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Published ahead of print on April 25, 2008, doi:10.1165/rcmb.2007-0330OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 440-447, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0330OC

The Role of High Mobility Group Box1 in Pulmonary Fibrosis

Naoki Hamada1, Takashige Maeyama1, Tomonobu Kawaguchi1, Michihiro Yoshimi1, Jyutaro Fukumoto1, Mizuho Yamada1, Singo Yamada2, Kazuyoshi Kuwano3 and Yoichi Nakanishi1

1 Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2 Central Institute, Shino-Test Corporation, Kanagawa, Japan; and 3 Division of Respiratory Diseases, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Kazuyoshi Kuwano, M.D., Division of Respiratory Diseases, Department of Internal Medicine, Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, 105-8641, Tokyo, Japan. E-mail: kkuwano{at}jikei.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
High mobility group box1 protein (HMGB1) was originally discovered as a nuclear binding protein, and is known to play an important role in acute lung injury. However, the role of HMGB1 in pulmonary fibrosis has not been addressed. Therefore, we measured the HMGB1 levels in serum and bronchoalveolar lavage fluids (BALF) from patients with idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia, interstitial pneumonia associated with collagen vascular diseases, and hypersensitivity pneumonitis (HP) by enzyme-linked immunosorbent assay. We also assessed the HMGB1 expression in bleomycin-induced pulmonary fibrosis in mice, and examined the effect of anti-HMGB1 antibody and ethyl pyluvate, which inhibits the HMGB1 secretion from alveolar macrophages. In addition, we examined the effect of HMGB1 on fibroblast proliferation, apoptosis, and collagen synthesis in vitro. Serum HMGB1 levels were not significantly increased in interstitial lung diseases compared with control subjects. BALF HMGB1 levels were significantly increased in IPF and HP compared with control subjects. HMGB1 protein was predominantly detected in inflammatory cells and hyperplasic epithelial cells in IPF. In bleomycin-induced pulmonary fibrosis in mice, HMGB1 protein was predominantly up-regulated in bronchiolar epithelial cells at early phase and in alveolar epithelial and inflammatory cells in fibrotic lesions at later phase. Intraperitoneal injection of anti-HMGB1 antibody or ethyl pyluvate significantly attenuated lung inflammation and fibrosis in this model. HMGB1 significantly induced proliferation, but not apoptosis or collagen synthesis on cultured fibroblasts. HMGB1 may be a promising target against pulmonary fibrosis as well as acute lung injury.

Key Words: HMGB1 • pulmonary fibrosis • fibroblast



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We demonstrated that high mobility group box1 protein (HMGB1) was expressed in pulmonary fibrosis, and that anti-HMGB1 therapy significantly attenuated animal models of pulmonary fibrosis. HMGB1 may be a promising target against pulmonary fibrosis as well as acute lung injury.

 
High mobility group box1 protein (HMGB1) is one of the nuclear proteins and was originally defined as a transcription factor–like protein (1). HMGB1 supports other proteins, such as nuclear hormone receptor family, Hox, and POU, to associate with DNA, and facilitates the expression of several genes (2). The C-terminal domain and A-box of HMGB1 are critical for the enhancement of the p53-mediated transactivation and p53-dependent apoptosis (3). In addition to its role as a transcription factor–like protein, HMGB1 is currently thought to be a cytokine-like molecule. HMGB1 is present in nuclei of all mammarian cells, and is released from necrotic cells, or actively secreted from activated macrophages, dendritic cells, and natural killer cells (1). Receptors for HMGB1 are thought to include receptor for advanced glycation products (RAGE), Toll-like receptor 2 (TLR2), and TLR4 (4). Therefore, HMGB1 has multiple functions in infection, tissue injury, inflammation, apoptosis, and immune responses (1).

It is well known that HMGB1 plays an important role in the development of sepsis-associated lung injury in mice and in patients with sepsis (5). The ligation of HMGB1 to its receptors activates endothelial cells to up-regulate adhesion molecules, and activates macrophages to release TNF-{alpha} and IL-1β. In contrast to the early inflammatory mediators TNF-{alpha} and IL-1β, HMGB1 is a late mediator of sepsis-associated lung injury, and is associated with the prognosis of patients (6).

Although HMGB1 is well known to be an important molecule in the pathogenesis of acute lung injury, little is known about its role in chronic lung diseases, especially in the development of pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic fibrosing interstitial pneumonia associated with the histopathologic appearance of usual interstitial pneumonia on surgical lung biopsies. The median survival of patients with IPF is reported to be 3 to 4 years from the onset of respiratory symptoms (1). In spite of such a poor prognosis, the pathogenesis of IPF remains to be elucidated, and no effective therapeutic strategy has been established.

To investigate the significance of HMGB1 in the pathogenesis of pulmonary fibrosis, we measured the levels of HMGB1 in serum and bronchoalveolar lavage fluid (BALF) from patients with fibrosing lung diseases. We also investigated the expression of HMGB1 in lung tissues from patients with IPF and nonspecific interstitial pneumonia (NSIP) by immunohistochemistry. To verify the involvement of HMGB1 in pulmonary fibrosis, we examined the expression of HMGB1 and the effect of a neutralizing anti-HMGB1 antibody in bleomycin-induced pulmonary fibrosis in mice, which is an animal model of acute lung injury followed by pulmonary fibrosis. We also examined whether ethyl pyruvate (EP), which is known to inhibit the release of HMGB1 from activated macrophages (7), has a therapeutic effect against pulmonary fibrosis. In addition, we investigated the effect of HMGB1 on the proliferation, apoptosis, and collagen synthesis of cultured fibroblasts.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Study Population
This study was approved by the Ethical Committee of Kyushu University Faculty of Medicine. The diagnosis of IPF and NSIP was established according to previously described criteria (1). The thoracoscopic lung biopsy specimens were obtained from eight patients with IPF and from all patients with NSIP. In interstitial pneumonia associated with collagen vascular diseases (CVD-IP), there were 11 cases of rheumatoid arthritis, 6 cases of polymyositis/dermatomyositis, 2 cases of Sjögren's syndrome, and 1 case of progressive systemic sclerosis. The diagnosis of hypersensitivity pneumonitis (HP) was based on previously described criteria (8). All control subjects were healthy volunteers. A transbronchial lung biopsy was performed in all cases to rule out competing diagnosis. In all patients, a current infection with bacteria, mycobacteria, or fungi was excluded by negative cultures of BALF and biopsied specimens. BALF and serum were obtained at the time of diagnosis and before steroid therapy.

Model of Bleomycin-Induced Pneumopathy
The present experiments were approved by the Committee on Ethics regarding Animal Experiments of Kyushu University Faculty of Medicine, and were performed according to the guidelines of the American Physiological Society. Seven- to 8-week-old C57Bl/6 male mice were purchased from KBT Oriental (Saga, Japan) and used in all experiments. The body weights of the mice were 20 to 25 g. The mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (Schering-Plough, Osaka, Japan). The anesthetized mice received 50 µl of bleomycin hydrochloride (Nippon Kayaku, Tokyo, Japan) solution containing 3 U bleomycin/kg body weight in sterile saline intratracheally. Polyclonal chicken IgY anti-HMGB1 neutralizing antibody and a control chicken IgY antibody were gifts from Shino-Test (Sagamihara, Kanagawa, Japan). The dose of a neutralizing antibody was determined according to the protocols as previously described (9). Anti-HMGB1 neutralizing antibody (200 µg/body) or a control chicken IgY antibody (200 µg/body) was injected intraperitoneally at 5 days after bleomycin instillation. The dose of ethyl pyruvate was determined according to the protocols as previously described (7). Briefly, ethyl pyruvate (EP) (40 mg/kg) was injected intraperitoneally every day from 3 to 13 days after bleomycin instillation. The mice were killed at 14 days after the bleomycin instillation, and whole blood was collected. The right lung tissues were fixed in 10% buffered formalin, while the left lung tissues were snap-frozen in liquid nitrogen and stored at –80°C until use.

BAL Procedure
In patients, BAL was performed as previously described (10). Briefly, BAL was performed using a total of 150 ml of sterile physiologic saline solution. In mice, a tracheotomy was performed in killed mice. After insertion of a tracheal tube, the trachea was lavaged twice with 1-ml volumes of sterile saline at room temperature. The recovered fluids were filtered through a single layer of gauze to remove the mucus. The cells present in the lavage fluid were counted using a hemocytometer. Differential counts of BAL cells were performed on 200 cells stained with Diff-Quick (Baxter Diagnostics, Dearfield, IL). The lavage fluid supernatant was stored at –80°C until the measurement of HMGB1.

Immunohistochemistry for HMGB1
Three-micrometer-thick paraffin sections were adhered to slides pretreated with poly-L-lysine. After deparaffinization, immunohistochemistry was performed by a modified streptavidin-biotinylated peroxidase technique using a Histofine SAB-PO kit (Nichirei Corporation, Tokyo, Japan). Nonspecific protein staining was blocked with goat serum for 30 minutes at room temperature. The sections were incubated with an anti-HMGB1 antibody (Upstate, Lake Placid, NY) at 4°C overnight. For control incubations, the specific antibodies were replaced by nonimmune serum. The sections were counterstained with methyl green and mounted.

Histopathologic Examination in Mice
After thoracotomy, the pulmonary circulation was flushed with saline and the lungs were explored. The lung samples were fixed with 10% formalin overnight and embedded in paraffin. The pathologic grade of inflammation and fibrosis in the whole area of the midsagittal section was evaluated under x40 magnification, and determined according to the following criteria: 0, no lung abnormality; 1, presence of inflammation and fibrosis involving less than 25% of the lung parenchyma; 2, lesions involving 25 to 50% of the lung; and 3, lesions involving more than 50% of the lung.

DNA Damage and Apoptosis in Lung Tissues
The number of TUNEL-positive cells is correlated with lung injury and fibrosis in this model (11–13). DNA damage and apoptosis were assessed by the TUNEL method using the DeadEnd Colorimetric Apoptosis Detection System (Promega, Madison, WI) as previously described (14). The number of TUNEL-positive cells was counted in 20 randomly selected fields per section under a microscope at 200-fold magnification.

Enzyme-Linked Immunosorbent Assay for HMGB1, IL-1β, and TGF-β1 in BALF
Enzyme-linked immunosorbent assay (ELISA) for HMGB1 was performed with the use of monoclonal antibodies to HMGB1 as previously described (15). The lower detection limit of HMGB1 was 2 ng/L. IL-1β levels were measured with a cytokine-specific ELISA obtained from BioSource International (Camarillo, CA), and activated TGF-β1 levels were measured with an ELISA obtained from R&D Systems (Minneapolis, MN), according to the manufacturers' instructions. We performed all assays in duplicate, and the mean of two data was determined for individual sample.

Collagen Assay
Collagen content in lung homogenates and in supernatant of cell culture medium was measured using Sircor Collagen Assay kit (Biocolor, Northern Ireland, UK) as previously described (16). Supernatant of unstimulated cells and cells treated with 10 or 30 ng/ml HMGB1 for 24 hours were stored at –80°C.

Apoptosis Analysis by Flow Cytometry
A human lung fibroblast cell line (WI-38) was purchased from DS Pharma Biomedical (Osaka, Japan). WI-38 was derived from normal embryonic lung tissue. These cells were grown in 75 cm2 tissue culture flasks (FALCON, Franklin Lakes, NJ) in growth medium that consisted of Minimum Essential Medium with 10% fetal bovine serum. These cultures were incubated at 37°C in a humidified, 95% air/5% CO2 atmosphere. When the cells were subconfluent, cells were harvested by trypsinization and plated in another flask in the same medium. For analysis of apoptosis on WI-38 cells, cells were removed from the plate with trypsin/EDTA. Detached and floating cells were also recovered and included with those that were adherent when testing for apoptosis. Apoptosis was analyzed by Annexin V–FLUOS staining kit (Roche Diagnostics, Penzberg, Germany). Cells (106) were washed in PBS and resuspended in incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) and Annexin V–FITC and propidium iodide. After 20 minutes of incubation on ice, fluorescence was analyzed by a Coulter EPICS XL flow cytometer (Coulter, Miami, FL). Five samples for each group were used for apoptosis and collagen assay.

Cell Proliferation Assay
The proliferation of WI-38 cells was analyzed using Tetra color ONE assay kit (Seikagaku Corporation, Tokyo, Japan). WI-38 cells were incubated for 24 hours in 96-well tissue culture plates and treated with 10 or 30 ng/ml HMGB1(eight samples each) in a humidified atmosphere of 5% CO2 incubator at 37°C. After 24 hours, 10 ml tetra color ONE was added, the plate was incubated for 4 hours at 37°C, and absorbance was measured using an automated microplate reader at a wavelength of 540 nm (Easy Reader EAR 340; LABEQUIP Ltd., Markham, ON, Canada).

Statistics
For statistical analysis regarding comparisons of the number of BAL cells in BALF, the number of TUNEL-positive cells, body weight, results of ELISA, collagen content, the results of flow cytometry, and the results of MTT assay, ANOVA followed by Scheffe's F test was used. For comparison of the pathologic grade, Kruskal-Wallis test followed by Mann-Whitney's U-test was used. P values of less than 0.05 were considered significant. Statistical analysis was performed with StatView J-4.5 (Abacus Concepts Inc., Berkeley, CA).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
HMGB1 Levels in Serum and BALF in Interstitial Lung Diseases
Serum HMGB1 levels (mean ± SE) were 3.7 ± 1.9 in IPF, 0.1 ± 0.1 in NSIP, 0.8 ± 0.5 in CVD-IP, 4.1 ± 3.4 in HP, and 0.5 ± 0.3 ng/ml in control subjects. There was no significant difference in serum HMGB1 levels between interstitial lung diseases and control subjects (Figure 1a). BALF HMGB1 levels (mean ± SE) were 3.5 ± 0.6 in IPF, 4.9 ± 2.4 in NSIP, 2.2 ± 0.5 in CVD-IP, 9.4 ± 4.2 in HP, and 1.6 ± 0.4 ng/ml in control subjects. BALF HMGB1 levels were significantly higher in patients with IPF and HP compared with control subjects (P < 0.05, P < 0.01, respectively) (Figure 1b).


Figure 1
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  		Figure 1. High mobility group box1 protein (HMGB1) levels in serum and bronchoalveolar lavage fluid (BALF) from patients with interstitial lung diseases and normal volunteers. Bars show the mean of HMGB1 levels in each group.

 
Expression of HMGB1 in Lung Tissues from Patients with IPF and NSIP
The results of immunohistochemistry show that HMGB1 expression is not detectable in normal lung parenchyma, except few alveolar macrophages (Figure 2a). HMGB1 was predominantly found in nuclei of infiltrating inflammatory cells, alveolar macrophages, and epithelial cells in affected lesions of IPF and NSIP, and positive staining in cytoplasm was also found in some of these cells (Figures 2b, 2c, and 2d).


Figure 2
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  		Figure 2. The representative results of immunohistochemistry for HMBG1 in (a) normal lung parenchyma, (b) lung tissues from patients with nonspecific interstitial pneumonia (NSIP), and those from patients with idiopathic pulmonary fibrosis (IPF) with (a) lower magnification and (d) higher magnification. Arrows in b show nuclear staining in cells of alveolar wall. Arrow in d shows nuclear staining of epithelial cells. Original magnification: a, x100; b and d, x200; c, x40.

 
Expression of HMGB1 in Bleomycin-Induced Pulmonary Fibrosis
The results of immunohistochemistry show that HMGB1 expression was detected in some nuclei of bronchiolar epithelial cells in untreated mice (Figure 3a). The expression of HMGB1 was up-regulated in bronchiolar epithelial cells at 1 day after bleomycin instillation (Figure 3b). The positive signals were found in cytoplasm as well as nuclei of alveolar epithelial cells and inflammatory cells as well as bronchiolar epithelial cells at 5 days (Figure 3c). Positively stained bronchiolar epithelial cells appeared to be decreased, whereas positively stained alveolar epithelial cells and inflammatory cells appeared to be increased at 7 to 14 days. The positively stained cells were predominantly detected in inflammatory and fibrotic lesions at 7 to 14 days (Figures 3d and 3e). Arrows in Figures 3f and 3g show positive staining on cytoplasm of macrophages and bronchiolar epithelial cells, respectively.


Figure 3
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  		Figure 3. The representative results of immunohistochemistry for HMBG1 in lung tissues from (a) untreated mice and from mice at (b) 1 d, (c) 5 d, (d) 7 d, and (e) 14 d, and (f) at 14 d with higher magnification, after bleomycin instillation. Arrows in f and g show cytoplasmic staining of macrophages and bronchiolar epithelial cells, respectively. Original magnification: a–e, x100; f and g, x400.

 
Effect of Neutralizing Anti-HMGB1 Antibody on Bleomycin-Induced Pulmonary Fibrosis
Body weight. The body weight at 14 days was significantly increased in mice treated with anti-HMGB1 antibody (n = 8) compared with those of mice treated with control antibody (n = 7) after bleomycin instillation (Figure 5a).

Histologic findings. The alveolar wall had begun to thicken with infiltration of neutrophils and lymphocytes at 7 days after the bleomycin instillation, compared with that of untreated mice. At 14 days, a large number of lymphocytes had infiltrated into the lung interstitium, and thickening of the alveolar septa, collapse of the alveolar spaces, and accumulation of fibroblasts were observed (Figure 4a). Anti-HMGB1 antibody attenuated the histopathologic findings at 14 days (Figure 4c). Semi-quantification of the histologic analysis showed that anti-HMGB1 antibody significantly decreased the pathologic grade at 14 days (n = 8) compared with mice treated with control antibody (n = 7) (Figure 5b).


Figure 4
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  		Figure 4. Effect of anti-HMGB1 antibody administration on pathological findings and the results of TUNEL staining. Representative results of (a) histologic findings and (b) TUNEL straining at Day 14 after the bleomycin instillation plus control antibody. Arrows in b show TUNEL positive cells. The effect of anti-HMGB1 antibody administration on the (c) histopathologic findings and (d) TUNEL stainings at 14 days. Original magnification: a and c, x40; b and d, x200.

 

Figure 5
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  		Figure 5. The effect of anti-HMGB1 antibody on bleomycin-induced pulmonary fibrosis in mice. (a) Shaded bar shows the body weight of untreated mice (n = 5). Solid and open bars show body weight of mice at 14 days after bleomycin instillation treated with anti-HMGB1 antibody (n = 8) or control antibody (n = 7), respectively. Data was shown as mean ± SE, *P < 0.01. (b) Solid and open circles show pathologic grade of each mouse at 14 days after bleomycin instillation treated with anti-HMGB1 antibody (n = 8) or control antibody (n = 7), respectively; {dagger}P < 0.05. (c) Solid and open bars show the number of TUNEL-positive cells in lung tissues of mice at 14 days after bleomycin instillation treated with anti-HMGB1 antibody (n = 8) or control antibody (n = 7), respectively. Data are shown as mean ± SE; *P < 0.01. (d) Solid and open bars show collagen content of lung homogenates from mice at 14 days after bleomycin instillation treated with anti-HMGB1 antibody (n = 8) or control antibody (n = 7), respectively. Shaded bar shows collagen content of untreated mice (n = 5). Data are shown as mean ± SE; {dagger}P < 0.05. (e) The effect of anti-HMGB1 antibody on the results of BAL cell analysis. Solid and open bars show the number of BAL cells from mice at 14 days after bleomycin instillation treated with anti-HMGB1 antibody (n = 5) or control antibody (n = 5), respectively. Shaded bar shows the results of untreated mice (n = 5). (f, g, h) The effect of anti-HMGB1 antibody on HMGB1, IL-1β, and TGF-β1 levels in BALF, respectively. Solid and open bars show the results of mice at 14 days after bleomycin instillation treated with anti-HMGB1 antibody (n = 8) or control antibody (n = 7), respectively. Shaded bars show the results of untreated mice (n = 5). Data are shown as mean ± SE; *P < 0.01, {dagger}P < 0.05.

 
TUNEL staining. Although the type of cells was not clearly identified, some of the bronchiolar and alveolar epithelial cells or inflammatory cells in the inflammatory lesions showed evidence of DNA damage and apoptosis at 14 days after the bleomycin instillation (Figure 4b). Anti-HMGB1 antibody markedly reduced the number of TUNEL-positive signals at 14 days (n = 8) compared with that of mice treated with control antibody (n = 7) (Figures 4d and 5c). There were a few positive cells in bronchiolar epithelial cells in whole lung field in untreated mice.

Collagen content. Collagen content in lung homogenates was significantly decreased in mice treated with anti-HMGB1 antibody (n = 8) compared with those of mice treated with control antibody (n = 7) (Figure 5d).

BALF analysis. The numbers of total cells, macrophages, neutrophils, and lymphocytes in BALF were significantly increased at 14 days in bleomycin-instilled mice compared with those in untreated mice. When anti-HMGB1 antibody was administered, the numbers of total cells and neutrophils were significantly decreased in the BALF at 14 days, whereas numbers of macrophages and lymphocytes were not significantly changed compared with those in mice treated with control antibody (Figure 5e). BAL procedure was performed in five mice for each group. The HMGB1 and IL-1β, but not TGF-β1, levels in BALF were significantly decreased in mice treated with anti-HMGB1 antibody (n = 8) compared with those in mice treated with control IgG (n = 7) (Figures 5f–5h).

Effect of Ethyl Pyruvate on Bleomycin-Induced Pulmonary Fibrosis
HMGB1 levels. HMGB1 levels in serum were not decreased in mice treated with ethyl pyruvate (n = 15) compared with those in mice treated with Ringer solution (n = 10) (Figure 6a). HMGB1 levels in BALF were significantly decreased in mice treated with ethyl pyruvate (n = 15) compared with those in mice treated with Ringer solution (n = 10) (Figure 6b).


Figure 6
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  		Figure 6. The effect of ethyl pyruvate (EP) on bleomycin-induced pulmonary fibrosis in mice. (a and b) Solid and open bars show HMGB1 levels in serum or BALF from mice at 14 days after bleomycin instillation treated with EP (n = 15) or Ringer solution (n = 10), respectively. HMGB1 levels in serum or BALF from untreated mice (n = 5) were not detectable in untreated mice (c) in panels a and b. Data are shown as mean ± SE; {dagger}P < 0.05. (c) Shaded bar shows the body weight of untreated mice (n = 5). Solid and open bars show body weight of mice at 14 days after bleomycin instillation treated with EP (n = 15) or Ringer solution (n = 10), respectively. Data are shown as mean ± SE; *P < 0.01. (d) Solid and open circles show pathologic grade of each mouse at 14 days after bleomycin instillation treated with EP (n = 11) or Ringer solution (n = 10), respectively; {dagger}P < 0.05. (e) The effect of EP on the results of BAL cell analysis. Solid and open bars show the number of BAL cells from mice at 14 days after bleomycin instillation treated with EP (n = 5) or Ringer solution (n = 5), respectively. Shaded bars show the results of untreated mice (n = 5). Data were shown as mean ± SE; *P < 0.01, {dagger}P < 0.05.

 
Body weight. The body weight at 14 days was significantly increased in mice treated with ethyl pyruvate (n = 15) compared with mice treated with Ringer solution (n = 10) (Figure 6c).

Histologic findings. The administration of ethyl pyruvate attenuated the histopathologic findings at 14 days compared with controls. Semi-quantification of the histologic analysis showed that the administration of ethyl pyruvate significantly decreased the pathologic grade at 14 days (n = 11) compared with controls (n = 10) (Figure 6d).

BALF analysis. When EP was administered, numbers of total cells, neutrophils, and lymphocytes were significantly decreased in the BALF at 14 days, whereas the number of macrophages was not significantly changed compared with those in controls. BAL procedure was performed in five mice for each group (Figure 6e).

Effect of HMGB1 on Proliferation, Apoptosis, and Collagen Synthesis of Fibroblasts In Vitro
HMGB1 did not induce apoptosis on WI-38 cells (Figure 7a). HMGB1 significantly increased the number of viable WI-38 cells as much as three to four times compared with controls at 24 hours (Figure 7b). HMGB1 did not affect collagen content of supernatant of WI-38 cells (Figure 7c).


Figure 7
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  		Figure 7. The effect of HMGB1 on fibroblast apoptosis, proliferation, and collagen synthesis. Open bars show the result of untreated cells at 24 hours. Shaded and solid bars show the results of 10 and 30 ng/ml HMGB1 treatments for 24 hours. Data are shown as mean ± SE; *P < 0.01, {dagger}P < 0.05.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We investigated the role of HMGB1 in the pathogenesis of pulmonary fibrosis. There was no significant difference in serum HMGB1 levels between patients with interstitial lung diseases and control subjects. HMGB1 levels in BALF were significantly increased in IPF and HP, although all patients with IPF had a stable disease and no patient with IPF was representing acute exacerbation of the disease. There were some patients with NSIP with high levels of HMGB1 levels in BALF compared with control subjects, although there was no significant difference between patients with NSIP and control subjects. Since the number of patients was small, it remains to be determined whether HMGB1 levels in BALF may be associated with the prognosis of patients with IPF and NSIP.

HMGB1 was predominantly found in nuclei of infiltrating inflammatory cells, alveolar macrophages, and epithelial cells in affected lesions of IPF and NSIP, and positive staining in cytoplasm was also found in some of these cells. These results suggest that HMGB1 expression is up-regulated in affected lesions and that secreted HMGB1 may play a role as a proinflammatory cytokine in interstitial lung diseases. The cytoplasmic staining for HMGB1 in lung tissues appears to be reflected by the high levels of HMGB1 in BALF and the inflammatory cell accumulation in strongly stained areas, as also found in bleomycin-induced pulmonary fibrosis in mice. In addition, the intratracheal administration of HMGB1 induces the increase of proinflammatory cytokines such as IL-1β, TNF-{alpha}, and MIP2 in lung tissues (6).

HMGB1 in nuclei may play as a transcription factor in interstitial lung diseases. We previously demonstrated that DNA damage and apoptosis were associated with the up-regulation of p53 and p21 protein in hyperplasic epithelial cells in IPF, but not in normal lung parenchyma (17). HMGB1 was reported to be critical for the enhancement of the p53-mediated transactivation and p53-dependent apoptosis (3). HMGB1 may play a role as a transcription factor through the p53-mediated mechanism.

We found that some nuclei of normal airway epithelial cells in mice were positively stained for HMGB1. HMGB1 expression was up-regulated in both nuclei and cytoplasm of bronchiolar epithelial cells after bleomycin instillation. HMGB1 expression in alveolar epithelial cells and macrophages was gradually increased after 5 days. HMGB1 expression was predominantly detected in nuclei and cytoplasm of infiltrating inflammatory cells and epithelial cells in inflammatory and fibrotic lesions at 7 to 14 days after bleomycin instillation. HMGB1 in cytoplasm of epithelial cells as well as macrophages may contribute to acute lung injury and fibrosis in this model. Similar results were also found in various diseases in other organs. The strong positive signals for HMGB1 were detected in nuclei and cytoplasm of mononuclear cells in inflammatory synoviocytes and chondrocytes from rat models of arthritis and from patients with rheumatoid arthritis (18, 19).

To investigate the role of HMGB1 in pulmonary fibrosis, we tried to inhibit HMGB1 signaling using neutralizing antibody in an animal model. Kim and coworkers demonstrated that anti-HMGB1 antibody prevented NF-{kappa}B activation, proinflammatory cytokine production, and lung permeability in acute lung injury after hemorrhage (9). Anti-HMGB1 antibody significantly reduced the body weight loss, pathologic grade, the number of TUNEL-positive cells, lung collagen content, the number of total cells and neutrophils in BALF, BALF HMGB1, and IL-1β levels at 14 days. Gasse and colleagues previously demonstrated that the production of inflammatory mediators such as IL-1β, KC, and IL-6, and the activation of MMP-9, MMP-2, and TIMP-1, are mediated by IL-1R1/MyD88 signaling. They concluded that IL-1β is a critical inflammatory mediator of acute inflammation, remodeling, and fibrosis upon bleomycin-induced lung injury (20). Although we could not find a significant decrease of TGF-β in BALF, the decrease of IL-1β could affect the expression and activation of so many factors associated with bleomycin-induced pulmonary fibrosis. The effects of HMGB1 upstream and downstream of the IL-1β signaling associated with apoptosis and fibrosis remain to be investigated more precisely in bleomycin-induced pulmonary fibrosis.

Furthermore, we investigated whether HMGB1 can directly stimulate fibroblast survival and collagen synthesis in vitro. We found that HMGB1 stimulated proliferation of human fibroblasts, although HMGB1 did not have any effect on apoptosis or collagen synthesis. HMGB1 was reported to induce the proliferation of human fibroblast cell line NIH/3T3 (21). In addition to its role as a trigger of inflammation, HMGB1 could directly stimulate fibroblast proliferation and participate in fibrogenesis.

In addition, we tried to suppress the release of HMGB1 from macrophages using ethyl pyruvate. Ulloa and coworkers demonstrated that ethyl pyruvate inhibited the release of TNF-{alpha} and HMGB1 from macrophages through interfering with the activation of both the p38MAPK and NF-{kappa}B signaling pathways. They also demonstrated that ethyl pyruvate treatment rescued animals from lethal sepsis (7). We added ethyl pyruvate at 3 to 13 days after bleomycin instillation to verify the effect of ethyl pyruvate on the development of this model. Ethyl pyruvate treatment significantly reduced HMGB1 levels, the number of total cells, lymphocytes, and neutrophils in BALF at 14 days. Finally, ethyl pyruvate treatment significantly attenuated body weight loss and pathologic grade in this model. Although it is not clear whether the attenuation of this model is due to the inhibition of release of HMGB1 from macrophages or the direct inhibition of p38 MAPK and NF-{kappa}B signaling pathway, these results suggest that ethyl pyruvate may not only be an effective inhibitor of in vivo model of lethal sepsis, but also has an inhibitory potential of lung injury and fibrosis.

There are other possibilities for anti-HMGB1 therapy to prevent the development of lung injury and fibrosis. First, Palumbo and colleagues demonstrated that extracellular HMGB1 and its receptor RAGE induce both migration and proliferation of vessel-associated stem cells, and thus may play a role in muscle tissue regeneration (22). Smooth muscle cell proliferation is one of characteristics of pulmonary fibrosis. Second, HMGB1 is known to activate macrophages to produce angiogenesis factors, such as vascular endothelial growth factor (VEGF), TNF-{alpha}, and IL-8 (23, 24). Schlueter and coworkers demonstrated that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner (25). Angiogenesis was reported to be involved in the pathogenesis of bleomycin-induced pulmonary fibrosis in mice (26, 27). Third, Dumitriu and colleagues demonstrated that dendritic cells actively release their own HMGB1 upon activation and induce the maturation of dendritic cells themselves. Dendritic cell maturation is required for T cell survival and proliferation (28). Although the involvement of T cells in the development of lung injury and fibrosis is controversial, anti-HMGB1 therapy may attenuate this model through inhibiting T cell activation.

In conclusion, we demonstrated here that HMGB1 levels in BALF are significantly increased in IPF and HP compared with control subjects. HMGB1 is predominantly expressed in alveolar macrophages, infiltrating inflammatory cells, and epithelial cells in lung tissues from patients with IPF. Anti-HMGB1 antibody or ethyl pyruvate protected mice from bleomycin-induced pulmonary fibrosis through attenuating inflammation, apoptosis, and fibrosis. HMGB1 could directly stimulate fibroblast proliferation in vitro. Although the precise role of HMGB1 in pulmonary fibrosis remains to be verified, inhibition of HMGB1 function may be an effective strategy against pulmonary fibrosis.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research (19390225) from the Ministry of Education, Science and Culture of Japan.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0330OC on April 25, 2008

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 10, 2007

Accepted in final form March 24, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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