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TGF-

1 Induces Progressive Pleural Scarring
and Subpleural Fibrosis
1
Nathalie Decologne,* Martin Kolb,
‡
Peter J. Margetts,
‡
Franck Menetrier,* Yves Artur,
†
Carmen Garrido,* Jack Gauldie,
‡
Philippe Camus,*
§
and Philippe Bonniaud
2
*
§
Pleural fibrosis is a misunderstood disorder which can cause severe restrictive lung disease with high morbidity and even mor-
tality. The condition can develop in response to a large variety of diseases and tissue injury, among them infectious disease,
asbestos, drugs, and radiation therapy. There is no efficient treatment to reverse established pleural fibrosis. TGF-

1 is suspected,
even if not proven, as a key cytokine in this process. In this study, we used adenoviral gene transfer of TGF-

1 to the pleural
mesothelium in rats. We show that local and transient TGF-

1 overexpression induces homogenous, prolonged, and progressive
pleural fibrosis without pleurodesis, associated with severe impairment of pulmonary function. We further demonstrate that
pleural fibrosis can expand into the lung parenchyma from the visceral layer, but not into the muscle from the parietal layer. We
provide evidence that matrix accumulation and fibrosis within the parenchyma evolved through a process involving “mesothelial-
fibroblastoid transformation” and suggest that the pleural mesothelial cell may be an important player involved in the develop-
ment of the subpleural distribution pattern known to be a hallmark of pulmonary fibrosis. This new model of pleural fibrosis will
allow us to better understand the mechanisms of progressive fibrogenesis, and to explore novel antifibrotic therapies in the pleural
cavity. The Journal of Immunology, 2007, 179: 6043– 6051.
P
leural fibrosis can cause severe restrictive lung disease. It
is usually considered as a complication of other disorders
involving the chest cavity. Pneumonia, with parapneumo-
nic effusion and empyema, tuberculosis and asbestos are among
the most common causes for pleural fibrosis (1). Furthermore, nu-
merous drugs can contribute to the development of pleural fibrosis,
the best known being ergot drugs, cytostatic agents, and thoracic
irradiation (Refs. 2 and 3, and www.pneumotox.com (“The drug-
induced lung diseases”)). Other potential reasons are systemic con-
nective tissue disease, hemothorax, and progressive postthoracot-
omy scarring after coronary bypass (4). Depending on disease
severity, pleural fibrosis can compromise respiratory function,
markedly impair quality of life, and can be associated with high
morbidity or even mortality. There is no effective therapy to re-
verse established pleural fibrosis.
The pleura is a metabolically active membrane involved in
maintaining a dynamic homeostasis of fluid within the chest cav-
ity. The homeostasis is important for the mechanical properties of
chest wall and lungs, and a breakdown of the fluid balance can lead
to pleural effusion and ultimately fibrosis (5, 6). Pleural fibrosis
can be defined as excessive deposition of matrix components re-
sulting in the destruction of regular pleural tissue architecture. The
disorder can manifest itself as discrete localized lesions (pleural
plaques) or diffuse pleural thickening (5). Most research related to
pleural scarring concerns the induction of pleurodesis as an ap-
proach to treat chronic effusion associated with metastasized can-
cer (7). Animal studies have shown that TGF-

plays an active role
in pleurodesis as well as in pleural fluid formation (8 –12).
TGF-

is a multifunctional cytokine critically involved in the
pathogenesis of fibrosis through its potent effects on fibroblast dif-
ferentiation, extracellular matrix formation (13, 14), and epithelial-
to-mesenchymal transition (EMT)
3
(15). Peritoneal mesothelial
cells may undergo mesenchymal conversion (16, 17) and TGF-

1
gene transfer to the peritoneal mesothelium induces peritoneal fi-
brosis with evidence of mesothelial-to-mesenchymal transition or
“mesothelial-fibroblastoid transformation” (MFT) (18).
In this study, we used transient transfer of the active TGF-

1
gene by adenoviral vectors to the pleural cavity and mesothelium.
We demonstrate that this approach induces homogenous, pro-
longed, and progressive pleural fibrosis without pleurodesis, asso-
ciated with severe impairment of pulmonary function. This new
model of pleural fibrosis will allow us to better understand the
mechanisms of progressive fibrogenesis, and to explore novel an-
tifibrotic therapies in the pleural cavity. We further show that pleu-
ral fibrosis, through a process involving MFT, can expand into the
lung parenchyma from the visceral layer, but not into the muscle
from the parietal layer, suggesting that a distinct local environment
is required for progressive fibrotic responses in the tissue.
*Faculty of Medicine and Pharmacy, Institut National de la Sante´ et de la Recherche
Me´dicale (INSERM), Unite´ Mixte de Recherche (UMR) 866, Dijon, France;
†
UMR
1129, Flaveur, Vision et Comportement du Consommateur, Institut National de la
Recherche Agronomique, Etablissement National d’Enseignement Supe´rieur
Agronomique de Dijon, University of Burgundy, Dijon, France;
‡
Department of Pa-
thology and Molecular Medicine, Centre for Gene Therapeutics, McMaster Univer-
sity, Hamilton, Ontario, Canada; and
§
Service de Pneumologie et Re´animation Res-
piratoire, Centre Hospitalier Universitaire du Bocage, Dijon, France
Received for publication June 12, 2007. Accepted for publication August 14, 2007.
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
N.D. was supported by the Comite´deCoˆte d’Or de la Ligue Contre le Cancer and
by the Socie´te´ de Pneumologie de Langue Franc¸aise. P.B. was supported by Pneu-
mologie De´veloppement. M.K. is a Parker B. Francis Fellow and was supported by a
Career Development Award of Department of Medicine, McMaster University.
P.J.M. is a Canadian Institutes for Health Research Clinician Scientist.
2
Address correspondence and reprint requests to Dr. Philippe Bonniaud, Service de
Pneumologie et Re´animation Respiratoire, Centre Hospitalier Universitaire du Bo-
cage, 21079 Dijon, France. E-mail address: philippe.bonniaud@chu-dijon.fr
3
Abbreviations used in this paper: EMT, epithelial-to-mesenchymal transition; MFT,
mesothelial-fibroblastoid transformation; PLF, pleural lavage fluid; BAL, bronchoal-
veolar lavage; BALF, BAL fluid; HSP, heat shock protein; SMA, smooth muscle
actin; MMP, matrix metalloproteinase.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org
Materials and Methods
Recombinant adenovirus
We used AdTGF-

1
223/225
, an adenovirus construct with a mutant TGF-

1
translated into spontaneously bioactive TGF-

1, AdLacZ (coding for

-ga-
lactosidase), and AdDL (control vectors) with no insert in the deleted E1
region for the experiments described. The construction of adenoviral vec-
tors is described in detail elsewhere (19, 20).
Animal treatment
Female Sprague-Dawley rats (Charles River Laboratories) weighing 200–
225 g were housed in special pathogen-free conditions. Rodent laboratory
food and water were provided ad libitum. The animals were treated in
accordance to the guidelines of the Ministe`re de la Recherche et de la
Technologie (Paris, France). All animal procedures were performed with
inhalation anesthesia with isoflurane (TEM). A total of 1.3 ⫻ 10
9
PFU of
AdTGF-

1, AdLaCZ, or AdDL were administered in a volume of 800
l
of NaCl 0.9%, without any surgery, by intrapleural injection on the right
side (sixth space) with a 20-G needle, animals in a left lateral decubitus
position. For coadministration experiments, rats received, in 800
lof
0.9% NaCl, 1 ⫻ 10
9
PFU AdLacZ plus 1.3 ⫻ 10
9
PFU AdTGF-

1or1⫻
10
9
PFU AdLacZ plus 1.3 ⫻ 10
9
PFU AdDL. Rats were euthanized by
abdominal aortic bleeding at days 4, 7, 14, 21, and 64 after adenoviral
administration. After slight incision through the diaphragm, 2.5 ml of 0.9%
NaCl were injected in the pleural space. The pleural lavage fluid (PLF) was
retrieved with a 1-ml needle and maintained on ice until further processing.
A canula was then placed into the trachea, the lungs were removed en bloc
and bronchoalveolar lavage (BAL) was performed as previously described
(21). Six milliliters of NaCl 0.9% was slowly injected intratracheally, re-
trieved, and maintained on ice. The lungs were connected through the
canula to a column filled with 4% formalin with a constant pressure of 20
cm H
2
O for 10 min. The total volume of formalin required to inflate the
lungs was considered as an estimate of total lung volume. Lungs, dia-
phragm, and chest wall were placed in 4% formalin for 24 h.
PLF and BAL fluid (BALF) were centrifuged at 2500 rpm for 15 min.
After removal of cell and debris pellets, supernatants were stored at ⫺80°C
until further use. The pellets were resuspended in 1 ml of 0.9% NaCl.
Cytospins (200
l, 300 rpm, 2 min) were done in a cytocentrifuge (Cyto-
spin 4; Thermo Shandon) and stained with Giemsa (Sigma-Aldrich) for
differential cell count.
Determination of TGF-

1 levels
Active TGF-

1 was determined from BALF and PLF supernatants using
an ELISA kit for human TGF-

1 (R&D Systems), performed according to
the recommendations of the manufacturer. The sensitivity of this assay is
7 pg/ml.

-galactosidase staining
Cytochemical staining for

-galactosidase was performed on samples ob-
tained from four animals per time point after intrapleural injection of
AdLacZ, AdLacZ plus AdTGF-

1, or AdLacZ plus AdDL. After1hin
fixative (2% formaldehyde/0.2% glutaraldehyde), fresh tissue samples
were stained for6hinasolution containing potassium ferrous cyanide,
potassium ferric cyanide, magnesium chloride, Triton X-100 and 5-bromo-
4-chloro-3-indolyl-

-D-galactopyranoside (X-Gal; Sigma-Aldrich). The
samples were stored in 70% ethanol and then paraffin-processed and
-embedded. Five-micrometer sections were counterstained with nuclear
fast red as previously described (22).
Histology
Immunohistochemistry. Transverse sections of the lung were paraffin-
embedded, 5-
m sectioned, and stained with H&E, Masson-Trichrome and
Picrosirius Red or processed for collagen and heat shock protein (HSP) 47
immunohistochemistry. The primary Abs were a mouse monoclonal
(COL-1) to colligin-I (Abcam) and a mouse anti-HSP47 (colligin) mAb
(Stressgen; TebuBio); the secondary Abs were, respectively, a goat anti-
mouse IgG biotin conjugated (Chemicon International) and a biotinylated
goat anti-mouse/rabbit Ig (DakoCytomation). After peroxidase inhibition
(PBS plus H
2
O
2
, 20 min), tissue sections were incubated with the primary
Ab (1/50 dilution for collagen I and 1/500 dilution for HSP47, overnight at
4°C in humidity chamber). Tissue sections were then incubated with the
secondary Ab (1/500 dilution for collagen-I and 1/250 dilution for HSP47,
1 h). The streptavidin-HRP complex (DakoCytomation) was applied (1/300
dilution for collagen-I and 1/500 dilution for HSP47) during 45 min at
room temperature. 3-Amino-9-ethylcarbazole/hydrogen peroxide was used
as chromogen substrate. Slides were counterstained with hematoxylin.
For the purpose of being as distant from the intrapleural injection site
as possible, we always used the left lung for histology and morpho-
metric analysis.
Immunofluorescence. Dual staining with Abs to
␣
-smooth muscle actin
(
␣
-SMA) and cytokeratin was performed using fluorescently labeled Abs.
After Ag retrieval in boiling 10 mM citrate buffer (pH 6.0) for 45 min,
sections were incubated with
␣
-SMA Ab (DakoCytomation) followed by a
secondary rabbit anti-mouse Ab labeled with Texas Red (Molecular
Probes). Sections were stained with a FITC-labeled Ab to pancytokeratin
(Sigma-Aldrich) and mounted with a 4⬘,6⬘-diamidino-2-phenylindole
(DAPI) nuclear stain (Vectashield; Vector Laboratories). Negative control
for immunofluorescent staining were conducted using IgG2a (DakoCyto-
mation) control Ab substituted for anti-
␣
-SMA or normal goat serum sub-
stituted for anti-pancytokeratin. These sections were viewed with a Leica
DMR fluorescence microscope (Leica Microsystems).
FIGURE 1. Adenovirus-mediated gene transfer to the pleural me-
sothelium is transient and limited to the pleura. Lungs were fixed and
stained for

-galactosidase activity 4, 10, 14, and 21 days after AdLacZ
administration. A, Extensive uniform expression (blue staining) of ad-
enoviral gene product was seen until day 10 but had completely disap-
peared by day 21. B, Four days after AdLacZ intrapleural injection,
section of the lung demonstrated

-galactosidase staining limited to the
mesothelial cell monolayer. Gene transfer was also effective in me-
sothelial cells from the chest wall and the diaphragm (chest side only).
Sections were counterstained with nuclear fast red. No

-galactosidase
activity was observed in control vector AdDL-treated animals (A and
B); n ⫽ 4 animals per day.
6044 TGF-

1-INDUCED PLEURAL FIBROSIS
Pleural thickness assessment
The pleural thickness was measured by histomorphometric measurement
on lung sections stained with Masson-Trichrome (200 times). Twenty-five
random measures per lung section were obtained for each animal using an
Eclipse E600 microscope (Nikon). Video images were captured with a 3
CCD color video camera (Sony/Nikon) and analyzed using an image an-
alyzing system (Archimed; Microvision Instruments).
Collagen quantitation
Collagen amount was analyzed on paraffin sections stained with Picrosirius
Red (20 times) as previously described (23, 24). Briefly, 25 random fields
for each animal were digitized under polarized transmission illumination.
The percentage of emission was quantified (morphometry software from
Histolab/Microvision Instruments) as a reflection of percentage collagen.
Collagen intensity in the pleura was measured within a rectangle deter-
mined by a constant length (200
m), the width depending on pleura thick-
ness. Collagen content was expressed as the percent of emission multiplied
by the surface of each rectangle. Collagen amount within the pulmonary
parenchyma was measured using circles randomly disposed at a constant
distance from the pleural surface (see Fig. 5A). Each circle had a constant
diameter of 640
m. Circle 1 was below the pleura. Circle 2 was deeper in
the parenchyma, directly underneath circle 1 in a perpendicular way to the
pleura. Large vessels and airways were excluded. The collagen content
within circles was expressed as the percent of emission.
Zymography
The hydrolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9
in PLF and BAL was measured by gelatin zymography. Samples were
separated by 10% SDS-PAGE containing 0.1% gelatin (Sigma-Aldrich).
After electrophoresis, the gels were incubated in 2.5% Triton X-100 (Sigma-
Aldrich) for 30 min and were then placed in the activating buffer (50
mM Tris-HCl (pH 8), 10 mM CaCl
2
,5
M ZnSO
4
, 150 mM NaCl; Sigma-
Aldrich), overnight at 37°C. The gels were stained with 0.1% Coomassie
brilliant blue-250 solution (Sigma-Aldrich), during 30 min at 37°C. The
gels were then destained with several changes of 40% methanol and 7%
acetic acid. Zones of enzymatic activity were evident as clear bands against
blue background. Reference standards used were MMP-2 and MMP-9
(Chemicon International).
Statistical analysis
Comparisons between the AdTGF-

1-treated group and the control group
(AdDL) were performed by Mann-Whitney U test and comparisons be-
tween rats of the same group were performed by Wilcoxon test.
Results
Bilateral and transient gene transfer in rat pleura after right
intrapleural injection
Lungs were harvested en bloc at various time points after AdLacZ
administration into the right pleural space and stained for

-ga-
lactosidase activity (Fig. 1A). Adenovirus was highly efficient
in transfecting mesothelial cells, as demonstrated by wide-
spread expression of

-galactosidase in the visceral pleura by
days 4 and 10 (Fig. 1B). Mesothelial cells from the chest wall
and the diaphragm were transfected as well. The transgenic pro-
tein was strictly limited to pleural mesothelial cells and was not
observed within the pulmonary parenchyma (Fig. 1B)orinthe
peritoneal cavity. Gene transfer was transient and disappeared
over a period of 14 days. No

-galactosidase activity was found
in control rats (Fig. 1). Gene transfer was bilateral and uniform
due to anatomical connections between the right and left pleural
space in rodents.
FIGURE 2. TGF-

1 pleural fibrosis is associated with a severe lung
volume restriction: AdTGF-

1-treated rat lungs appeared much smaller
than their controls by day 64. For inflation of the lungs from AdTGF-

1-
treated rats, the volume of formalin needed at a constant pressure of 20 cm
H
2
O for 10 min, was significantly smaller than that of control rats by day
64; p ⫽ 0.034; n ⫽ 5 AdTGF-

1 and 3 AdDL.
FIGURE 3. TGF-

1 gene transfer to the
pleural mesothelium induces transient active
TGF-

1 production in PLF (ELISA): a
strong TGF-

1 increase in PLF was detected
by days 4 and 7 after AdTGF-

1 adminis-
tration but no longer by day 14. No TGF-

1
overexpression was detected in PLF from
control rats. In BALF from AdTGF-

1-
treated rats, only a slight TGF-

1 increase
was detected by day 7. ⴱ, p ⫽ 0.034 com-
pared with AdDL animals; n ⫽ 5 AdTGF-

1
and 3–4 AdDL per day.
Table I. Cell count of BALF and PLF from rats treated with AdDL
or AdTGF-

1
a
Day
Total Cell Count (⫻10
4
)
AdDL AdTGF-

1
PLF BALF PLF BALF
4 767 ⫾ 22 37 ⫾ 10 1953 ⫾ 1510 43 ⫾ 3
7 4900 ⫾ 1600 33 ⫾ 7 1133 ⫾ 402 44 ⫾ 9
14 2900 ⫾ 1888 58 ⫾ 9 1143 ⫾ 933 45 ⫾ 5
21 600 ⫾ 164 77 ⫾ 22 373 ⫾ 190 88 ⫾ 15
64 255 ⫾ 100 97 ⫾ 23 180 ⫾ 59 85 ⫾ 17
a
The cell pellets remaining after centrifugation of the BALF or PLF samples
were resuspended in 0.9% NaCl. The numbers of cells were counted by using a
hemocytometer. Cell differentials were determined with at least 300 cells. No
significant difference was observed between AdTGF-

1 and AdDL in total cell
count in PLF as well as in BALF. No difference was observed in differential count
in PLF with a majority of mononucleated cells.
6045The Journal of Immunology
TGF-

1 adenovector-mediated gene transfer to the pleura
Rats treated with the control virus (AdDL) were healthy and steadily
gained weight. In contrast, rats treated with AdTGF-

1 appeared sick
with ruffled fur and lost up to 15% of body weight in the first 2 wk
before recovering. Some animals had to be euthanized after
AdTGF-

1 administration due to poor condition and slow recovery.
These animals demonstrated fibrosis of pleura but were not included
in data analysis, because they did not reach the determined endpoints.
FIGURE 4. AdTGF-

1 transient gene transfer to the pleura induces a progressive pleural fibrosis. A, Four days after AdTGF-

1 administration, there
was already a slight collagen deposition with a thickened pleura. The collagen accumulation was increasing up to day 64 with a thick, dense pleura and
thickened alveolar wall underneath the pleura. In control rats, the pleura appeared normal with no collagen deposition at any time point (Masson-Trichrome,
200 times). HSP47 is strongly overexpressed from days 4 to 64 in AdTGF-

1-treated rats compared with control rats (HSP47 immunohistochemistry, 200
times). B, By histomorphometric measurement (collagen intensity, Picrosirius Red, 20 times, under polarized transmission illumination), the collagen
accumulation within the pleura was progressive up to day 64 (bar chart). The increased thickness (Masson-Trichrome, 200 times) was sustained up to day
64 (line). n ⫽ 5 AdTGF-

1 and 3– 4 AdDL per day; p ⬍ 0.01 for each day in both experiments when ADTGF-

1 was compared with AdDL rats from
the corresponding day. ⴱ, p ⬍ 0.05 compared with day 4 for collagen accumulation and thickness. C, Sixty-four days after intrapleural AdTGF-

1
administration, there was a collagen accumulation on the chest surface of the diaphragm. The chest wall surface demonstrated a moderate collagen
accumulation strictly limited to the surface.
6046 TGF-

1-INDUCED PLEURAL FIBROSIS
Macroscopically, the visceral pleural surface of AdTGF-

1-
treated lungs appeared at any examined time point homogenously
white. AdTGF-

1-treated lungs looked abnormal with shrinkage
when compared with control, an observation which was progres-
sive until day 64 (Fig. 2). Interestingly, the parietal pleura on the
chest wall did not look different between AdTGF-

1- and AdDL-
treated rats. Adhesions were rare and blunt dissection between
parietal and visceral pleura was easy. Lung volumes were assessed
by measuring the volume of formalin that drained into the lungs
after 10 min at a constant pressure of 20 cm H
2
O. Volumes were
reduced by 30% in AdTGF-

1-treated lungs compared with con-
trol by day 64 (7.45 ⫾ 0.76 ml and 10.17 ⫾ 0.17 ml, respectively,
p ⫽ 0.034).
Intrapleural administration of AdTFG-

1 induced local and
transient overexpression of active TGF-

1
The concentration of transgenic protein was measured by ELISA
for active human TGF-

1 protein in the supernatants of PLF
and BALF. Analysis of PLF from AdTGF-

1-treated rats (Fig.
3) revealed significantly increased levels of active TGF-

1in
the pleural space by days 4 and 7 (4717 ⫾ 785 pg/ml and
2872 ⫾ 594 pg/ml, respectively) compared with AdDL control
rats (61 ⫾ 9 and 48 ⫾ 2 pg/ml, respectively). The transgenic
product was no longer detectable after day 14. In BALF, a small
increase in TGF-

1 was detected by day 7 in AdTGF-

1-
treated rats compared with control (332 ⫾ 103 pg/ml and 60 ⫾
1 pg/ml, respectively, p ⫽ 0.034).
AdTGF-

1-induced pleural effusion
By day 4, we found significant pleural effusion associated with
high concentration of TGF-

1 (3.3 ⫾ 1.1 ml, p ⫽ 0.025 compared
with control). The volume of pleural effusion decreased over time
(1.1 ⫾ 0.6 ml by day 7, p ⫽ 0.025 when compared with controls),
and was no longer present by day 14. No pleural effusion was
observed in control vector-treated animals at any time point. Total
cells in PLF were increased by days 4, 7, and 14 compared with
later time points, with no significant difference between AdDL-
and AdTGF-

1-treated rats (Table I). There was a predominance
of mononucleated cells in the differential cell count (⬎98%) at
each time point in all AdDL- and AdTGF-

1-exposed animals. No
difference was observed in BALF cell count at any time point
between AdDL- and AdTGF-

1-treated animals.
TGF-

1 gene transfer induces bilateral and progressive fibrosis
Sections of left and right lung from the same animal were assessed
by histomorphometry to compare visceral pleura thickness be-
tween both sides after intrapleural AdTGF-

1 administration (into
FIGURE 5. AdTGF-

1 transient gene transfer to the pleural mesothe-
lium induces a progressive increase in collagen amount within the pulmo-
nary parenchyma. A, Sixty-four days after AdTGF-

1 administration, col-
lagen I (brown color) is strongly overexpressed within the pleura and
within the pulmonary parenchyma (collagen I immunohistochemistry, 200
times). B, Collagen levels were randomly measured at constant distance
from the pleura (Picrosirius Red staining, 20 times, under polarized trans-
mission illumination, as shown on the picture). Each circle has a 640-
m
diameter. Circle 1 is the one against the pleura. Circle 2 is deeper in the
parenchyma, directly underneath circle 1 in a perpendicular way to the
pleura. Large vessels and airways were excluded. C, Results are expressed
as mean quantity of collagen in AdTGF-

1-treated rats reported to these of
control rats (n ⫽ 5 AdTGF-

1 and 3 AdDL); p ⬍ 0.01 for each day when
ADTGF-

1 was compared with AdDL rats from the corresponding day.
ⴱ, p ⬍ 0.05 compared with AdTGF-

1 animals from days 4, 7, and 14.
FIGURE 6. TGF-

1 induces mesothelial cell migration within the fibrotic tissue. After AdLacZ/AdDL coadministration, transfected mesothelial cells
expressing

-galactosidase by day 6 are on a single monolayer as normally observed. After AdLacZ/AdTGF-

1 coadministration, mesothelial cells
transfected by day 0 with AdLacZ (blue staining) are still localized by day 2 on a single monolayer but clearly start to change the phenotype from a flat
to a more spindle like shape (please compare with Fig. 1B which shows cells exposed to AdLacZ but not AdTGF-

1 at the same ⫻400 magnification).
These cells migrate into the fibrotic tissue by day 6 when TGF-

1 was overexpressed.
6047The Journal of Immunology
the right chest cavity). The findings demonstrate that pleural fi-
brosis after intrapleural administration of AdTGF-

1 is bilateral
and homogeneous (data not shown), due to anatomical connections
between the two cavities in rodent lung. Consequently, we decided
to perform all histomorphometric measurements on the left lung to
be distant from the injection side and potential local irritation of
the pleura by inflammatory cells or hemorrhage. We found that
pleural thickening began as early as at day 4 and progressively
increased through days 21 and 64 (Fig. 4A). Similarly, the amount
of collagen within the thickened pleura as assessed by picrosirius
red staining (a marker specific for collagen) was progressively
increased up to day 64 (Fig. 4B). The pleura appeared normal in
AdDL-treated rats at any time point without any indication for
collagen accumulation. The parietal pleura of the diaphragm
showed fibrotic changes with a uniform collagen deposition de-
spite normal aspect on visual examination (Fig. 4C). There was
no fibrosis on the peritoneal side of the diaphragm. The parietal
pleura of the chest wall showed even less collagen accumula-
tion (Fig. 4C).
HSP47, a collagen-specific chaperon and closely associated with
de novo synthesis of collagen, was present in the fibrotic tissue
from day 4 on through to day 64 indicating an active fibroprolif-
erative process (Fig. 4A).
Increase in the collagen level in the pulmonary parenchyma
Although fibrotic changes were strictly limited to the surface of
diaphragm and chest wall and exclusively pleural, they appeared
markedly different on the visceral pleura (Fig. 4A). Indeed, the
fibrotic changes invaded the lung parenchyma adjacent to the pleu-
ral surface (Fig. 5, A and B). We measured collagen density within
the parenchyma at a constant distance from the pleural surface and
demonstrated that intrapleural TGF-

1 gene transfer induces a
moderate but significant and progressive fibrotic response within
the pulmonary parenchyma. Collagen accumulation started right
underneath the pleura where it was most intense (Fig. 5C, circle 1)
and it increased progressively up to day 64. The amount of paren-
chymal collagen accumulation decreased with increasing distance
from the surface (Fig. 5C, circle 2) but was still progressive
over time.
Mesothelial-fibroblastoid transformation
In experiments with coadministration of AdLacZ and AdTGF-

1,
we observed that mesothelial cells transfected by day 0 with
AdLacZ (blue staining) progressively migrated into the fibrotic
tissue when TGF-

1 was overexpressed (Fig. 6). The mesothelial
cell layer contained abundant cytokeratin-positive cells (Fig. 7,
thin arrow) from day 4 to 64. By day 4 after AdTGF-

1, but not
AdDL, mesothelial cells became round and lost their intercellular
connection (Figs. 6 and 7). Seven and 14 days after AdTGF-

1,
immunofluorescence demonstrated the appearance of dual-labeled
cytokeratin and
␣
-SMA-positive cells (Fig. 7, thick arrow) within
the fibrotic tissue. By day 64 numerous
␣
-SMA-positive cells were
observed within the parenchyma underneath the strong pleural fi-
brotic tissue.
These phenotypical changes observed in mesothelial cells were
accompanied by a strong increase in gelatinolytic activity in the
FIGURE 7. TGF-

1 induces phenotypic transition. Immunofluores-
cence from pleural sections stained for cytokeratin (green),
␣
-SMA (red),
with nuclear counterstain (blue, DAPI). The mesothelial cell layer demon-
strates cytokeratin-positive cells (thin arrows) from days 4 to 64. By days
7 and 14 after AdTGF-

1, there was dual-stained cytokeratin and
␣
-SMA-
positive cells (thick arrows). By day 64, numerous
␣
-SMA-positive cells
(dotted arrows) were observed within the parenchyma. Magnification,
⫻400 for days 4, 7, and 14; ⫻200 for day 64.
FIGURE 8. TGF-

1 induces a local MMP activity (gel-
atin zymography from PLF): AdTGF-

1 induces an in-
crease in MMP-2 activity by days 4, 7, and 14 and in
MMP-9 activity by day 4, compared with control rats (n ⫽
5 AdTGF-

1 and 3 AdDL per day).
6048 TGF-

1-INDUCED PLEURAL FIBROSIS
pleural fluid, which is a critical factor in the process of mesothe-
lial-to-mesenchymal transition. MMP-2 activity was markedly in-
creased as determined by gelatin zymography in PLF from
AdTGF-

1 compared with control-treated rats from days 4 to 14
and MMP-9 activity was increased by day 4 (Fig. 8).
Discussion
Pleural fibrosis is usually considered as complication of other dis-
orders involving the chest cavity, and it is unclear why only some
individuals develop progressive pleural scarring in response to in-
jury. In this study, we show that active TGF-

1 administered to the
rat pleural mesothelium by adenoviral gene transfer leads to pro-
gressive pleural fibrosis without pleurodesis, but with severe lung
volume restriction. We further report that pleural fibrotic changes
are associated with MFT and advance significantly into the lung
parenchyma, but not into adjacent chest wall or diaphragmatic
muscle.
Pleural fibrosis can develop in response to a large variety of
diseases and tissue injury. These are frequently but not exclusively
associated with inflammation. The majority of pleural inflamma-
tory processes resolves with treatment of the underlying cause or
improves spontaneously. However, sometimes chronic scarring
and fibrosis develops despite obvious resolution of the inflamma-
tory phase (1). Progressive scarring can also occur without major
inflammation, such as after surgical thoracotomy for coronary by-
pass (25). The trigger for switching a resolving to a progressively
fibrotic tissue response in the pleural cavity has not yet been iden-
tified. Several studies have demonstrated a potential role of the
growth factors basic fibroblast growth factor, platelet-derived
growth factor, and TGF-

1 (26). TGF-

1 has been widely studied
in the context of fibrotic diseases. This growth factor is known for
its anti-inflammatory effects, is chemotactic for fibroblasts and
promotes the accumulation of extracellular matrix (27). Transient
overexpression of active TGF-

1 in rat lungs by adenoviral gene
transfer causes progressive lung fibrosis without major inflamma-
tion, characterized by extensive deposition of extracellular matrix
proteins such as collagen and fibronectin and by accumulation of
myofibroblasts (20). In contrast, adenovector-mediated overex-
pression of IL-1

induces a severe inflammatory response in the
lung followed by progressive fibrosis, likely mediated through en-
dogenous up-regulation of TGF-

1 (21, 24). The profibrotic effects
of TGF-

do not only apply to bronchial and alveolar epithelial
cells but also to mesothelial cells (28). TGF-

stimulation causes
mesothelial cells to synthesize excessive collagen and other matrix
proteins in vitro (28). In the study reported here, we administered
the gene for spontaneously active TGF-

1 into the right pleural
cavity of rats and generated high levels of active protein in the
pleural fluid, with peak expression between days 4 and 7. This
approach resulted in severe and progressive pleural fibrosis. There
was a moderate mononuclear inflammatory response to the ade-
novirus with no difference between AdTGF-

1 and control vector-
treated rats. AdTGF-

1-induced fibrosis was progressive up to 64
days, long after the transgenic protein has disappeared from the
pleural fluid. We assume that this progressive fibrogenic response
is partly mediated by TGF-

autoinduction similar to what has
been discussed in lung fibrosis (20). It has recently been shown
that intrapleural injection of TGF-

2 stimulates mesothelial cells
to produce collagen and endogenous TGF-

, further increasing the
production of matrix proteins (10). Human mesothelial cells are
not only able to synthesize TGF-

but also have receptors for this
cytokine (11, 29), further supporting the hypothesis of autocrine
activation of TGF-

in pleural fibrosis.
The focus of most pleural fibrosis research is related to induc-
tion of adhesions and fibrotic changes with the clinical purpose of
pleurodesis, mostly for treatment of malignant or chronic and ther-
apy resistant pleural effusion (7). Talc and tetracycline instillation
into the chest cavity are the most common approaches to pleu-
rodesis (30). However, they are associated with significant clinical
problems such as high fever and pain. Even adult respiratory dis-
tress syndrome has been recognized as a complication in up to 9%
of patients receiving talc pleurodesis (31, 32). Hence, profibrotic
growth factors are considered as potential alternate therapies and
investigated in animal models. Lee et al. (8, 10) showed in a rabbit
or a sheep model that rTGF-

2 administration into the pleural
space induces initially marked fluid effusion, which is followed by
effective pleurodesis with multiple adhesions. In contrast to this
study, we noticed in our rat model only transient pleural effusion
at very early time points, followed by progressive fibrosis of the
pleura, but adhesions were almost absent (Fig. 2 and 4A). Both
these models support a major role for TGF-

in the development
and progression of pleural fibrosis, although the difference be-
tween TGF-

1 and 2 is somewhat unexpected. TGF-

1 and 2 are
thought to have similar effects on the formation of matrix, but the
route of administration may account for differences in the forma-
tion of adhesions (TGF-

1 through adenoviral gene transfer as
opposed to recombinant protein for the TGF-

2 studies) (8 –10).
We believe that the technique applied for intrapleural injection
may have had the most impact on the pleurodesis. Our experimen-
tal approach does not involve surgery and allows avoiding major
bleeding and additional inflammatory factors which can lead to
adhesions and pleurodesis (33). Finally, pleural adhesion and pleu-
ral thickening may be governed by different mechanisms.
Surprisingly, the value of anti-TGF-

strategies in the treatment
of pleural fibrosis and adhesions has not been investigated thor-
oughly. TGF-

is one of the major targets in the development of
antifibrotic therapies for pulmonary fibrosis, and a number of ex-
perimental studies in animals strongly support this approach (34,
35). In our model, we induced severe and homogenous bilateral
fibrosis of the visceral pleura, leading to significant restriction of
lung volumes. Pleural fibrosis in humans becomes clinically sig-
nificant with a restrictive pattern in lung function when it involves
major parts of the visceral pleura. This can cause substantial mor-
bidity and chronic respiratory failure, e.g., in asbestos lung, after
thoracic surgery, or hemothorax, in drug-induced pleural fibrosis
(ergots drug, previous chemotherapy for cancer, radiation therapy)
or in familial idiopathic pleural fibrosis. No effective medical treat-
ment is available for these conditions and surgical decortication is
frequently required, with limited therapeutic success (1). Anti-
TGF-

strategies are a promising therapeutic approach for preven-
tion or cure these complications. One study has shown that in-
trapleural injection of TGF-

Abs can reduce empyema-induced
pleural fibrosis in a rabbit model (11, 12). Although the role of
TGF-

in other pleural fibrotic disorders is not definitively proven,
we believe that our model could be very useful to further under-
stand how to halt the progression or even cure this chronic
condition.
The pattern and distribution of pleural fibrosis in our model was
different than expected, raising some interesting questions and hy-
potheses that need further exploration. Fibrotic changes were
strongest on the visceral pleura, characterized by increasing accu-
mulation of collagen by day 64. Less but still substantial fibrotic
thickening was seen on the parietal pleura of the diaphragm, and
only minor collagen was detectable on the parietal chest wall
pleura. Although no fibrosis was seen in the muscle of diaphragm
and chest wall, we interestingly found a significant increase of
collagen within the pulmonary parenchyma adjacent to the pleural
surface (Fig. 5) which diminished with farther distance from the
pleura. The presence of a strong positivity of subpleural HSP47 up
6049The Journal of Immunology
to day 64 (Fig. 4A) supports an ongoing fibroproliferative process
at this time point (36). It may be that diffusion of transgenic TGF-

from the pleural space into alveoli immediately underneath the
pleura contributed to this phenomenon. However, this would only
partly explain the progression of these changes over time as the
transgenic protein disappears within less than two weeks. We pro-
pose that the intrapulmonary collagen accumulation seen after in-
trapleural overexpression of TGF-

1 may also result from a pro-
gression of pleural fibrosis into the alveolar structure of the lungs.
Subpleural fibrosis may be result of a response characteristic for
the compartment lung, as Sime et al. (20) suggested earlier when
intrapulmonary overexpression of active TGF-

1 in rats resulted
in severe interstitial fibrosis starting centrally in the lung and even-
tually involved the visceral, but not parietal, pleura. Subpleural
fibrosis is one of the hallmarks of usual interstitial pneumonia (37).
Fibroblastic foci dominate in the earlier stages of disease, being a
sign for an ongoing fibrotic process, whereas honeycombing is
more indicative for advanced and “burned out” disease (38). It has
just been reported that fibroblastic foci may communicate much
closer with each other than anticipated. Cool et al. (39) suggest that
they may even form a network of fibroblasts, a “fibroblastic retic-
ulum” extending from the pleura to the underlying parenchyma.
The interconnecting fibroblasts described by this group were not
monoclonal, thus they likely result from a reactive rather than neo-
plastic response to tissue injury. The observation in our study fits
well into this concept, and we believe that our model will be ex-
tremely useful to investigate this hypothesis in detail. Further in-
dication for a potential modulating role of the pleura in fibrotic
lung disease is the ability of pleural mesothelial cells to transform
into myofibroblast-like cells. EMT is a central mechanism for di-
versifying the cells found in complex tissues (40). It is involved in
a variety of normal physiological as well as pathological processes
such as cancer progression and renal fibrosis (16, 17). Recent ev-
idence suggests a potential role for EMT in the pathogenesis of
pulmonary fibrosis (14, 15, 41– 43). MFT is a similar process and
has been described as a major factor in the development of peri-
toneal fibrosis (18). We demonstrate clear evidence that MFT oc-
curs in our model of AdTGF-

1-induced pleural fibrosis, and that
the mesenchymal cells derived through this process invade into the
lung parenchyma. We did not find obvious fibroblastic foci in our
model. Further research is needed to investigate whether the sub-
pleural distribution pattern of pulmonary fibrosis may be in part
related to a pleural process or involvement of mesothelial cells in
this disease.
In summary, we here present a novel model to examine the
pathogenesis of pleural fibrosis using transient adenoviral vector-
mediated overexpression of TGF-

1. The fibrotic process is pro-
gressive and interestingly invades into the subpleural zone of the
lung parenchyma. This is associated with MFT, and it can be spec-
ulated that the pleural mesothelium may be involved in the sub-
pleural distribution pattern of pulmonary fibrosis.
Acknowledgments
We thank V. San Giorgio and all the team from the animal quarter for their
invaluable and professional help.
Disclosures
The authors have no financial conflict of interest.
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6051The Journal of Immunology
