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The Journal of Immunology, 2007, 179: 708-714.
Copyright © 2007 by The American Association of Immunologists, Inc.
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*BLEOMYCIN
*THALIDOMIDE
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*Pulmonary Fibrosis

Thalidomide Prevents Bleomycin-Induced Pulmonary Fibrosis in Mice1

Chiharu Tabata2,*,{dagger}, Rie Tabata{ddagger}, Yoshio Kadokawa*, Shigeo Hisamori*, Meiko Takahashi*, Michiaki Mishima§, Takashi Nakano{dagger} and Hajime Kubo*

* Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan; {dagger} Department of Internal Medicine, Respiratory Division, Hyogo College of Medicine; {ddagger} Department of Internal Medicine, Hyogo Prefectural Tsukaguchi Hospital, Hyogo, Japan; and § Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pulmonary fibrosis in humans can occur as a result of a large number of conditions. In idiopathic pulmonary fibrosis (IPF), pulmonary function becomes progressively compromised resulting in a high mortality rate. Currently there are no proven effective treatments for IPF. We have recently reported that IL-6 and TGF-beta1 plays an important role in proliferation and differentiation of lung fibroblasts, and all-trans-retinoic acid (ATRA) prevented bleomycin-induced lung fibrosis through the inhibition of these cytokines. Thalidomide (Thal) has been used in the treatment of multiple myeloma through the inhibitory effect on IL-6-dependent cell growth and angiogenesis. In this study, we examined the preventive effect of Thal on bleomycin-induced pulmonary fibrosis in mice. We performed histological examinations and quantitative measurements of IL-6, TGF-beta1, collagen type I{alpha}1 (COL1A1), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in bleomycin-treated mouse lung tissues with or without the administration of Thal. Thal histologically ameliorated bleomycin-induced fibrosis in mouse lung tissues. Thal decreased the expressions of IL-6, TGF-beta1, VEGF, Ang-1 Ang-2, and COL1A1 mRNA in mouse lung tissues. In addition, Thal inhibited angiogenesis in the lung. In vitro studies disclosed that Thal reduced 1) production of IL-6, TGF-beta1, VEGF, Ang-1, and collagen synthesis from human lung fibroblasts, and 2) both IL-6-dependent proliferation and TGF-beta1-dependent transdifferentiation of the cells, which could be the mechanism underlying the preventive effect of Thal on pulmonary fibrosis. These data may provide a rationale to explore clinical use of Thal for the prevention of pulmonary fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation of fibroblasts and deposition of extracellular matrixes and is frequently associated with collagen diseases, rheumatoid arthritis, radiotherapy to the thoracic region, and drugs including anticancer agents. Idiopathic pulmonary fibrosis (IPF)3 is the most common type with a prevalence of 16–18 per 100,000 persons, leading to high incidence of death (>50% 5-year mortality rate) due to ultimate respiratory failure (1). There are currently few effective therapeutic and preventive strategies (2, 3, 4). However, there have been a number of reports on cytokines being associated with lung fibrosis in animal models, including IL-6, TGF-beta1, and platelet-derived growth factor, by overexpression of cytokine genes (5). We have recently reported that IL-6 and TGF-beta1 play an important role in proliferation and differentiation of lung fibroblasts in vitro. Irradiation stimulated IL-6 production through the protein kinase C (PKC)-{delta}/NF-{kappa}B pathway and IL-6-dependent proliferation of fibroblasts. The production of TGF-beta1 was also increased by irradiation through p38 MAPK and/or NF-{kappa}B pathways, and the fibroblasts were stimulated and differentiated to myofibroblasts by irradiation. All-trans-retinoic acid (ATRA) activated PKC-{delta} and inhibited p38 MAPK and/or NF-{kappa}B, resulting in the inhibition of both pathways and the decrease in collagen synthesis. ATRA prevented radiation- or bleomycin-induced lung fibrosis through the inhibition of these cytokines in mice (6, 7).

ATRA is well known to inhibit the growth of myeloma cells by the reduction of both IL-6 production and its receptor expression (8, 9). Recently, the usefulness of thalidomide (Thal) has been shown in the therapy for multiple myeloma similarly by the reduction of IL-6 production (10). Thal was originally introduced in 1954 as a sedative/hypnotic agent but was removed from the market for its teratogenic effects. In contrast, its unique effects have been reported such as anti-inflammatory (11), immunomodulatory (12, 13, 14), and antiangiogenic activity (15). Several clinical trials have demonstrated the efficacy of Thal or its analogs in the treatment of a variety of diseases including erythema nodosum leprosum (16), rheumatoid arthritis (17, 18), Castleman’s disease (19), Crohn’s disease (20), and HIV-associated aphthous ulceration (21). In addition, Thal has been used in the treatment of malignancies like AIDS-related Kaposi sarcoma (22), renal cell carcinoma (23), Waldenstrom’s macroglobulinemia (24), and myelodysplastic disease (25).

In this study, we investigated whether Thal had preventive or therapeutic effects on bleomycin-induced pulmonary fibrosis in mice. Moreover, we examined the mechanism underlying the preventive effect of Thal on pulmonary fibrosis. Here we show that Thal prevents bleomycin-induced pulmonary fibrosis in mice through the inhibition of the production of proinflammatory, profibrotic cytokines and the reduction of angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

WI38VA-13, a human embryonal lung fibroblastic cell line transformed by SV40, and IMR-90, a cell line derived from human fetal lung fibroblasts, were cultured in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS. All cells were cultured with antibiotics in a humidified incubator with 5% CO2 at 37°C. In some experiments, cells were stimulated with human recombinant TGF-beta1 (PeproTech) at a dose from 0.5 to 5 ng/ml for 24 h.

When cells were treated with Thal (Sigma-Aldrich), Thal was diluted in DMSO and added to the growth medium to yield the final DMSO solvent concentration <0.05% (v/v). For control, cells were treated with the same concentration of DMSO. In preliminary experiments, this final concentration of DMSO had no gross effect on WI38VA-13 and IMR-90 cells.

Animal studies

C57BL/6 female mice were purchased from Japan SLC and maintained in our specific pathogen-free animal facility. All animals were kept according to the Animal Protection Guidelines of Kyoto University. All protocols for animal use and euthanasia were reviewed and approved by the Institute of Laboratory Animals (Graduate School of Medicine, Kyoto University, Japan). Eight-week-old mice were injected i.p. with bleomycin sulfate (2 mg/mouse/day; Nippon Kayaku) on days 1, 8, and 15. In some experiments, mice were injected i.p. with 4 mg of Thal dissolved in 0.1 ml of 0.5% carboxymethylcellulose (CMC; Sigma-Aldrich) or 0.1 ml of 0.5% CMC (controls). Injections were repeated five times weekly: 1) throughout the course, 2) for the first 14 days; and 3) for the last 14 days. The mice were sacrificed at 4 wk after the first bleomycin treatment, and histological examination was performed by staining with H&E or Azan using an aniline blue method. For a quantitative analysis of the severity of fibrosis, the level of fibrosis was measured as the blue pixel number in Azan staining by analytical digital photomicroscopy technique using Adobe Photoshop (Adobe Systems).

Quantitative real-time RT-PCR

RNA preparation and quantitative real-time RT-PCR was performed as previously described (6) using TaqMan Gene expression products for mouse IL-6, TGF-beta1, collagen type I{alpha}1 (COL1A1), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). 18S rRNA served as an endogenous control (Applied Biosystems).

Immunofluorescence staining

Immunofluorescence staining was performed as previously described (7). The fixed cells were stained with mouse anti-human {alpha}-smooth muscle actin ({alpha}-SMA) mAb (1:100) (Sigma-Aldrich) followed by Alexa 488-conjugated donkey anti-mouse Ab (1:1000; Molecular Probes). Hoechst 33258 fluorochrome (Sigma-Aldrich) was used for nuclear staining.

Immunohistochemistry

Mice were sacrificed at day 28 after the first injection of bleomycin, and the lungs were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin and sectioned. Sections were immunostained as described (26) by using anti-mouse CD31 Ab (1:100; BD Pharmingen). TSA Biotin System (PerkinElmer Life and Analytical Sciences) was used to enhance staining, and peroxidase activity was envisioned with diaminobenzidine kit (DakoCytomation). The sections were counterstained with hematoxylin. The CD31-positive vessels were counted in 10 fields of vision (magnification; x200) in each of three consecutive sections by three investigators.

Measurement of IL-6, sIL-6R, TGF-beta1, TNF-{alpha}, IL-1beta, VEGF, Ang-1, and Ang-2

The concentrations of IL-6, sIL-6R, TGF-beta1, TNF-{alpha}, IL-1beta, VEGF, Ang-1, and Ang-2 in the culture supernatants with or without Thal (50 µg/ml) for 24 h were measured by ELISA kit (BioSource; R&D Systems).

Cell proliferation assay

Cell proliferation assay was performed as described (6). Cells were cultured in 96-well flat-bottom culture plates for 4 days with or without IL-6 (1–10,000 pg/ml), Thal (50 µg/ml), and/or mouse anti-human IL-6R mAb (20 ng/ml) (DakoCytomation) to inhibit the binding of IL-6 to its receptor. Cell Counting Kit-8 (Dojindo, Tokyo, Japan) was used to characterize the growth of cells.

Statistical analysis

Results are given as the mean ± SD of values. Statistical analysis was performed using Bonferroni/Dunn multiple comparison tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Prevention of bleomycin-induced pulmonary fibrosis by Thal

We examined the effect of Thal on bleomycin-induced lung fibrosis in mice. The histological changes at 28 days are shown in Fig. 1A. In the lung tissues from the bleomycin-treated mice without Thal, pulmonary interalveolar septa became thickened and infiltrated by inflammatory cells, with collagen depositions in the interstitium disclosed by Azan staining. Intraperitoneal administration of Thal five times per week inhibited the collagen deposition in the bleomycin-treated mouse lung tissues. Mice treated by Thal without bleomycin-treatment showed no changes at all (data not shown).


Figure 1
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  		FIGURE 1. Effect of Thal on bleomycin-induced pulmonary fibrosis and the expressions of IL-6, TGF-beta1, and COL1A1 mRNA in mouse lung tissues. Eight-week-old mice were injected i.p. with bleomycin sulfate (2 mg/mouse/day) (Bleo, Bleo+Thal) on days 1, 8, and 15. Thal (4 mg/mouse/day) dissolved in 0.1 ml of 0.5% CMC (Bleo+Thal) or 0.1 ml of 0.5% CMC alone (control, Bleo) was administered i.p. five times per week during the time course. On day 28, mice (n = 3 in each experiments) were sacrificed. A, Histological changes were demonstrated by H & E and Azan staining (original magnification, x100). B–D, Real-time RT-PCR was performed to determine the changes in mRNA levels from lung tissues of mice for IL-6 (B), TGF-beta1 (C), and COL1A1 (D) as described in Materials and Methods. The levels of mRNA are represented as the ratio to 18S rRNA. The results are indicated as the mean ± SD of three separate experiments.

 
Effect of Thal on the expressions of IL-6, TGF-beta1, and COL1A1 mRNA in mouse lung tissues

We previously reported that IL-6 played an important role in pulmonary fibrosis (6). In this report, mRNA levels of IL-6 from lung tissues of mice 28 days after first injection of bleomycin were analyzed by real-time RT-PCR and shown to be ~21.8-fold elevated compared with control mice, which were significantly suppressed by the administration of Thal (p = 0.0002) (Fig. 1B). TGF-beta1 is a well-known key cytokine in the process of human pulmonary fibrogenesis (27). We examined the effect Thal on TGF-beta1 production in bleomycin-treated mouse lung tissues. The mRNA levels of TGF-beta1 (Fig. 1C) and COL1A1 (Fig. 1D), which reflects collagen synthesis, in the lung tissues of bleomycin-treated mice were highly elevated (~10.2- and 16.2-fold, respectively) compared with control mice without bleomycin treatment or Thal. Notably, in bleomycin-treated mice with Thal, the mRNA levels of TGF-beta1 and COL1A1 were markedly decreased (p < 0.0001 and p < 0.0001, respectively).

Effect of Thal on IL-6 production from human lung fibroblasts and the IL-6-mediated proliferation of lung fibroblasts

In vitro, human lung fibroblasts secreted IL-6, and IL-6 stimulated the proliferation of the cells in an autocrine manner (6). Thus, we examined the effect of Thal on IL-6 production of lung fibroblasts and IL-6-mediated cell proliferation. WI38VA-13 cells were cultured with or without Thal for 48 h and the concentrations of IL-6 in the culture supernatants were measured. The concentration of IL-6 in the culture supernatant was decreased by the addition of Thal (p = 0.006) (Fig. 2A). According to our previous study, addition of IL-6 stimulated cell growth in a dose-dependent manner, and reached a plateau at the concentration of 1000 pg/ml at 96 h of culture (6). 1000 pg/ml IL-6-mediated cell proliferation was blocked in the presence of neutralizing Ab against IL-6. Thal also inhibited the IL-6-mediated proliferation to a similar extent as the neutralizing Ab. An additive effect was observed when both the neutralizing Ab and Thal were simultaneously added to the culture (Fig. 2B). Similar examinations were studied at 48 and 72 h, but the apparent cell proliferation was not seen by the addition of IL-6 (data not shown).


Figure 2
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  		FIGURE 2. Effect of Thal on production of IL-6 and TGF-beta1 from human lung fibroblasts, IL-6-dependent proliferation, and TGF-beta1-induced transdifferentiation of lung fibroblasts to myofibroblast. A, IL-6 concentration in the supernatants of confluent WI38VA-13 cells, cultured with 50 µg/ml Thal or DMSO alone (control) for 48 h was measured by ELISA. At the time of harvest, cells were counted and the IL-6 values were corrected as proportion to cell numbers. B, WI38VA-13 cells were cultured in the presence ({blacksquare}) or absence ({square}) of IL-6 (1000 pg/ml) with or without mouse anti-human IL-6R mAb (20 ng/ml) which can inhibit the binding of IL-6 to its receptor and/or Thal (50 µg/ml) for 96 h. All cultures contained the same concentration of DMSO. Cell proliferation was assayed as described in Materials and Methods. C, TGF-beta1 concentration in the supernatants of confluent IMR-90 cells, cultured in the presence or absence of Thal (50 µg/ml) for 24 h was measured by ELISA. At the time of harvest, cells were counted and the TGF-beta1 values were corrected as proportion to cell numbers. D, Inhibitory effect of Thal on TGF-beta1-induced {alpha}-SMA expression in IMR-90 cells. IMR-90 cells were preconditioned with Thal or DMSO alone (control) and then stimulated with TGF-beta1 (5 ng/ml) for 24 h. Immunofluorescence staining for expression of cytoplasmic {alpha}-SMA was performed as described in Materials and Methods. Representative immunofluorescence staining for the cytoplasmic expression of {alpha}-SMA was demonstrated. The representative results from three independent experiments are shown. E, Cytoplasmic {alpha}-SMA expression was measured (%positive cells) in the presence of various concentrations of TGF-beta1 (0–5 ng/ml) with or without Thal (50 µg/ml). In the experiments A–E all cultures contained the same concentration of DMSO. The values in A–C, and E represent the mean ± SD of three separate experiments.

 
Effect of Thal on TGF-beta1 production and transdifferentiation of lung fibroblasts

We performed experiments to study the effect of Thal on TGF-beta1 production of lung fibroblasts. IMR-90 cells were cultured with or without Thal for 24 h and the concentrations of TGF-beta1 in the culture supernatants were measured. The concentration of TGF-beta1 in the culture supernatant was decreased by the addition of Thal (p = 0.0267) (Fig. 2C). TGF-beta1 is known to promote collagen synthesis by transdifferentiation of fibroblasts to myofibroblasts (28). We next investigated the impact of Thal on TGF-beta1-induced transdifferentiation of IMR90 cells. To study whether Thal influenced this transdifferentiation process as well as cell proliferation, we examined the expression of {alpha}-smooth muscle actin ({alpha}-SMA) in TGF-beta1 stimulated IMR-90 cells. IMR-90 cells were preconditioned overnight with Thal, and then stimulated with TGF-beta1 (5 ng/ml) for 24 h. The cytoplasmic expression of {alpha}-SMA was significantly up-regulated by TGF-beta1 treatment, which was significantly decreased by the addition of Thal (p < 0.0001) (Fig. 2D). The stimulative effect of TGF-beta1 on transdifferentiation of IMR-90 cells was observed in a dose-dependent manner, and Thal suppressed the effect of TGF-beta1 even at the concentration of 5 ng/ml (Fig. 2E).

Thal inhibited angiogenesis in bleomycin-treated mouse lung tissues

It is well established that Thal possesses antiangiogenic activities. To evaluate the effect of Thal on angiogenesis in bleomycin-treated lung tissues, we counted the number of vessels in the lung by immunostaining for pan-endothelial marker CD31. The number of CD31-positive vessels was increased in bleomycin-treated lung, which was decreased by the administration of Thal to the control levels (Fig. 3, A and B).


Figure 3
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  		FIGURE 3. Effect of Thal on bleomycin-induced angiogenesis and the expressions of VEGF, Ang-1, and Ang-2 mRNA in mouse lung tissues. The samples from mouse lung were common with the experiments shown in Fig. 1. A, Representative results of immunohistochemistry for CD31 in mouse lung tissues at 28 days after bleomycin instillation with or without Thal (original magnification; x200). B, The numbers of vessels, which showed positive reactivity for CD31, in three separate sections were counted and the values represent the mean ± SD. C–E, Real-time RT-PCR was performed to determine the changes in mRNA levels of VEGF (C), Ang-1 (D), and Ang-2 (E) from lung tissues of mice with or without Thal. The levels of mRNA are represented as the ratio to 18S rRNA. The results are indicated as the mean ± SD of three separate experiments.

 
mRNA levels of VEGF, Ang-1, and Ang-2 in mouse lung tissues

To study a more precise mechanism for Thal-mediated inhibition of angiogenesis, we analyzed mRNA levels of VEGF, Ang-1, and Ang-2, which are known to be important regulators of angiogenesis (29, 30, 31, 32), from lung tissues of mice 28 days after the first injection of bleomycin by real-time RT-PCR. All of their levels were ~12.0, 7.2, and 7.4-fold elevated, respectively, by bleomycin treatment compared with control mice, which were significantly suppressed by the administration of Thal (p = 0.0002, p < 0.0001, and p < 0.0001, respectively) (Fig. 3, C–E).

Effect of Thal on VEGF, Ang-1, and Ang-2 productions from human lung fibroblasts and myofibroblast

We performed in vitro experiments to examine whether the effect of Thal on VEGF, Ang-1, and Ang-2 production was related to cell differentiation. The angiogenetic cytokines have been reported to be produced by myofibroblasts (33). IMR-90 cells were cultured with or without Thal, IL-6 (10 ng/ml), or TGF-beta1 (5 ng/ml) for 24 h, and the concentrations of VEGF, Ang-1, and Ang-2 in the culture supernatants of lung "fibroblasts" were measured. The concentrations of VEGF and Ang-1 in the culture supernatant were increased by TGF-beta1 stimulation (p < 0.0001, p < 0.0001, respectively), which were decreased by the addition of Thal (p = 0.0005, p = 0.0005, respectively) (Fig. 4, A and B). In contrast, preconditioned IMR-90 cells with TGF-beta1 (5 ng/ml) for 24 h, which were transdifferentiated to myofibroblasts as shown in Fig. 4, were cultured with or without Thal, IL-6, or TGF-beta1 for 24 h, and the concentrations of VEGF, Ang-1, and Ang-2 in the culture supernatants of lung "myofibroblasts" were measured. The concentrations of VEGF and Ang-1 in the culture supernatant were increased by TGF-beta1 stimulation (p = 0.0001, p = 0.0001, respectively), which were decreased by the addition of Thal (p = 0.0028, p = 0.0054) (Fig. 4, C and D). Although the basal productions of VEGF and Ang-1 were increased after the lung fibroblasts were transdifferentiated to myofibroblasts by the addition of TGF-beta1 (from 30 to 100 pg/ml and from 100 to 1000 pg/ml, respectively), the stimulant effects of TGF-beta1 on their productions in myofibroblasts were less than in fibroblasts. IL-6 had almost no effect on VEGF and Ang-1 productions of these cells (Fig. 4). Ang-2 was not detected in the culture supernatants of both fibroblasts and myofibroblasts with or without Thal, IL-6, or TGF-beta1 (data not shown).


Figure 4
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  		FIGURE 4. Effect of Thal on VEGF and Ang-1 productions from human lung fibroblasts and myofibroblast. A and B, IMR-90 cells were cultured for 24 h with or without Thal (50 µg/ml), IL-6 (10 ng/ml), or TGF-beta1 (5 ng/ml) and the concentrations of VEGF (A) and Ang-1 (B) in the culture supernatants were measured by ELISA. C and D, IMR-90 cells were preconditioned with TGF-beta1 (5 ng/ml) for 24 h, which were transdifferentiated to myofibroblasts as shown in Fig. 2, D and E, and then were cultured with or without Thal, IL-6, or TGF-beta1 for 24 h. The concentrations of VEGF (C) and Ang-1 (D) in the culture supernatants were measured by ELISA. In the experiments A–D, all cultures contained the same concentration of DMSO. The results are indicated as the mean ± SD of three separate experiments.

 
Both early and late preventive effects of Thal on bleomycin-induced lung fibrosis

To study the "preventive" and "therapeutic" effects of Thal on lung fibrosis, we examined the early (probably the inflammatory responses are mainly demonstrated) and late (probably the postinflammatory, fibrotic changes are mainly observed) effects of Thal by transient administration to bleomycin-treated mice. The administration throughout the course of Thal most effectively prevented bleomycin-induced pulmonary fibrosis and the administration for the first 14 days or the last 14 days ameliorated it compared with control (Fig. 5). Taken together, these results demonstrate the "late," namely therapeutic effect of Thal in bleomycin-induced lung fibrosis models, in addition to the "early," namely preventive effect.


Figure 5
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  		FIGURE 5. Both early and late preventive effects of Thal on bleomycin-induced pulmonary fibrosis. Mice (n = 3 in each experiment) were sacrificed at 28 days after first injection i.p. with bleomycin sulfate on days 1, 8, and 15 in the presence or absence of Thal, and the level of fibrosis was measured as the blue pixel number as described in Materials and Methods. Thal was administrated in three ways: 1) for the first 14 days; 2) for the last 14 days; and 3) throughout the course. The results are indicated as the mean ± SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Several factors have been reported to be associated with pulmonary fibrosis, including TGF-beta1, TNF-{alpha}, IL-1, and IL-6, platelet-derived growth factor, VEGF and fibroblast growth factor (7, 34, 35, 36, 37). We previously demonstrated that both IL-6 and TGF-beta1 played important roles in pulmonary fibrosis (7). In this study, we showed that markedly increased mRNA levels of IL-6, TGF-beta1, and COL1A1, which represents collagen synthesis, in the bleomycin-treated mouse lung tissues were decreased by the addition of Thal. As we previously reported (6), human lung fibroblasts were proliferated by IL-6 in a dose-dependent manner. In this study, Thal inhibited both IL-6 production and IL-6-induced cell proliferation of lung fibroblasts. In contrast, it is well known that fibroblasts transdifferentiate to myofibroblasts, which express elevated levels of {alpha}-SMA and, consequently, display a markedly enhanced ability to contract extracellular matrix (38, 39). IL-6 did not induce transdifferentiation of fibroblasts (data not shown). In this study, we demonstrated that the expression of {alpha}-SMA was increased by TGF-beta1 in a dose-dependent manner, with a maximum effect at 5 ng/ml, which was the dose used throughout this study. Thal decreased both TGF-beta1 production from cultured fibroblasts and TGF-beta1-induced {alpha}-SMA expression, namely transdifferentiation process. In addition, bleomycin treatment increased the level of TGF-beta1 mRNA in mouse lung tissues, which was decreased by the addition of Thal. There have been some reports concerning the interaction between IL-6 and TGF-beta1 (40, 41, 42). However, IL-6 failed to induce TGF-beta1 production and vice versa in IMR-90 cells (data not shown). In addition, TNF-{alpha} and IL-1beta, both of which are cytokines reported to stimulate IL-6 (43, 44) and TGF-beta1 production (45, 46, 47), were undetected in culture supernatants of both WI38VA-13 and IMR-90 cells using ELISA (6). Because TGF-beta1 does not stimulate the proliferation of {alpha}-SMA-positive myofibroblasts, we propose a dual inhibitory effect of Thal on IL-6-dependent proliferation and TGF-beta1-dependent transdifferentiation of fibroblasts to myofibroblast, which may be the mechanism underlying the preventive and therapeutic effect of Thal on pulmonary fibrosis. Recently it has been reported that Thal reduced the production of TNF-{alpha}, IL-8, and IL-18 from alveolar macrophages in pulmonary fibrosis (48). Especially as to TNF-{alpha}, Thal has been reported to reduce its production in damaged lung (49, 50). We mainly discussed IL-6 and TGF-beta1 in the present study, because TNF-{alpha} was undetected in culture supernatants of lung fibroblasts as mentioned above. However, Thal may have effects on TNF-{alpha} production from other cells such as alveolar macrophages in vivo.

Currently the usefulness of Thal for the treatments of multiple myeloma or myelofibrosis has been reported. Some reports show that the mechanism underlying the preventive effect of Thal on multiple myeloma and myelofibrosis is related to the suppression of angiogenesis, which is associated with poor prognosis of these diseases (10, 37, 51, 52). Furthermore, the association between pulmonary fibrosis and neovascularization has been recently demonstrated in the lung tissues of patients with IPF (53) or in a rat bleomycin-treated model (54). In addition to the effect on cytokine production such as IL-6 and TGF-beta1, the suppression of angiogenesis might be one of the mechanisms for the inhibitory effect of Thal on pulmonary fibrosis. Therefore, in this study, we investigated whether Thal had the preventive effect on angiogenesis associated with pulmonary fibrosis and demonstrated that the increased number of vessels in bleomycin-treated fibrous mouse lung tissues was decreased by the administration of Thal. In other words, the association between pulmonary fibrosis and angiogenesis was demonstrated in our bleomycin-treated mouse model.

To elucidate further the precise mechanisms involved, we next examined the expression of several vascular-specific growth factors in pulmonary fibrosis. VEGF has an important role in endothelial cell proliferation, vascular permeability, and angiogenesis in several inflammatory lesions (31). VEGF expression is known to be up-regulated by various stimuli, such as low oxygen tension and cytokines. Ang-1 and Ang-2 are also important regulators of blood vessel growth, maturation, and function. Ang-1 promotes angiogenesis, induces vascular maturation, and decrease vascular permeability. Ang-2 has the ability to destabilize blood vessels, enhance vascular leak, and antagonize Ang-1 (29, 30, 33). Here we demonstrated that VEGF, Ang-1, and Ang-2 mRNA level were increased in the bleomycin-treated mouse lung tissues compared with control, which were inhibited by Thal. These vascular-specific growth factors are expressed by several cells such as alveolar epithelial cells, macrophages, smooth muscle cells, and myofibroblasts. To study the importance of these factors especially in fibroblasts, we examined the effect of Thal on VEGF, Ang-1, and Ang-2 productions of human lung fibroblasts and myofibroblasts cultured with or without IL-6 or TGF-beta1. The concentrations of VEGF and Ang-1 in the culture supernatants of lung fibroblasts were increased by the addition of TGF-beta1, which were decreased by Thal. Although the basal productions of VEGF and Ang-1 were more increased after the lung fibroblasts were transdifferentiated to myofibroblasts by the addition of TGF-beta1 (from 30 to 100 pg/ml and from 100 to 1000 pg/ml, respectively), the stimulant effects of TGF-beta1 on their productions in myofibroblasts were less than in fibroblasts. IL-6 had almost no effect on VEGF and Ang-1 production of these cells. Therefore, one of the possible mechanisms by which Thal exerts its inhibitory effects on angiogenesis in pulmonary fibrosis may be through the suppression of TGF-beta1-mediated productions of both VEGF and Ang-1 in fibroblasts and myofibroblasts. Ang-2 was not detected in culture supernatants of fibroblasts and myofibroblasts with or without Thal, IL-6, or TGF-beta1 (data not shown). Although the precise cellular suppressive mechanism of Thal in Ang-2 production has not been fully investigated, the inhibitory effect of Thal on Ang-2 mRNA expression in mouse fibrotic lung tissues might be dependent on other cells such as lung epithelial cells associated with pulmonary fibrosis.

It has been previously reported that a platelet-derived growth factor receptor/cAbl/cKit kinase inhibitor, imatinib mesylate was beneficial in the inhibition of lung fibrosis both by anti-inflammatory and antifibrotic mechanism in rat bleomycin model (55). It is noteworthy that in this report we showed the late namely therapeutic effect of Thal in bleomycin-induced lung fibrosis models in addition to the early namely preventive effect, because in the clinical use, the therapeutic effect is often more important when the clinicians find that the fibrotic changes of various etiology are already apparent in their patients. The histopathological fibrosis in our animal studies was entirely interstitial and without the significant consolidation or architectural remodeling found in even the earliest example of human IPF. Whether the changes we have documented for bleomycin-induced injury after 28 days translate to potential benefits in patients with IPF remains to be shown in clinical trials. Furthermore, the oral administration of the drug results in good compliance. Our data may lead to the development of novel strategies incorporating Thal for the prevention and treatment of various types of lung fibrosis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and the Special Coordination Funds for Promoting Science and Technology. Back

2 Address correspondence and reprint requests to Dr. Chiharu Tabata, Department of Internal Medicine, Respiratory Division, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, Japan. E-mail address: ctabata{at}hyo-med.ac.jp Back

3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; PKC, protein kinase C; ATRA, all-trans-retinoic acid; Thal, thalidomide; COL1A1, collagen type I{alpha}1; VEGF, vascular endothelial growth factor; Ang-1, angiopoietin-1; Ang-2, angiopoietin-2; CMC, carboxymethylcellulose; {alpha}-SMA, {alpha} smooth muscle actin. Back

Received for publication February 21, 2007. Accepted for publication April 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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P. Rogliani, M. Mura, M. Assunta Porretta, and C. Saltini
Review: New perspectives in the treatment of idiopathic pulmonary fibrosis
Therapeutic Advances in Respiratory Disease, April 1, 2008; 2(2): 75 - 93.
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