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Interferon-ß Inhibits Bleomycin-Induced Lung Fibrosis by Decreasing Transforming Growth Factor-ß and Thrombospondin

Fourth Department of Internal Medicine, Nippon Medical School, and First Department of Internal Medicine, Nihon University School of Medicine, Tokyo; Pharmaceutical Research Laboratories, Toray Industries, Inc.; and Department of BioResearch, Kamakura TechnoScience, Inc., Kanagawa, Japan

Address correspondence to: Arata Azuma, M.D., Ph.D., Fourth Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan. E-mail: a-azuma{at}nms.ac.jp

	Abstract

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Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-ß is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-ß did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-ß is a potent inhibitor of fibrogenesis, beneficial effects of IFN-ß have been expected. We therefore attempted to determine effects of IFN-ß and investigated the mechanism of action of IFN-ß in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-ß for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-ß. Tissue distribution and amounts of TGF-ß1 and thrombospondin (TSP)-1/2 were analyzed. IFN-ß attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-ß1 in epithelial cells and extracellular matrix was attenuated by IFN-ß. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-ß is inhibition of TGF-ß and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.

Key Words: interstitial pneumonia • TGF-ß • thrombospondin • interferon-ß • bleomycin

	Introduction

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Pulmonary fibrosis is thought to result from a type of abnormal repair that occurs after various sorts of lung injury. Although the pathogenesis of pulmonary fibrosis is not well understood, transforming growth factor (TGF)-ß is a key molecule in the progression of the type of pulmonary fibrosis typically termed idiopathic pulmonary fibrosis (IPF) (1). The ATS/ERS International Consensus Statement on idiopathic interstitial pneumonias was issued based on recent developments in and worldwide use of high-resolution computed tomography (HRCT) and histopathologic observation of lung biopsy specimens through video-assisted thoracoscopic surgery (VATS) (2). Although diagnosis with these modalities has made considerable progress, understanding of the entire course of disease of IPF has yet to be achieved and no appropriate treatment has been established for this disease despite a number of clinical trials. Interferon (IFN)-ß is expected to be a potent inhibitor of fibrotic disorders, as strongly suggested by findings for it such as inhibition of fibroblast proliferation (3, 4) and attenuation of procollagen mRNA and collagen synthesis (5, 6), although a clinical trial of IFN-ß 6,000,000 units three times a week did not yield improvement of survival of patients with IPF (7).

Bleomycin-induced pulmonary fibrosis in animals is a widely used experimental model of pulmonary fibrosis in humans (8–10). Although the mechanisms of bleomycin-induced pulmonary fibrosis are not understood in detail, it has been reported that fibrotic cytokines including TGF-ß1 play important roles in the process of proliferation of fibroblasts in lung tissue (11). Thrombospondin (TSP)-1/2 are potent activators of TGF-ß usually located in platelets at rest, and are induced in response to injury (12).

In this study, we investigated the effects of IFN-ß on bleomycin-induced pulmonary fibrosis in mice and evaluated the mechanisms of action of IFN-ß in promoting fibrosis, and obtained findings that may be useful for determining how IFN-ß can be used to treat IPF in humans.

	MATERIALS AND METHODS

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Animal Model of Pulmonary Fibrosis
Male ICR mice (6–8 wk old) and male C57BL/6 mice (6–8 wk old) were purchased from Charles River Japan (Yokohama, Japan). ICR mice were used for histologic and chemical evaluations, while C57BL/6 mice were used for immunohistochemical analysis and Western blot evaluation. They were maintained in specific pathogen–free conditions and provided with food and water ad libitum. To induce pulmonary fibrosis, mice were treated with a single intravenous injection of 100 or 150 mg/kg bleomycin (Nippon Kayaku, Tokyo, Japan) on Day 0. Control animals received only sterile saline. IFN-ß was administered at 10,000 U/head intravenously every day for 4 wk. At 28 d after injection, animals were killed by exsanguination under anesthesia. Then the left lungs were removed for homogenization for chemical analysis or Western blot analysis and the right lungs excised for histologic or immunohistochemical analysis.

Lung Tissue Preparation
Bleomycin- or saline-treated left lungs that had been stored at –70°C were homogenized in lysis buffer solution including 0.5 M acetic acid. Specimens were centrifuged at 1,000 x g for 30 min, and frozen at –70°C until thawed for collagen and prolyl hydroxylase assays. Right lungs were fixed in 4% paraformaldehyde and embedded in paraffin for histologic and immunohistochemical analysis.

Collagen and Prolyl Hydroxylase Assay
Collagen contents in right lung tissue of ICR mice were measured as soluble collagen with Sircol Assay kits (Biocolor Ltd., Belfast, Northern Ireland). Prolyl hydroxylase activity in the right lung was determined with PANASSAY PH FYK kits (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). Each measurement was performed using the procedure indicated by the manufacturer.

Histologic and Immunohistochemical Analysis
C57BL/6 mice were killed, and their right lungs were dissected out and fixed in 4% paraformaldehyde in 0.1 mM phosphate buffer (pH 7.4). Slices 2 mm in thickness were obtained and then embedded in paraffin at 62°C. Sections (4 m) were cut on a microtome and mounted on slides. Paraffin sections were used for hematoxylin-eosin and immunohistochemical staining.

In histologic analysis using ICR mice, fibrosis was assessed using the Ashcroft method (13) as a scoring system. The degree of fibrosis in lung specimens was graded on a scale from 0 to 8, using the average of microscopic field scores. This system allows fibrosis to be measured in small samples of tissue (1 cm), and thus enables detailed determination of fibrotic changes in a lung. All assessments were performed in blind fashion by three independent investigators.

For immunohistochemical analysis, the following primary antibodies were used: goat anti–TSP-1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and rabbit anti–TGF-ß1 (Santa Cruz Biotechnology, Inc.). Deparaffinized sections were blocked for 30 min in 10% normal goat serum and 0.1% Triton X-100 in PBS, and then incubated with a primary antibody diluted in the same blocking solution overnight at 4°C. After three washes with PBS, bound antibodies were visualized by incubation with fluorochrome-conjugated secondary antibodies, fluorescein isothiocyanate (FITC)-labeled sheep anti-goat IgG or FITC-labeled sheep anti-rabbit IgG. After another three washes, the sections were mounted and examined by fluorescein microscopy.

Western Blot Analysis
Western blot analysis was performed for measurement of TGF-ß and TSP-1/2 in murine lung tissues in which vessels had been irrigated with normal saline to wash out remaining blood. Homogenized murine lung was lysed in 6 M urea and 2% SDS by sonication. Crude protein extracts were prepared by centrifugation to remove cell debris. After protein quantification using a BCA protein Assay Reagent (Pierce, Rockford, IL), protein samples (10 µg) were fractionated by SDS-polyacrylamide (12%) gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes.

On the membrane, TGF-ß and TSP-1/2 were probed with an anti-murine TGF-ß rabbit polyclonal antibody (sc-7892; Santa Cruz Biotechnology) and an anti-murine TSP-1/2 goat polyclonal antibody (sc-7653; Santa Cruz Biotechnology), respectively. The complexes were then incubated with horseradish peroxidase–conjugated rabbit anti-goat IgG (sc-2056; Santa Cruz Biotechnology). TGF-ß and TSP-1/2 were detected by chemiluminescence using ECL pulse (RPN2132; Amersham Biosciences, Buckinghamshire, UK) and visualized on X-ray film.

MTT Assay for Growth of MLg2908 Fibroblasts
A murine fibroblast cell line, MLg2908, originating from ddY mice, was assessed for TGF-ß enhanced growth under treatment with IFN-ß in the MTT assay. MLg2908 cells (1 x 10⁵ cells/ml) in RPMI 1640 with 10% FCS were incubated with 100 ng/ml of TGF-ß and IFN-ß at various concentrations (0, 10, 100, 1,000 U/ml) for 3 d. Dimethyl sulfoxide was added to all wells to dissolve the dark blue crystals. Optical density (560 nm) was measured on a Microplate Reader after incubation with [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (10 µl/100 µl medium) at 37°C for 4 h (14).

Statistical Analysis
Data were analyzed using JMP statistical software. Comparisons were performed using Dunnett's multiple test. Differences were considered significant at P < 0.05. All values are means ± SD.

	RESULTS

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Histopathologic Assessment of Pulmonary Fibrosis
Bleomycin induced pulmonary fibrosis predominantly in the subpleural area of ICR mouse lung. MuIFN-ß markedly attenuated bleomycin-induced pulmonary fibrosis at 28 d. The Ashcroft score (mean ± SD) of the MuIFN-ß–treated group (1.3 ± 1.3) was significantly lower than that of the bleomycin-alone group (3.0 ± 1.9)(P < 0.05) (Figure 1).

View larger version (42K): [in this window] [in a new window]   Figure 1. Histopathologic assessment of bleomycin-induced pulmonary fibrosis in ICR mouse lung. MuIFN-ß markedly attenuated bleomycin-induced pulmonary fibrosis at 28 d. Ashcroft score was significantly decreased in mice with combined treatment with MuIFN-ß (P < 0.05).

View larger version (38K): [in this window] [in a new window]   Figure 2. (a) Prolyl hydroxylase activity was significantly lower in the MuIFN-ß group than in the bleomycin-alone group (P < 0.05). (b) Soluble collagen content in the bleomycin + MuIFN-ß group was significantly lower than that in the bleomycin-alone group (P < 0.05).

Immunofluorescence Staining of TGF-ß1 in Lung Tissue
TGF-ß1 was stained with antibody conjugated with FITC (green). TGF-ß1 was strongly expressed in bronchiolar epithelial cells but only weakly in alveolar epithelial cells and extracellular matrix (ECM) in unstimulated condition (Figures 3a and 3b, and Table 1). The intensity of expression of TGF-ß1 was increased in ECM of bleomycin-treated murine lung (Figure 3c). The bleomycin-alone group exhibited prominent expression of TGF-ß1 in alveolar epithelial cells and ECM in pulmonary tissue (Figure 3c, Table 1). However, this expression was decreased to basal level by treatment with IFN-ß (Figure 3d, Table 1). These findings suggested that IFN-ß decreased the amount of TGF-ß1 in fibrotic tissue of murine lung, especially in ECM.

View larger version (28K): [in this window] [in a new window]   Figure 3. Immunohistochemical staining for TGF-ß1 (indicated by arrows). (a) TGF-ß1 expression was detected in bronchiolar epithelial cells and (b) also detected in alveolar epithelial cells in normal condition. (c) TGF-ß1 was strongly expressed in alveolar epithelial cells and extracellular matrix of bleomycin-injected lung. (d) However, bleomycin-induced immunofluorescense intensity of TGF-ß1 in alveolar epithelial cells and extracellular matrix was attenuated in lungs of mice treated with bleomycin and IFN-ß. Groups A, B, and C indicate the "normal saline group," "bleomycin-alone group," and "bleomycin and IFN-ß treatment group," respectively. a, bronchiolar epithelial cells, BLM(–); b, alveolar epithelial cells, BLM (–); c, extracellular matrix, BLM(+); d, decreased TGF-ß expression, BLM(+) and IFN-ß.

View larger version (58K): [in this window] [in a new window]   Figure 4. Immunohistologic staining for TSP-1/2. Green color indicates TSP-1/2 expression. In a, TSP-1/2 is constitutively expressed in platelets found in lumens of vessels in a normal mouse. (b and c) TSP-1/2 appeared in foamy cells (b, hematoxylin-eosin stain), but (d) disappeared from platelets in mouse lung after treatment with bleomycin alone. (e) TSP-1/2 expression also appeared in foamy cells, and further accumulated in platelets in vascular lumens of lungs of mice treated with IFN-ß. Groups A, B, and C indicate the "normal saline group," "bleomycin-alone group," and "bleomycin and IFN-ß treatment group," respectively. a, platelet, BLM (–); b and c, foamy cell, BLM(+).

View larger version (83K): [in this window] [in a new window]   Figure 5. Western blot analysis of TGF-ß and TSP-1/2 in murine lung tissues. (a) TGF-ß expression increased in the "bleomycin-alone" group, and increased expression of TGF-ß in lung tissue was decreased in the "bleomycin and IFN-ß treatment" group. (b) TSP-1/2 expression in lung tissue was increased by treatment with bleomycin. Increase in expression of TSP-1/2 by bleomycin was attenuated by combined treatment with IFN-ß.

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of IFN-ß on TGF-ß–induced growth of MLg2908 fibroblasts. Although IFN-ß (10–1,000 ng/ml) did not inhibit the enhancement of growth of murine MLg2908 fibroblasts by TGF-ß, a high dose of IFN-ß (1,000 U/ml) inhibited the proliferation of MLg2908 in the absence of TGF-ß.

	DISCUSSION

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Morphologic and chemical improvements of bleomycin-induced pulmonary fibrosis were observed when we injected MuIFN-ß daily intravenously in ICR mice without adverse effects (Figures 1 and 2).

Effects of IFN-ß on TSP-1/2 and TGF-ß Production and Its Activation in Pulmonary Fibrosis Induced by Bleomycin
Effects of IFN-ß on fibroblasts and their functions have usually been evaluated in vitro, by examination of the inhibitory effects of IFN-ß on fibroblast proliferation (3), collagen synthesis (4), and human lung fibroblast glycosaminoglycan production (19). In this study, we for the first time investigated the antifibrotic effect of MuIFN-ß on bleomycin-induced pulmonary fibrosis in vivo. This effect of MuIFN-ß was found to be associated with attenuation of expression of TGF-ß and TSP-1/2. Attenuation of TSP-1/2 in turn decreased TGF-ß activation.

Immunohistochemical examination revealed weak expression of TGF-ß in alveolar epithelial cells and extracellular matrix (ECM) in control mice (Figures 3a and 3b, Table 1). After 4 wk of treatment with bleomycin, TGF-ß was intensely and moderately expressed in ECM and alveolar epithelial cells, respectively (Figure 3c, Table 1). These findings indicate promotion of proliferation of fibroblasts and production of collagen in lung tissue. TGF-ß enhances Fas-mediated apoptosis of epithelial cells and subsequent progression of remodeling (20). In contrast, MuIFN-ß decreased the amount of TGF-ß in lung tissue at 28 d after bleomycin administration (Figure 5a). MuIFN-ß also attenuated expression of TSP-1/2 in lung tissue (Figure 5b) and subsequently inhibited the activation of TGF-ß in ECM and alveolar epithelial cells to an extent similar to that in control mice (Figure 3 d, Table 1).

We performed Western blot analysis of murine lung tissues in which vessels had been irrigated with normal saline to wash out remaining blood. We therefore acknowledge that results of Western blot analysis are accurate for TGF-ß and TSP-1/2 only in lung tissues without platelets in vessels. In Group A (control), TSP-1/2 was located in platelets; in Group B (bleomycin alone), TSP-1/2 was increased in lung tissue (Figure 5b), but decreased in platelets (Table 2); and in Group C (bleomycin + IFN), IFN decreased TSP-1/2 in lung tissue (Figure 5b) but accumulated them in platelets and foamy cells (Table 2). Unfortunately, we did not detect TSP-1/2 in lung tissue by immunohistochemistry, suggesting that the concentration of TSP-1/2 in lung tissue may be too low to detect by immunohistochemical methods.

MuIFN-ß has been reported to inhibit fibroblast proliferation (18) and collagen synthesis by a human fibroblast cell line in vitro (4, 21). These direct effects of MuIFN-ß on fibroblasts indicate decrease in the volume of fibrosis in mouse lung challenged with bleomycin. It also appeared that TSP-1/2 is spread into lung tissue by bleomycin to activate TGF-ß. In our study, however, the location of TGF-ß changed after bleomycin administration, but TGF-ß had returned to its original location before challenge with bleomycin (Figures 3c and 3d, Table 1). Enhancement of growth of MLg2908 fibroblasts by TGF-ß was not directly inhibited by concomitant administration of IFN-ß (Figure 6). These results suggest that IFN-ß decreased amounts of TGF-ß and TSP-1/2 in lung tissue, and also restored the location of TGF-ß and TSP-1/2 before bleomycin administration, but did not inhibit the effects of TGF-ß signal transduction on proliferation of fibroblasts.

Inhibition of TSP-1/2 by IFN-ß
TSP-1 and TSP-2, the most potent activators of TGF-ß, cleave a masking protein from the latent form of TGF-ß (22, 23). TSP-1/2 was markedly expressed on platelets in vascular lumens of normal control mouse lung (Figure 4a). IFN-ß decreased the total amount of TSP-1/2 in lung tissue (Figure 5b). Under bleomycin challenge, TSP-1/2 expression was attenuated in platelets. However, immunofluorescence representing TSP-1/2 appeared in foamy cells but not platelets (Figures 4b and 4c). It is possible that a large amount of TSP-1/2 in platelets disappeared and that a portion of it was subsequently withdrawn by foamy cells after bleomycin administration (Figure 4d). Further, combined treatment with MuIFN-ß restored the expression of TSP-1/2 in platelets to some extent (Figure 4e, Table 2). These observations may indicate that (1) IFN-ß inhibits release of TSP-1/2 from platelets, and (2) released TSP-1/2 is absorbed by foamy cells. It appears that few TSP-1/2 molcules in lung tissue activate TGF-ß. Because MuIFN-ß decreased free TSP-1/2 in lung tissue, activation of TGF-ß was minimized and subsequent pulmonary fibrosis was attenuated.

Clinical Application of IFN-ß
Although interferons have been widely used for treatment of pulmonary diseases (24), IFN-ß has been reported to be ineffective for patients with IPF (7). In a well designed, placebo-controlled, randomized clinical trial, neither survival time nor pulmonary function parameters determined as primary endpoints differed significantly between the IFN-ß and placebo groups. However, we believe, based on the findings of the present study, that IFN-ß is able to directly inhibit mechanisms of fibrosis. Although the clinical efficacy of IFN-ß in humans may not equal that in the bleomycin mouse model, detailed investigation will be helpful in development of IFN-ß treatment for fibrotic disorders in humans. Sufficient concentrations of IFN-ß in lung tissue will also be required for improvement of fibrosis. A recent study suggested that the early stage of interstitial pneumonia was treatable with pirfenidone, but that the advanced stage of interstitial pneumonia in Hermansky-Pudlak Syndrome was not (25). IFN-ß might be efficacious if a good system of drug delivery enabling sufficiently localized administration of it can be designed for treatment of interstitial fibrosis when pulmonary function still exhibits only mild disturbance.

Conclusions
We demonstrated the effect of IFN-ß on bleomycin-induced pulmonary fibrosis. The antifibrotic effect of IFN-ß was due to inhibition of TGF-ß and TSP-1/2 expression in lung tissue, which resulted in attenuation of TGF-ß activation in foci of fibrosis. Knowledge of the biological activity of IFN-ß will be useful for treatment with it in combination with other candidate antifibrotic agents, such as IFN-, pirfenidone, and N-acetyl cysteine, which exhibit other types of antifibrotic effects.

Received in original form October 20, 2003

Received in final form November 2, 2004

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This Article Abstract Full Text (PDF) All Versions of this Article: 2003-0374OCv1 32/2/93    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Azuma, A. Articles by Kudoh, S. Search for Related Content PubMed PubMed Citation Articles by Azuma, A. Articles by Kudoh, S.