes 21 days after the instillation of saline or BLM. In WT mice (open bars), the score was significantly higher in those treated with BLM than in those with saline. In IL-6–deficient mice (solid bars), BLM-induced increase revealed significantly decreased the score after BLM challenge. All values are expressed as the mean ± SE (n = 6). *P < 0.05 was considered to be significantly different from the corresponding value of the control mice with saline instillation. {dagger}P < 0.01 was considered to be significantly different from the corresponding value of the WT mice.

 
Collagen Contents in the Lung in Chronic Phase
The lungs harvested on Day 21 were analyzed for collagen content (Figure 5). Intratracheal BLM caused significant increase in the collagen content of the lungs compared with those administered with saline (P < 0.05). The collagen contents in the lungs were significantly less in IL-6–/– mice than in WT mice (P < 0.05).


Figure 5
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  		Figure 5. Collagen contents in the lung 21 days after the instillation of saline or BLM. Intratracheal BLM caused significant increase in the collagen content of the lungs, which was significantly less in IL-6–/– mice (solid bars) than in WT mice (open bars). All values are expressed as the mean + SE (n = 6). *P < 0.05 was considered to be significantly different from the corresponding value of the control mice with saline instillation. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the WT mice.

 
Levels of TGF-β1 and CCL3 in the Lung
To validate the effect of IL-6 deficiency on BLM-induced up-regulation of the mediators associated with fibrosis, the levels of TGF-β1 and CCL3 were measured in lung homogenate with ELISA, and the results are shown in Figure 6. On Day 2, the TGF-β1 level in the lung was significantly increased in the BLM-treated mice (Figure 6A). The increase in the lung TGF-β1 level after BLM challenge was completely suppressed in the IL-6–/– mice. On Day 7 and after, the TGF-β level in the lung did not differ between the study groups.


Figure 6
Figure 6
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  		Figure 6. The levels of TGF-β1 and CCL3 in the lung sampled 2, 7, or 21 days after intratracheal challenge of saline or BLM. (A) TGF-β1 level was elevated only in WT mice 2 days after the BLM instillation, which was similar to the levels in the saline controls on Day 7 and after. (B) CCL3 level in the lung was not significantly changed by the BLM challenge. The levels were normalized to total protein and are expressed as the mean + SE (n = 6). *P < 0.05 was considered to be significantly different from the corresponding value of the control mice with saline instillation. {dagger}P < 0.05 was considered to be significantly different from the corresponding value of the WT mice.

 
In the BLM-treated mice, the levels of CCL3 in the lung were elevated 7 days after the challenge, but there was no difference between the genotypes (Figure 6B). In WT mice, the BLM-induced elevation of CCL3 level was still significant 21 days after the administration, whereas the CCL3 levels in IL-6–/– mice were not different between the BLM-treated mice and those with saline instillation.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, it was found that IL-6 deficiency attenuated BLM-induced inflammatory cell accumulation and subsequent fibrotic changes of the lung. Decreased expression of the fibrosis-related mediators TGF-β1 and CCL3 was observed in IL-6–/– mice after BLM administration, although the differences were observed at different time points. These results indicate a critical role for IL-6 in the development of the pulmonary inflammatory response and fibrosis after a nonbacterial stimulus.

IL-6 has been shown to be involved in the pathogenesis of various inflammatory disorders of the lung, such as ARDS, pulmonary fibrosis, and chronic obstructive pulmonary disease (7, 8, 10, 17). IL-6–deficient mice demonstrated attenuation in ozone-induced neutrophil accumulation and subepithelial fibrosis after exposure to aerosolized antigen, whereas IL-6 deficiency resulted in enhancement of endotoxin-induced pulmonary hemorrhage with neutrophilic inflammation (9, 18, 19). In this study, we demonstrated for the first time the contribution of IL-6 to the development of BLM-induced lung injury using IL-6–deficient mice. We speculate that the role of IL-6 in inflammatory disorders may be different between the types of the insult.

In the present study, BLM-induced accumulation of macrophages and neutrophils on Day 2 was significantly reduced by IL-6 deficiency. Alveolar macrophages are known to play a critical role in events associated with BLM-induced pulmonary fibrosis (20). On the other hand, it was reported that granulocyte colony-stimulating factor, which enhanced neutrophil infiltration in the lungs, exacerbated BLM-induced lung injury and subsequent fibrosis in rats (21). In this study, since the proportion of neutrophils in the total inflammatory cells that accumulated into the alveolar space after BLM was small, the reduction of macrophage infiltration is likely to be a key mechanism of the attenuation of the pathologic changes in IL-6–deficient mice.

The BLM toxicity is related to the production of free radicals that result in endothelial and epithelial cell damage, the appearance of DNA damage–inducible proteins, increased microvascular permeability, and respiratory distress (22). It has been shown that the chemokine/cytokine network is capable of modulating the different phases of lung fibrosis pathogenesis, namely inflammation and fibrogenesis (6, 14). Among the several cytokines and chemokines that have been implicated in the pathogenesis of BLM toxicity, particular relevance has been given to TGF-β1 (13, 23, 24). BLM produces an initial inflammatory response marked by elevated levels of TGF-β1 (25), a finding that is compatible with the results of the present study. TGF-β1, which is elevated in BAL fluid from patients with ARDS, causes enhanced alveolar epithelial repair in vitro (26, 27). Given that fibroproliferative ARDS is a pathologic form of tissue repair, TGF-β1 is also considered as a key participant of the progressive lung fibrosis in ARDS (28). Although its precise role is still under investigation, TGF-β1 may contribute to the repair of lung injury, but, when in excess, to subsequent fibroproliferative changes as well.

In this study, we revealed that IL-6 deficiency significantly suppressed the BLM-induced up-regulation of TGF-β1 on Day 2. In a previous study, it was reported that there was a difference in the fibrogenic response between the BLM-sensitive and -insensitive genotypes, which could be related in part to the different expression of IL-6 and TGF-β1 (29). Although there has been no report showing the induction of TGF-β1 by IL-6 in vitro, it has been demonstrated that TGF-β1 can be a stimulus for IL-6 release from fibroblasts (30, 31). Although it remains to be determined whether IL-6 acts directly on fibrocytes or via up-regulation of TGF-β1 or other mediator, the results indicated that BLM-induced TGF-β1 expression might be dependent, at least in part, on IL-6.

In addition to TGF-β1, a relative contribution to lung fibrosis of members of the CC chemokines, such as monocyte chemotactic protein-1 (MCP-1/CCL2) and CCL3, has been proposed (14, 32, 33). In this study, CCL3 was not elevated in the lung 2 days after the BLM challenge, but it significantly rose by Day 7. CCL3, a chemokine responsible for mononuclear cell recruitment, is secreted by a variety of cell types such as lymphocytes, macrophages, fibroblasts, and endothelial cells (14). CCL3 is significantly up-regulated both in lung homogenate and BAL fluid during BLM-induced lung fibrosis (33). It has also been demonstrated that neutralization of IL-6 resulted in a significant decrease in CCL3 expression at 2 days, but not 10 days, after BLM challenge (34). Recently, Ishida and colleagues reported that CCL3 is expressed mainly in infiltrating granulocytes and macrophages after BLM administration, whereas TGF-β1 is detected in macrophages and myofibroblasts (35). They also revealed that, in CCL3-deficient mice, BLM-induced collagen accumulation and the increases in intrapulmonary macrophage and fibrocyte numbers were attenuated (35). In the chronic phase, we observed IL-6–dependent elevation of CCL3 levels with increased collagen content in the lung, whereas the TGF-β1 level was not different. Taken together, IL-6 may contribute to up-regulation of CCL3, resulting in chronic infiltration of macrophages and fibrocytes.

One of the limitations of this study is that the effect of IL-6 deficiency on the molecular pathways remains unclear. Although BLM-induced up-regulation of TGF-β1 and CCL3 was suppressed in IL-6–deficient mice, the change is not considered particularly compelling. Since IL-6 has been shown to up-regulate various mediators, it is possible that IL-6 deficiency might influence the production of mediators other than those examined in this study. In addition, molecular pathways other than the inflammatory mediators could also be affected by IL-6 deficiency. For example, there have been several reports concerning the effect of IL-6 on proliferation and apoptosis of fibroblasts. Shahar and coworkers reported that blockade of IL-6 with monoclonal antibody suppressed the proliferative capacity of fibroblasts in patients with pulmonary fibrosis (36). Whereas IL-6 promotes apoptosis and inhibits proliferation of fibroblasts from normal subjects, it has mitogenic and anti-apoptotic effects on fibroblasts from patients with pulmonary fibrosis (37, 38). These findings suggest that altered IL-6 signaling may contribute to a profibrotic effect of IL-6 in patients with pulmonary fibrosis.

In conclusion, IL-6 may play an important role in the pathogenesis of BLM-induced lung injury and subsequent fibrotic changes. IL-6 deficiency attenuated BLM-induced up-regulation of TGF-β1 and CCL3. Specific inhibitors of IL-6, such as tocilizumab, could be considered as a candidate of a therapeutic modality for noninfectious lung injury and subsequent fibrotic changes.


   Acknowledgments
 
The authors thank Miho Sakurai and Eiko Koike for their technical assistance.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2007-0299OC on December 20, 2007

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 August 4, 2007

Accepted in final form November 13, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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