And Subpleural Fibrosis 1 Induces Progressive Pleural Scarring Β TGF

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TGF-β1 Induces Progressive Pleural Scarring and Subpleural Fibrosis Nathalie Decologne, Martin Kolb, Peter J. Margetts, Franck Menetrier, Yves Artur, Carmen Garrido, Jack Gauldie, This information is current as Philippe Camus and Philippe Bonniaud of September 29, 2021. J Immunol 2007; 179:6043-6051; ; doi: 10.4049/jimmunol.179.9.6043 http://www.jimmunol.org/content/179/9/6043 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TGF-␤1 Induces Progressive Pleural Scarring and Subpleural Fibrosis1

Nathalie Decologne,* Martin Kolb,‡ Peter J. Margetts,‡ Franck Menetrier,* Yves Artur,† Carmen Garrido,* Jack Gauldie,‡ Philippe Camus,*§ and Philippe Bonniaud2*§

Pleural fibrosis is a misunderstood disorder which can cause severe restrictive lung disease with high morbidity and even mortality. 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 Downloaded from 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 development 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. http://www.jimmunol.org/ leural fibrosis can cause severe restrictive lung disease. It to pleural effusion and ultimately fibrosis (5, 6). Pleural fibrosis is usually considered as a complication of other disorders can be defined as excessive deposition of matrix components re- P involving the chest cavity. Pneumonia, with parapneumo- sulting in the destruction of regular pleural tissue architecture. The nic effusion and empyema, tuberculosis and asbestos are among disorder can manifest itself as discrete localized lesions (pleural the most common causes for pleural fibrosis (1). Furthermore, nu- plaques) or diffuse pleural thickening (5). Most research related to merous drugs can contribute to the development of pleural fibrosis, pleural scarring concerns the induction of pleurodesis as an ap- the best known being ergot drugs, cytostatic agents, and thoracic proach to treat chronic effusion associated with metastasized can- irradiation (Refs. 2 and 3, and www.pneumotox.com (“The drug- cer (7). Animal studies have shown that TGF-␤ plays an active role by guest on September 29, 2021 induced lung diseases”)). Other potential reasons are systemic con- in pleurodesis as well as in pleural fluid formation (8–12). nective tissue disease, hemothorax, and progressive postthoracot- TGF-␤ is a multifunctional cytokine critically involved in the omy scarring after coronary bypass (4). Depending on disease pathogenesis of fibrosis through its potent effects on fibroblast dif- severity, pleural fibrosis can compromise respiratory function, ferentiation, extracellular matrix formation (13, 14), and epithelial- markedly impair quality of life, and can be associated with high to-mesenchymal transition (EMT)3 (15). Peritoneal mesothelial morbidity or even mortality. There is no effective therapy to re- cells may undergo mesenchymal conversion (16, 17) and TGF-␤1 verse established pleural fibrosis. gene transfer to the peritoneal mesothelium induces peritoneal fi- The pleura is a metabolically active membrane involved in brosis with evidence of mesothelial-to-mesenchymal transition or maintaining a dynamic homeostasis of fluid within the chest cav- “mesothelial-fibroblastoid transformation” (MFT) (18). ity. The homeostasis is important for the mechanical properties of In this study, we used transient transfer of the active TGF-␤1 chest wall and lungs, and a breakdown of the fluid balance can lead gene by adenoviral vectors to the pleural cavity and mesothelium. We demonstrate that this approach induces homogenous, prolonged, and progressive pleural fibrosis without pleurodesis, asso- *Faculty of Medicine and Pharmacy, Institut National de la Santeét de la Recherche Me´dicale (INSERM), Unite´Mixte de Recherche (UMR) 866, Dijon, France; †UMR ciated with severe impairment of pulmonary function. This new 1129, Flaveur, Vision et Comportement du Consommateur, Institut National de la model of pleural fibrosis will allow us to better understand the Recherche Agronomique, Etablissement National d’Enseignement Supe´rieur Agronomique de Dijon, University of Burgundy, Dijon, France; ‡Department of Pa- mechanisms of progressive fibrogenesis, and to explore novel an- thology and Molecular Medicine, Centre for Gene Therapeutics, McMaster Univer- tifibrotic therapies in the pleural cavity. We further show that pleu- sity, Hamilton, Ontario, Canada; and §Service de Pneumologie et Reánimation Res- ral fibrosis, through a process involving MFT, can expand into the piratoire, Centre Hospitalier Universitaire du Bocage, Dijon, France lung parenchyma from the visceral layer, but not into the muscle Received for publication June 12, 2007. Accepted for publication August 14, 2007. from the parietal layer, suggesting that a distinct local environment 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 is required for progressive fibrotic responses in the tissue. 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 Française. P.B. was supported by Pneu- mologie De´veloppement. M.K. is a Parker B. Francis Fellow and was supported by a 3 Abbreviations used in this paper: EMT, epithelial-to-mesenchymal transition; MFT, Career Development Award of Department of Medicine, McMaster University. mesothelial-fibroblastoid transformation; PLF, pleural lavage fluid; BAL, bronchoal- P.J.M. is a Canadian Institutes for Health Research Clinician Scientist. veolar lavage; BALF, BAL fluid; HSP, heat shock protein; SMA, smooth muscle 2 Address correspondence and reprint requests to Dr. Philippe Bonniaud, Service de actin; MMP, matrix metalloproteinase. Pneumologie et Reánimation Respiratoire, Centre Hospitalier Universitaire du Bo- cage, 21079 Dijon, France. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 6044 TGF-␤1-INDUCED PLEURAL FIBROSIS

Materials and Methods Recombinant adenovirus We used AdTGF-␤1223/225, an adenovirus construct with a mutant TGF-␤1 translated into spontaneously bioactive TGF-␤1, AdLacZ (coding for ␤-galactosidase), and AdDL (control vectors) with no insert in the deleted E1 region for the experiments described. The construction of adenoviral vectors 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 ϫ 109 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 ϫ 109 PFU AdLacZ plus 1.3 ϫ 109 PFU AdTGF-␤1or1ϫ 9 ϫ 9 10 PFU AdLacZ plus 1.3 10 PFU AdDL. Rats were euthanized by Downloaded from 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, retrieved, and maintained on ice. The lungs were connected through the http://www.jimmunol.org/ canula to a column filled with 4% formalin with a constant pressure of 20 cm H2O for 10 min. The total volume of formalin required to inflate the lungs was considered as an estimate of total lung volume. Lungs, diaphragm, 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 Ϫ80°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. by guest on September 29, 2021 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 FIGURE 1. Adenovirus-mediated gene transfer to the pleural me- 7 pg/ml. sothelium is transient and limited to the pleura. Lungs were fixed and stained for ␤-galactosidase activity 4, 10, 14, and 21 days after AdLacZ ␤-galactosidase staining administration. A, Extensive uniform expression (blue staining) of adenoviral gene product was seen until day 10 but had completely disap- Cytochemical staining for ␤-galactosidase was performed on samples obtained from four animals per time point after intrapleural injection of peared by day 21. B, Four days after AdLacZ intrapleural injection, ␤ AdLacZ, AdLacZ plus AdTGF-␤1, or AdLacZ plus AdDL. After1hin section of the lung demonstrated -galactosidase staining limited to the fixative (2% formaldehyde/0.2% glutaraldehyde), fresh tissue samples mesothelial cell monolayer. Gene transfer was also effective in me- were stained for6hinasolution containing potassium ferrous cyanide, sothelial cells from the chest wall and the diaphragm (chest side only). potassium ferric cyanide, magnesium chloride, Triton X-100 and 5-bromo- Sections were counterstained with nuclear fast red. No ␤-galactosidase 4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal; Sigma-Aldrich). The activity was observed in control vector AdDL-treated animals (A and samples were stored in 70% ethanol and then paraffin-processed and B); n ϭ 4 animals per day. -embedded. Five-micrometer sections were counterstained with nuclear fast red as previously described (22). Histology Immunohistochemistry. Transverse sections of the lung were paraffin- For the purpose of being as distant from the intrapleural injection site embedded, 5-␮m sectioned, and stained with H&E, Masson-Trichrome and as possible, we always used the left lung for histology and morpho- Picrosirius Red or processed for collagen and heat shock protein (HSP) 47 metric analysis. immunohistochemistry. The primary Abs were a mouse monoclonal Immunofluorescence. Dual staining with Abs to ␣-smooth muscle actin (COL-1) to colligin-I (Abcam) and a mouse anti-HSP47 (colligin) mAb (␣-SMA) and cytokeratin was performed using fluorescently labeled Abs. (Stressgen; TebuBio); the secondary Abs were, respectively, a goat anti- After Ag retrieval in boiling 10 mM citrate buffer (pH 6.0) for 45 min, mouse IgG biotin conjugated (Chemicon International) and a biotinylated ␣ goat anti-mouse/rabbit Ig (DakoCytomation). After peroxidase inhibition sections were incubated with -SMA Ab (DakoCytomation) followed by a secondary rabbit anti-mouse Ab labeled with Texas Red (Molecular (PBS plus H2O2, 20 min), tissue sections were incubated with the primary Ab (1/50 dilution for collagen I and 1/500 dilution for HSP47, overnight at Probes). Sections were stained with a FITC-labeled Ab to pancytokeratin 4°C in humidity chamber). Tissue sections were then incubated with the (Sigma-Aldrich) and mounted with a 4Ј,6Ј-diamidino-2-phenylindole secondary Ab (1/500 dilution for collagen-I and 1/250 dilution for HSP47, (DAPI) nuclear stain (Vectashield; Vector Laboratories). Negative control 1 h). The streptavidin-HRP complex (DakoCytomation) was applied (1/300 for immunofluorescent staining were conducted using IgG2a (DakoCyto- dilution for collagen-I and 1/500 dilution for HSP47) during 45 min at mation) control Ab substituted for anti-␣-SMA or normal goat serum sub- room temperature. 3-Amino-9-ethylcarbazole/hydrogen peroxide was used stituted for anti-pancytokeratin. These sections were viewed with a Leica as chromogen substrate. Slides were counterstained with hematoxylin. DMR fluorescence microscope (Leica Microsystems). The Journal of Immunology 6045

Table I. Cell count of BALF and PLF from rats treated with AdDL or AdTGF-␤1a

Total Cell Count (ϫ104)

AdDL AdTGF-␤1

Day PLF BALF PLF BALF

4 767 Ϯ 22 37 Ϯ 10 1953 Ϯ 1510 43 Ϯ 3 7 4900 Ϯ 1600 33 Ϯ 7 1133 Ϯ 402 44 Ϯ 9 ␤ FIGURE 2. TGF- 1 pleural fibrosis is associated with a severe lung 14 2900 Ϯ 1888 58 Ϯ 9 1143 Ϯ 933 45 Ϯ 5 volume restriction: AdTGF-␤1-treated rat lungs appeared much smaller 21 600 Ϯ 164 77 Ϯ 22 373 Ϯ 190 88 Ϯ 15 than their controls by day 64. For inflation of the lungs from AdTGF-␤1- 64 255 Ϯ 100 97 Ϯ 23 180 Ϯ 59 85 Ϯ 17 treated rats, the volume of formalin needed at a constant pressure of 20 cm a The cell pellets remaining after centrifugation of the BALF or PLF samples H2O for 10 min, was significantly smaller than that of control rats by day were resuspended in 0.9% NaCl. The numbers of cells were counted by using a 64; p ϭ 0.034; n ϭ 5 AdTGF-␤1 and 3 AdDL. 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 Pleural thickness assessment in PLF with a majority of mononucleated cells. The pleural thickness was measured by histomorphometric measurement on lung sections stained with Masson-Trichrome (200 times). Twenty-five Downloaded from random measures per lung section were obtained for each animal using an gels were then destained with several changes of 40% methanol and 7% Eclipse E600 microscope (Nikon). Video images were captured with a 3 acetic acid. Zones of enzymatic activity were evident as clear bands against CCD color video camera (Sony/Nikon) and analyzed using an image an- blue background. Reference standards used were MMP-2 and MMP-9 alyzing system (Archimed; Microvision Instruments). (Chemicon International). Collagen quantitation Statistical analysis Collagen amount was analyzed on paraffin sections stained with Picrosirius Comparisons between the AdTGF-␤1-treated group and the control group http://www.jimmunol.org/ Red (20 times) as previously described (23, 24). Briefly, 25 random fields (AdDL) were performed by Mann-Whitney U test and comparisons be- for each animal were digitized under polarized transmission illumination. tween rats of the same group were performed by Wilcoxon test. 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- Results mined by a constant length (200 ␮m), the width depending on pleura thick- Bilateral and transient gene transfer in rat pleura after right ness. Collagen content was expressed as the percent of emission multiplied intrapleural injection by the surface of each rectangle. Collagen amount within the pulmonary parenchyma was measured using circles randomly disposed at a constant Lungs were harvested en bloc at various time points after AdLacZ ␤ distance from the pleural surface (see Fig. 5A). Each circle had a constant administration into the right pleural space and stained for -ga- by guest on September 29, 2021 diameter of 640 ␮m. Circle 1 was below the pleura. Circle 2 was deeper in lactosidase activity (Fig. 1A). Adenovirus was highly efficient the parenchyma, directly underneath circle 1 in a perpendicular way to the in transfecting mesothelial cells, as demonstrated by wide- pleura. Large vessels and airways were excluded. The collagen content ␤ within circles was expressed as the percent of emission. spread expression of -galactosidase in the visceral pleura by days 4 and 10 (Fig. 1B). Mesothelial cells from the chest wall Zymography and the diaphragm were transfected as well. The transgenic pro- The hydrolytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 tein was strictly limited to pleural mesothelial cells and was not in PLF and BAL was measured by gelatin zymography. Samples were observed within the pulmonary parenchyma (Fig. 1B)orinthe separated by 10% SDS-PAGE containing 0.1% gelatin (Sigma-Aldrich). peritoneal cavity. Gene transfer was transient and disappeared After electrophoresis, the gels were incubated in 2.5% Triton X-100 (Sigma- over a period of 14 days. No ␤-galactosidase activity was found Aldrich) for 30 min and were then placed in the activating buffer (50 ␮ in control rats (Fig. 1). Gene transfer was bilateral and uniform mM Tris-HCl (pH 8), 10 mM CaCl2,5 M ZnSO4, 150 mM NaCl; Sigma- Aldrich), overnight at 37°C. The gels were stained with 0.1% Coomassie due to anatomical connections between the right and left pleural brilliant blue-250 solution (Sigma-Aldrich), during 30 min at 37°C. The space in rodents.

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 administration 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 -p ϭ 0.034 com ,ء .was detected by day 7 pared with AdDL animals; n ϭ 5 AdTGF-␤1 and 3–4 AdDL per day. 6046 TGF-␤1-INDUCED PLEURAL FIBROSIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

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 ϭ 5 AdTGF-␤1 and 3–4 AdDL per day; p Ͻ 0.01 for each day in both experiments when ADTGF-␤1 was compared with AdDL rats from p Ͻ 0.05 compared with day 4 for collagen accumulation and thickness. C, Sixty-four days after intrapleural AdTGF-␤1 ,ء .the corresponding day 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.

␤ TGF- 1 adenovector-mediated gene transfer to the pleura before recovering. Some animals had to be euthanized after Rats treated with the control virus (AdDL) were healthy and steadily AdTGF-␤1 administration due to poor condition and slow recovery. gained weight. In contrast, rats treated with AdTGF-␤1 appeared sick These animals demonstrated fibrosis of pleura but were not included with ruffled fur and lost up to 15% of body weight in the first 2 wk in data analysis, because they did not reach the determined endpoints. The Journal of Immunology 6047

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 progressive 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 H2O. Volumes were reduced by 30% in AdTGF-␤1-treated lungs compared with control by day 64 (7.45 Ϯ 0.76 ml and 10.17 Ϯ 0.17 ml, respectively, p ϭ 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. Downloaded from 3) revealed signiﬁcantly increased levels of active TGF-␤1in the pleural space by days 4 and 7 (4717 Ϯ 785 pg/ml and 2872 Ϯ 594 pg/ml, respectively) compared with AdDL control rats (61 Ϯ 9 and 48 Ϯ 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- http://www.jimmunol.org/ treated rats compared with control (332 Ϯ 103 pg/ml and 60 Ϯ 1 pg/ml, respectively, p ϭ 0.034).

AdTGF-␤1-induced pleural effusion By day 4, we found significant pleural effusion associated with high concentration of TGF-␤1 (3.3 Ϯ 1.1 ml, p ϭ 0.025 compared with control). The volume of pleural effusion decreased over time (1.1 Ϯ 0.6 ml by day 7, p ϭ 0.025 when compared with controls), by guest on September 29, 2021 FIGURE 5. AdTGF-␤1 transient gene transfer to the pleural mesothe- and was no longer present by day 14. No pleural effusion was lium induces a progressive increase in collagen amount within the pulmo- observed in control vector-treated animals at any time point. Total nary parenchyma. A, Sixty-four days after AdTGF-␤1 administration, col- cells in PLF were increased by days 4, 7, and 14 compared with lagen I (brown color) is strongly overexpressed within the pleura and later time points, with no significant difference between AdDL- within the pulmonary parenchyma (collagen I immunohistochemistry, 200 and AdTGF-␤1-treated rats (Table I). There was a predominance times). B, Collagen levels were randomly measured at constant distance of mononucleated cells in the differential cell count (Ͼ98%) at from the pleura (Picrosirius Red staining, 20 times, under polarized trans- each time point in all AdDL- and AdTGF-␤1-exposed animals. No mission illumination, as shown on the picture). Each circle has a 640-␮m difference was observed in BALF cell count at any time point 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 between AdDL- and AdTGF- 1-treated animals. 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 TGF- 1 gene transfer induces bilateral and progressive fibrosis control rats (n ϭ 5 AdTGF-␤1 and 3 AdDL); p Ͻ 0.01 for each day when Sections of left and right lung from the same animal were assessed ADTGF-␤1 was compared with AdDL rats from the corresponding day. by histomorphometry to compare visceral pleura thickness be- p Ͻ 0.05 compared with AdTGF-␤1 animals from days 4, 7, and 14. tween both sides after intrapleural AdTGF-␤1 administration (into ,ء

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 ϫ400 magnification). These cells migrate into the fibrotic tissue by day 6 when TGF-␤1 was overexpressed. 6048 TGF-␤1-INDUCED PLEURAL FIBROSIS

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 despite 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 accumulation (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 fibroproliferative 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 Downloaded from markedly different on the visceral pleura (Fig. 4A). Indeed, the ﬁbrotic changes invaded the lung parenchyma adjacent to the pleural 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 http://www.jimmunol.org/ 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. 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- Mesothelial-fibroblastoid transformation by guest on September 29, 2021 strates cytokeratin-positive cells (thin arrows) from days 4 to 64. By days In experiments with coadministration of AdLacZ and AdTGF-␤1, 7 and 14 after AdTGF-␤1, there was dual-stained cytokeratin and ␣-SMA- we observed that mesothelial cells transfected by day 0 with positive cells (thick arrows). By day 64, numerous ␣-SMA-positive cells (dotted arrows) were observed within the parenchyma. Magnification, AdLacZ (blue staining) progressively migrated into the fibrotic ␤ ϫ400 for days 4, 7, and 14; ϫ200 for day 64. 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 the right chest cavity). The findings demonstrate that pleural fi- AdDL, mesothelial cells became round and lost their intercellular brosis after intrapleural administration of AdTGF-␤1 is bilateral connection (Figs. 6 and 7). Seven and 14 days after AdTGF-␤1, and homogeneous (data not shown), due to anatomical connections immunofluorescence demonstrated the appearance of dual-labeled between the two cavities in rodent lung. Consequently, we decided cytokeratin and ␣-SMA-positive cells (Fig. 7, thick arrow) within to perform all histomorphometric measurements on the left lung to the fibrotic tissue. By day 64 numerous ␣-SMA-positive cells were be distant from the injection side and potential local irritation of observed within the parenchyma underneath the strong pleural fi- the pleura by inflammatory cells or hemorrhage. We found that brotic tissue. pleural thickening began as early as at day 4 and progressively These phenotypical changes observed in mesothelial cells were increased through days 21 and 64 (Fig. 4A). Similarly, the amount accompanied by a strong increase in gelatinolytic activity in the

FIGURE 8. TGF-␤1 induces a local MMP activity (gelatin zymography from PLF): AdTGF-␤1 induces an increase in MMP-2 activity by days 4, 7, and 14 and in MMP-9 activity by day 4, compared with control rats (n ϭ 5 AdTGF-␤1 and 3 AdDL per day). The Journal of Immunology 6049 pleural fluid, which is a critical factor in the process of mesothe- pleurodesis, mostly for treatment of malignant or chronic and ther- lial-to-mesenchymal transition. MMP-2 activity was markedly in- apy resistant pleural effusion (7). Talc and tetracycline instillation creased as determined by gelatin zymography in PLF from into the chest cavity are the most common approaches to pleu- AdTGF-␤1 compared with control-treated rats from days 4 to 14 rodesis (30). However, they are associated with significant clinical and MMP-9 activity was increased by day 4 (Fig. 8). problems such as high fever and pain. Even adult respiratory dis- tress syndrome has been recognized as a complication in up to 9% Discussion of patients receiving talc pleurodesis (31, 32). Hence, profibrotic Pleural fibrosis is usually considered as complication of other dis- growth factors are considered as potential alternate therapies and orders involving the chest cavity, and it is unclear why only some investigated in animal models. Lee et al. (8, 10) showed in a rabbit individuals develop progressive pleural scarring in response to in- or a sheep model that rTGF-␤2 administration into the pleural jury. In this study, we show that active TGF-␤1 administered to the space induces initially marked fluid effusion, which is followed by rat pleural mesothelium by adenoviral gene transfer leads to pro- effective pleurodesis with multiple adhesions. In contrast to this gressive pleural fibrosis without pleurodesis, but with severe lung study, we noticed in our rat model only transient pleural effusion volume restriction. We further report that pleural fibrotic changes at very early time points, followed by progressive fibrosis of the are associated with MFT and advance significantly into the lung pleura, but adhesions were almost absent (Fig. 2 and 4A). Both parenchyma, but not into adjacent chest wall or diaphragmatic these models support a major role for TGF-␤ in the development muscle. and progression of pleural fibrosis, although the difference be- Pleural fibrosis can develop in response to a large variety of tween TGF-␤1 and 2 is somewhat unexpected. TGF-␤1 and 2 are diseases and tissue injury. These are frequently but not exclusively thought to have similar effects on the formation of matrix, but the Downloaded from associated with inflammation. The majority of pleural inflamma- route of administration may account for differences in the forma- tory processes resolves with treatment of the underlying cause or tion of adhesions (TGF-␤1 through adenoviral gene transfer as improves spontaneously. However, sometimes chronic scarring opposed to recombinant protein for the TGF-␤2 studies) (8–10). and fibrosis develops despite obvious resolution of the inflamma- We believe that the technique applied for intrapleural injection tory phase (1). Progressive scarring can also occur without major may have had the most impact on the pleurodesis. Our experimen-

inflammation, such as after surgical thoracotomy for coronary by- tal approach does not involve surgery and allows avoiding major http://www.jimmunol.org/ pass (25). The trigger for switching a resolving to a progressively bleeding and additional inflammatory factors which can lead to fibrotic tissue response in the pleural cavity has not yet been iden- adhesions and pleurodesis (33). Finally, pleural adhesion and pleu- tified. Several studies have demonstrated a potential role of the ral thickening may be governed by different mechanisms. growth factors basic fibroblast growth factor, platelet-derived Surprisingly, the value of anti-TGF-␤ strategies in the treatment growth factor, and TGF-␤1 (26). TGF-␤1 has been widely studied of pleural fibrosis and adhesions has not been investigated thor- in the context of fibrotic diseases. This growth factor is known for oughly. TGF-␤ is one of the major targets in the development of its anti-inflammatory effects, is chemotactic for fibroblasts and antifibrotic therapies for pulmonary fibrosis, and a number of ex- promotes the accumulation of extracellular matrix (27). Transient perimental studies in animals strongly support this approach (34, overexpression of active TGF-␤1 in rat lungs by adenoviral gene 35). In our model, we induced severe and homogenous bilateral by guest on September 29, 2021 transfer causes progressive lung fibrosis without major inflamma- fibrosis of the visceral pleura, leading to significant restriction of tion, characterized by extensive deposition of extracellular matrix lung volumes. Pleural fibrosis in humans becomes clinically sig- proteins such as collagen and fibronectin and by accumulation of nificant with a restrictive pattern in lung function when it involves myofibroblasts (20). In contrast, adenovector-mediated overex- major parts of the visceral pleura. This can cause substantial mor- pression of IL-1␤ induces a severe inflammatory response in the bidity and chronic respiratory failure, e.g., in asbestos lung, after lung followed by progressive fibrosis, likely mediated through en- thoracic surgery, or hemothorax, in drug-induced pleural fibrosis dogenous up-regulation of TGF-␤1 (21, 24). The profibrotic effects (ergots drug, previous chemotherapy for cancer, radiation therapy) of TGF-␤ do not only apply to bronchial and alveolar epithelial or in familial idiopathic pleural fibrosis. No effective medical treat- cells but also to mesothelial cells (28). TGF-␤ stimulation causes ment is available for these conditions and surgical decortication is mesothelial cells to synthesize excessive collagen and other matrix frequently required, with limited therapeutic success (1). Anti- proteins in vitro (28). In the study reported here, we administered TGF-␤ strategies are a promising therapeutic approach for preven- the gene for spontaneously active TGF-␤1 into the right pleural tion or cure these complications. One study has shown that in- cavity of rats and generated high levels of active protein in the trapleural injection of TGF-␤ Abs can reduce empyema-induced pleural fluid, with peak expression between days 4 and 7. This pleural fibrosis in a rabbit model (11, 12). Although the role of approach resulted in severe and progressive pleural fibrosis. There TGF-␤ in other pleural fibrotic disorders is not definitively proven, was a moderate mononuclear inflammatory response to the ade- we believe that our model could be very useful to further under- novirus with no difference between AdTGF-␤1 and control vector- stand how to halt the progression or even cure this chronic treated rats. AdTGF-␤1-induced fibrosis was progressive up to 64 condition. days, long after the transgenic protein has disappeared from the The pattern and distribution of pleural fibrosis in our model was pleural fluid. We assume that this progressive fibrogenic response different than expected, raising some interesting questions and hy- is partly mediated by TGF-␤ autoinduction similar to what has potheses that need further exploration. Fibrotic changes were been discussed in lung fibrosis (20). It has recently been shown strongest on the visceral pleura, characterized by increasing accu- that intrapleural injection of TGF-␤2 stimulates mesothelial cells mulation of collagen by day 64. Less but still substantial fibrotic to produce collagen and endogenous TGF-␤, further increasing the thickening was seen on the parietal pleura of the diaphragm, and production of matrix proteins (10). Human mesothelial cells are only minor collagen was detectable on the parietal chest wall not only able to synthesize TGF-␤ but also have receptors for this pleura. Although no fibrosis was seen in the muscle of diaphragm cytokine (11, 29), further supporting the hypothesis of autocrine and chest wall, we interestingly found a significant increase of activation of TGF-␤ in pleural fibrosis. collagen within the pulmonary parenchyma adjacent to the pleural The focus of most pleural fibrosis research is related to induc- surface (Fig. 5) which diminished with farther distance from the tion of adhesions and fibrotic changes with the clinical purpose of pleura. The presence of a strong positivity of subpleural HSP47 up 6050 TGF-␤1-INDUCED PLEURAL FIBROSIS to day 64 (Fig. 4A) supports an ongoing fibroproliferative process 3. Camus, P., P. Bonniaud, A. Fanton, C. Camus, N. Baudaun, and P. Foucher. at this time point (36). It may be that diffusion of transgenic TGF-␤ 2004. Drug-induced and iatrogenic infiltrative lung disease. Clin. Chest Med. 25: 479–519, vi. from the pleural space into alveoli immediately underneath the 4. Huggins, J. T., and S. A. Sahn. 2004. Causes and management of pleural fibrosis. pleura contributed to this phenomenon. However, this would only Respirology 9: 441–447. 5. Mutsaers, S. E., C. M. Prele, A. R. Brody, and S. Idell. 2004. Pathogenesis of partly explain the progression of these changes over time as the pleural fibrosis. Respirology 9: 428–440. transgenic protein disappears within less than two weeks. We pro- 6. Antony, V. B. 2003. Immunological mechanisms in pleural disease. Eur. Respir. pose that the intrapulmonary collagen accumulation seen after in- J. 21: 539–544. ␤ 7. Lee, Y. C., and R. W. Light. 2004. Management of malignant pleural effusions. trapleural overexpression of TGF- 1 may also result from a pro- Respirology 9: 148–156. gression of pleural fibrosis into the alveolar structure of the lungs. 8. Lee, Y. C., K. B. Lane, R. E. Parker, D. S. Ayo, J. T. Rogers, R. W. Diters, ␤ ␤ Subpleural fibrosis may be result of a response characteristic for P. J. Thompson, and R. W. Light. 2000. Transforming growth factor 2 (TGF 2) produces effective pleurodesis in sheep with no systemic complications. Thorax the compartment lung, as Sime et al. (20) suggested earlier when 55: 1058–1062. intrapulmonary overexpression of active TGF-␤1 in rats resulted 9. Lee, Y. C., C. J. Devin, L. R. Teixeira, J. T. Rogers, P. J. Thompson, K. B. Lane, ␤ in severe interstitial fibrosis starting centrally in the lung and even- and R. W. Light. 2001. Transforming growth factor 2 induced pleurodesis is not inhibited by corticosteroids. Thorax 56: 643–648. tually involved the visceral, but not parietal, pleura. Subpleural 10. Lee, Y. C., D. Malkerneker, C. J. Devin, P. J. Thompson, J. E. Johnson, fibrosis is one of the hallmarks of usual interstitial pneumonia (37). K. B. Lane, and R. W. Light. 2001. 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