Thalidomide Prevents Bleomycin-Induced Pulmonary Fibrosis
Chiharu Tabata,2*†Rie Tabata,‡Yoshio Kadokawa,* Shigeo Hisamori,* Meiko Takahashi,*
Michiaki Mishima,§Takashi Nakano,†and Hajime Kubo*
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-?1plays 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-?1, collagen type I?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-?1, 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-?1,
VEGF, Ang-1, and collagen synthesis from human lung fibroblasts, and 2) both IL-6-dependent proliferation and TGF-?1-
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.
of Immunology, 2007, 179: 708–714.
with collagen diseases, rheumatoid arthritis, radiotherapy to the
thoracic region, and drugs including anticancer agents. Idiopathic
pulmonary fibrosis (IPF)3is the most common type with a prev-
alence 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 pre-
ventive strategies (2–4). However, there have been a number of
reports on cytokines being associated with lung fibrosis in animal
models, including IL-6, TGF-?1, and platelet-derived growth fac-
tor, by overexpression of cytokine genes (5). We have recently
ulmonary fibrosis is a progressive and lethal lung disease
characterized by the proliferation of fibroblasts and depo-
sition of extracellular matrixes and is frequently associated
reported that IL-6 and TGF-?1play an important role in prolifer-
ation and differentiation of lung fibroblasts in vitro. Irradiation
stimulated IL-6 production through the protein kinase C (PKC)-
?/NF-?B pathway and IL-6-dependent proliferation of fibroblasts.
The production of TGF-?1was also increased by irradiation
through p38 MAPK and/or NF-?B pathways, and the fibroblasts
were stimulated and differentiated to myofibroblasts by irradiation.
All-trans-retinoic acid (ATRA) activated PKC-? and inhibited p38
MAPK and/or NF-?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 re-
duction of IL-6 production (10). Thal was originally introduced in
1954 as a sedative/hypnotic agent but was removed from the mar-
ket for its teratogenic effects. In contrast, its unique effects have
been reported such as anti-inflammatory (11), immunomodulatory
(12–14), and antiangiogenic activity (15). Several clinical trials
have demonstrated the efficacy of Thal or its analogs in the treat-
ment of a variety of diseases including erythema nodosum lepro-
sum (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 malig-
nancies like AIDS-related Kaposi sarcoma (22), renal cell carci-
noma (23), Waldenstrom’s macroglobulinemia (24), and myelo-
dysplastic 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 pre-
ventive effect of Thal on pulmonary fibrosis. Here we show that
*Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto
University, Kyoto, Japan;†Department of Internal Medicine, Respiratory Division,
Hyogo College of Medicine;‡Department of Internal Medicine, Hyogo Prefectural
Tsukaguchi Hospital, Hyogo, Japan; and§Department of Respiratory Medicine, Grad-
uate School of Medicine, Kyoto University, Kyoto, Japan
Received for publication February 21, 2007. Accepted for publication April
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.
1This study was supported by grants from the Ministry of Education, Culture, Sports,
Science and Technology and the Special Coordination Funds for Promoting Science
2Address correspondence and reprint requests to Dr. Chiharu Tabata, Department of
Internal Medicine, Respiratory Division, Hyogo College of Medicine, 1-1
3Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; PKC, protein
kinase C; ATRA, all-trans-retinoic acid; Thal, thalidomide; COL1A1, collagen type
I?1; VEGF, vascular endothelial growth factor; Ang-1, angiopoietin-1; Ang-2, an-
giopoietin-2; CMC, carboxymethylcellulose; ?-SMA, ? smooth muscle actin.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
Thal prevents bleomycin-induced pulmonary fibrosis in mice
through the inhibition of the production of proinflammatory, pro-
fibrotic cytokines and the reduction of angiogenesis.
Materials and Methods
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% CO2at 37oC. In some experiments, cells were stimu-
lated with human recombinant TGF-?1(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 concentra-
tion of DMSO had no gross effect on WI38VA-13 and IMR-90 cells.
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, Ja-
pan). 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 experi-
ments, 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
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-?1, collagen type I?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 was performed as previously described (7).
The fixed cells were stained with mouse anti-human ?-smooth muscle actin
(?-SMA) mAb (1:100) (Sigma-Aldrich) followed by Alexa 488-conju-
gated donkey anti-mouse Ab (1:1000; Molecular Probes). Hoechst 33258
fluorochrome (Sigma-Aldrich) was used for nuclear staining.
Mice were sacrificed at day 28 after the first injection of bleomycin, and
the lungs were fixed in 4% paraformaldehyde, dehydrated, and embed-
ded in paraffin and sectioned. Sections were immunostained as de-
scribed (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 counter-
stained with hematoxylin. The CD31-positive vessels were counted in
10 fields of vision (magnification; ?200) in each of three consecutive
sections by three investigators.
Measurement of IL-6, sIL-6R, TGF-?1, TNF-?, IL-1?, VEGF,
Ang-1, and Ang-2
The concentrations of IL-6, sIL-6R, TGF-?1, TNF-?, IL-1?, VEGF,
Ang-1, and Ang-2 in the culture supernatants with or without Thal (50
?g/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 cul-
tured in 96-well flat-bottom culture plates for 4 days with or without IL-6
(1–10,000 pg/ml), Thal (50 ?g/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.
Results are given as the mean ? SD of values. Statistical analysis was
performed using Bonferroni/Dunn multiple comparison tests.
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
pulmonary fibrosis and the expressions of IL-6, TGF-
?1, and COL1A1 mRNA in mouse lung tissues. Eight-
week-old mice were injected i.p. with bleomycin sul-
fate (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 sacri-
ficed. A, Histological changes were demonstrated by
H&E and Azan staining (original magnification,
?100). 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-?1(C), and
COL1A1 (D) as described in Materials and Meth-
ods. The levels of mRNA are represented as the ra-
tio to 18S rRNA. The results are indicated as the
mean ? SD of three separate experiments.
Effect of Thal on bleomycin-induced
709The Journal of Immunology
and infiltrated by inflammatory cells, with collagen depositions in
the interstitium disclosed by Azan staining. Intraperitoneal admin-
istration of Thal five times per week inhibited the collagen depo-
sition in the bleomycin-treated mouse lung tissues. Mice treated by
Thal without bleomycin-treatment showed no changes at all (data
Effect of Thal on the expressions of IL-6, TGF-?1, and COL1A1
mRNA in mouse lung tissues
We previously reported that IL-6 played an important role in pul-
monary 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 ele-
vated compared with control mice, which were significantly sup-
pressed by the administration of Thal (p ? 0.0002) (Fig. 1B).
TGF-?1is a well-known key cytokine in the process of human
pulmonary fibrogenesis (27). We examined the effect Thal on
TGF-?1production in bleomycin-treated mouse lung tissues. The
mRNA levels of TGF-?1(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-?1and 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 cul-
tured 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 man-
ner, 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).
Effect of Thal on TGF-?1production and transdifferentiation of
We performed experiments to study the effect of Thal on
TGF-?1production of lung fibroblasts. IMR-90 cells were cul-
tured with or without Thal for 24 h and the concentrations of
TGF-?1in the culture supernatants were measured. The con-
centration of TGF-?1in the culture supernatant was decreased
by the addition of Thal (p ? 0.0267) (Fig. 2C). TGF-?1is
known to promote collagen synthesis by transdifferentiation of
fibroblasts to myofibroblasts (28). We next investigated the im-
pact of Thal on TGF-?1-induced transdifferentiation of IMR90
cells. To study whether Thal influenced this transdifferentiation
transdifferentiation of lung fibroblasts to myofibroblast. A, IL-6 concentration in the supernatants of confluent WI38VA-13 cells, cultured with 50
?g/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 (f) or absence (?) 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 ?g/ml) for 96 h. All cultures contained the
same concentration of DMSO. Cell proliferation was assayed as described in Materials and Methods. C, TGF-?1concentration in the supernatants
of confluent IMR-90 cells, cultured in the presence or absence of Thal (50 ?g/ml) for 24 h was measured by ELISA. At the time of harvest, cells
were counted and the TGF-?1values were corrected as proportion to cell numbers. D, Inhibitory effect of Thal on TGF-?1-induced ?-SMA
expression in IMR-90 cells. IMR-90 cells were preconditioned with Thal or DMSO alone (control) and then stimulated with TGF-?1(5 ng/ml) for
24 h. Immunofluorescence staining for expression of cytoplasmic ?-SMA was performed as described in Materials and Methods. Representative
immunofluorescence staining for the cytoplasmic expression of ?-SMA was demonstrated. The representative results from three independent
experiments are shown. E, Cytoplasmic ?-SMA expression was measured (%positive cells) in the presence of various concentrations of TGF-?1(0–5
ng/ml) with or without Thal (50 ?g/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 production of IL-6 and TGF-?1from human lung fibroblasts, IL-6-dependent proliferation, and TGF-?1-induced
710THALIDOMIDE PREVENTS LUNG FIBROSIS
process as well as cell proliferation, we examined the expres-
sion of ?-smooth muscle actin (?-SMA) in TGF-?1stimulated
IMR-90 cells. IMR-90 cells were preconditioned overnight with
Thal, and then stimulated with TGF-?1(5 ng/ml) for 24 h. The
cytoplasmic expression of ?-SMA was significantly up-regu-
lated by TGF-?1treatment, which was significantly decreased
by the addition of Thal (p ? 0.0001) (Fig. 2D). The stimulative
effect of TGF-?1on transdifferentiation of IMR-90 cells was
observed in a dose-dependent manner, and Thal suppressed the
effect of TGF-?1even at the concentration of 5 ng/ml (Fig. 2E).
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; ?200). 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.
Effect of Thal on bleomycin-induced angiogenesis and the expressions of VEGF, Ang-1, and Ang-2 mRNA in mouse lung tissues. The
24 h with or without Thal (50 ?g/ml), IL-6 (10 ng/ml), or TGF-?1(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-?1(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-?1for 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.
Effect of Thal on VEGF and Ang-1 productions from human lung fibroblasts and myofibroblast. A and B, IMR-90 cells were cultured for
711The Journal of Immunology
Thal inhibited angiogenesis in bleomycin-treated mouse lung
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).
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 angiogen-
esis (29–32), 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-?1(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 con-
centrations of VEGF and Ang-1 in the culture supernatant were
increased by TGF-?1stimulation (p ? 0.0001, p ? 0.0001, re-
spectively), 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-?1(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-?1for 24 h,
and the concentrations of VEGF, Ang-1, and Ang-2 in the culture
supernatants of lung “myofibroblasts” were measured. The con-
centrations of VEGF and Ang-1 in the culture supernatant were
increased by TGF-?1stimulation (p ? 0.0001, p ? 0.0001, re-
spectively), which were decreased by the addition of Thal (p ?
0.0028, p ? 0.0054) (Fig. 4, C and D). Although the basal pro-
ductions of VEGF and Ang-1 were increased after the lung fibro-
blasts were transdifferentiated to myofibroblasts by the addition of
TGF-?1(from 30 to 100 pg/ml and from 100 to 1000 pg/ml, re-
spectively), the stimulant effects of TGF-?1on 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 fibro-
blasts and myofibroblasts with or without Thal, IL-6, or TGF-?1
(data not shown).
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 re-
sponses are mainly demonstrated) and late (probably the postin-
flammatory, fibrotic changes are mainly observed) effects of Thal
by transient administration to bleomycin-treated mice. The admin-
istration 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
Several factors have been reported to be associated with pulmo-
nary fibrosis, including TGF-?1, TNF-?, IL-1, and IL-6, platelet-
derived growth factor, VEGF and fibroblast growth factor (7, 34–
37). We previously demonstrated that both IL-6 and TGF-?1
played important roles in pulmonary fibrosis (7). In this study, we
showed that markedly increased mRNA levels of IL-6, TGF-?1,
and COL1A1, which represents collagen synthesis, in the bleomy-
cin-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 prolif-
eration of lung fibroblasts. In contrast, it is well known that fibro-
blasts transdifferentiate to myofibroblasts, which express elevated
levels of ?-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 ?-SMA was increased by
TGF-?1in a dose-dependent manner, with a maximum effect at 5
ng/ml, which was the dose used throughout this study. Thal de-
creased both TGF-?1production from cultured fibroblasts and
TGF-?1-induced ?-SMA expression, namely transdifferentiation
process. In addition, bleomycin treatment increased the level of
TGF-?1mRNA 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-?1(40–42). However, IL-6
failed to induce TGF-?1production and vice versa in IMR-90 cells
(data not shown). In addition, TNF-? and IL-1?, both of which are
cytokines reported to stimulate IL-6 (43, 44) and TGF-?1produc-
tion (45–47), were undetected in culture supernatants of both
WI38VA-13 and IMR-90 cells using ELISA (6). Because TGF-?1
does not stimulate the proliferation of ?-SMA-positive myofibro-
blasts, we propose a dual inhibitory effect of Thal on IL-6-depen-
dent proliferation and TGF-?1-dependent transdifferentiation of fi-
broblasts to myofibroblast, which may be the mechanism
underlying the preventive and therapeutic effect of Thal on pul-
monary fibrosis. Recently it has been reported that Thal reduced
induced pulmonary fibrosis. Mice (n ? 3 in each experiment) were sacri-
ficed 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.
Both early and late preventive effects of Thal on bleomycin-
712 THALIDOMIDE PREVENTS LUNG FIBROSIS
the production of TNF-?, IL-8, and IL-18 from alveolar macro-
phages in pulmonary fibrosis (48). Especially as to TNF-?, Thal
has been reported to reduce its production in damaged lung (49,
50). We mainly discussed IL-6 and TGF-?1in the present study,
because TNF-? was undetected in culture supernatants of lung
fibroblasts as mentioned above. However, Thal may have effects
on TNF-? production from other cells such as alveolar macro-
phages 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 dem-
onstrated 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-?1, the suppression of angio-
genesis might be one of the mechanisms for the inhibitory effect of
Thal on pulmonary fibrosis. Therefore, in this study, we investi-
gated whether Thal had the preventive effect on angiogenesis as-
sociated with pulmonary fibrosis and demonstrated that the in-
creased 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 angiogen-
esis 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 fac-
tors in pulmonary fibrosis. VEGF has an important role in endo-
thelial 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-spe-
cific growth factors are expressed by several cells such as alveolar
epithelial cells, macrophages, smooth muscle cells, and myofibro-
blasts. To study the importance of these factors especially in fi-
broblasts, 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-?1. The concentrations of
VEGF and Ang-1 in the culture supernatants of lung fibroblasts
were increased by the addition of TGF-?1, 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-?1(from 30 to 100 pg/ml
and from 100 to 1000 pg/ml, respectively), the stimulant effects of
TGF-?1on their productions in myofibroblasts were less than in
fibroblasts. IL-6 had almost no effect on VEGF and Ang-1 pro-
duction of these cells. Therefore, one of the possible mechanisms
by which Thal exerts its inhibitory effects on angiogenesis in pul-
monary fibrosis may be through the suppression of TGF-?1-me-
diated 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-?1(data not shown). Although the precise cellular suppres-
sive mechanism of Thal in Ang-2 production has not been fully
investigated, the inhibitory effect of Thal on Ang-2 mRNA expres-
sion in mouse fibrotic lung tissues might be dependent on other
cells such as lung epithelial cells associated with pulmonary
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 mod-
els 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 signif-
icant consolidation or architectural remodeling found in even the
earliest example of human IPF. Whether the changes we have doc-
umented for bleomycin-induced injury after 28 days translate to
potential benefits in patients with IPF remains to be shown in clin-
ical 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 treat-
ment of various types of lung fibrosis.
The authors have no financial conflict of interest.
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714 THALIDOMIDE PREVENTS LUNG FIBROSIS