Conditional expression of transforming growth factor-{alpha} in adult mouse lung causes pulmonary fibrosis

William D. Hardie,1 Timothy D. Le Cras,2 Kenny Jiang,1 Jay W. Tichelaar,3 Mohamad Azhar,4 and Thomas R. Korfhagen2

Divisions of 1Pulmonary Medicine and 2Pulmonary Biology, Children's Hospital Medical Center, Cincinnati 45229-3039; and 3Department of Environmental Health, Division of Toxicology, 4Division of Molecular Genetics, University of Cincinnati, Cincinnati, Ohio 45267-0056

Submitted 30 June 2003 ; accepted in final form 26 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To determine whether overexpression of transforming growth factor (TGF)-{alpha} in the adult lung causes remodeling independently of developmental influences, we generated conditional transgenic mice expressing TGF-{alpha} in the epithelium under control of the doxycycline (Dox)-regulatable Clara cell secretory protein promoter. Two transgenic lines were generated, and following 4 days of Dox-induction TGF-{alpha} levels in whole lung homogenate were increased 13- to 18-fold above nontransgenic levels. After TGF-{alpha} induction, transgenic mice developed progressive pulmonary fibrosis and body weight loss, with mice losing 15% of their weight after 6 wk of TGF-{alpha} induction. Fibrosis was detected within 4 days of TGF-{alpha} induction and developed initially in the perivascular, peribronchial, and pleural regions but later extended into the interstitium. Fibrotic regions were composed of increased collagen and cellular proliferation and were adjacent to airway and alveolar epithelial sites of TGF-{alpha} expression. Fibrosis progressed in the absence of inflammatory cell infiltrates as determined by histology, without changes in bronchiolar alveolar lavage total or differential cell counts and without changes in proinflammatory cytokines TNF-{alpha} or IL-6. Active TGF-{beta} in whole lung homogenate was not altered 1 and 4 days after TGF-{alpha} induction, and immunostaining was not increased in the peribronchial/perivascular areas at all time points. Chronic epithelial expression of TGF-{alpha} in adult mice caused progressive pulmonary fibrosis associated with increased collagen and extracellular matrix deposition and increased cellular proliferation. Induction of pulmonary fibrosis by TGF-{alpha} was independent of inflammation or early activation of TGF-{beta}.

collagen; proliferation; epidermal growth factor receptor; transforming growth factor-{beta}


PULMONARY FIBROSIS CONTRIBUTES to morbidity and mortality in a number of pediatric and adult lung diseases. Fibrosis may develop secondarily to acute lung injury such as in acute respiratory distress syndrome, from chronic inflammatory diseases such as in cystic fibrosis (CF), or may arise with no defined cause as in idiopathic pulmonary fibrosis (IPF). Although the pathologic features of pulmonary fibrosis may vary depending on the underlying disease process, a number of common features are present including mesenchymal cell proliferation, expansion of the extracellular matrix, and extensive remodeling of the lung parenchyma (6). The molecular pathways and cellular mechanisms leading to fibrosis remain poorly understood, and current therapy is often ineffective or prohibitive due to side effects, emphasizing the need to identify new therapeutic targets.

The epidermal growth factor receptor (EGFR) is a ubiquitous, highly conserved 170-kDa membrane-spanning glycoprotein that is expressed by many cell types in the lung, including the epithelium, smooth muscle cells, endothelium, and fibroblasts (23). Ligands for the EGFR found in the lung include transforming growth factor-{alpha} (TGF-{alpha}), EGF, amphiregulin, and heparin-binding EGF. Several experimental studies have identified a role for EGFR and its ligands in the pathogenesis of pulmonary fibrosis. Madtes et al. (21) demonstrated that TGF-{alpha} knockout mice have significantly reduced lung collagen accumulation compared with wild-type mice following bleomycin injury. A tyrophostin inhibitor of EGFR tyrosine kinase reduced metal-induced pulmonary fibrosis in rats (29). EGFR and its ligands have also been associated with clinical disorders of pulmonary fibrosis. TGF-{alpha} was increased in the bronchoalveolar lavage (BAL) in patients with IPF and immunolocalized to type II epithelial cells, fibroblasts, and the vascular endothelium (1, 22). Lung biopsies from patients with end-stage CF showed increased immunostaining for TGF-{alpha} in alveolar macrophages, airway epithelial cells, and fibrosed submucosal areas (12). Bronchial mucosal biopsy specimens from patients with asthma demonstrate increased EGFR mRNA and protein in the airway epithelium with the level of EGFR expression directly correlating with subbasement membrane thickening (27, 28, 34). TGF-{alpha}, EGF, and EGFR were all increased in the airway and alveolar epithelial cells, macrophages, and vascular smooth muscle of infants with bron-chopulmonary dysplasia (32, 33).

Fibrosis of the pleural surface, alveolar septa, peribronchial and perivascular regions, and alveolar emphysema were detected in transgenic mice that overexpressed TGF-{alpha} under control of the human surfactant protein C (SP-C) promoter (18). The degree of remodeling was directly proportional to the levels of TGF-{alpha} expressed in different lines of SP-C/TGF-{alpha} transgenic mice and was initiated only during the postnatal saccular and alveolar phases of development (13, 14). As the SP-C promoter is constitutively active throughout pre- and postnatal phases of lung development as well as in the adult lung, previous studies did not determine whether TGF-{alpha} over-expression in the adult lung is sufficient to cause emphysema and pulmonary fibrosis. To determine whether TGF-{alpha} causes remodeling in the adult lung independently of developmental influences, we generated transgenic mice in which TGF-{alpha} was conditionally regulated with tetracycline-responsive promoters (11). We demonstrate that induction of TGF-{alpha} expression in the respiratory epithelium of adult transgenic mice produced progressive fibrosis in the absence of increased inflammatory cells, proinflammatory cytokines, or early TGF-{beta} activation.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid construction and oocyte microinjection. Transgenic mouse lines were previously generated bearing the reverse tetracycline-responsive transactivator (rtTA) fusion protein under control of the 2.3-kb rat Clara cell secretory protein (CCSP) gene promoter (35). Separate transgenic mouse lines were generated by microinjection of a (TetO)7-cmv TGF-{alpha} transgene. The (TetO)7-cmv TGF-{alpha} transgene consists of seven copies of tet operator DNA binding sequence linked to a minimal cytomegalovirus (CMV) promoter, the human TGF-{alpha} cDNA, and SV40 polyadenylation signal.

PCR and Southern genotyping. Transgenic mice were identified using PCR primers specific for each transgene as follows: 5' primer in the rat CCSP promoter, 5'-ACT GCC CAT TGC CCA AAC AC-3'; 3' primer in rtTA coding sequence, 5'-AAA ATC TTG CCA GCT TTC CCC-3'. Amplification of PCR products for CCSP-rtTA was performed by denaturation at 94°C for 5 min and then 30 cycles of amplification at 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s, followed by a 7-min extension at 72°C. The (TetO)7-cmv TGF transgene was initially identified by a diagnostic 1.4-kb band on genomic Southern blots of Pst1-digested tail DNA as previously described (13). Mice were subsequently identified for (TetO)7-cmv TGF-{alpha} transgene with PCR with 5' primer in human TGF-{alpha} coding region 5'-CCT GTT CGC TCT GGG TAT TGT GTT and 3' primer in human TGF-{alpha} coding region 5'-CGT GGT CCG CTG ATT TCT TCT CTA. Amplification of PCR products for (TetO)7-cmv TGF-{alpha} was performed by denaturation at 94°C for 2 min and then 28 cycles of amplification at 94°C for 30 s, 62°C for 30 s, and 72°C for 90 s, followed by a 7-min extension at 72°C.

Animal use and administration of doxycycline. Animals were housed under specific pathogen-free conditions and handled in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Children's Hospital Research Foundation and the University of Cincinnati Medical Center. Bitransgenic mice were generated by mating CCSP rtTA mice with (TetO)7-cmv TGF-{alpha} mice. To induce TGF-{alpha} expression, we administered bitransgenic mice doxycycline (Dox) in the drinking water at a final concentration of 0.5 mg/ml with 1% ethanol and replaced the Dox three times per week. Body weights were measured at 2 mo of age and then weekly.

Lung histology and immunohistochemistry. Lungs were inflated fixed using 4% paraformaldehyde at 25 cm of pressure, fixed overnight at 4°C, washed with phosphate-buffered saline (PBS), dehydrated through a graded series of ethanols, and processed for paraffin embedding. Sections (5 µm) were loaded onto polysine slides for immunostaining. Sections were stained with hematoxylin and eosin, Gomori's trichrome stain, or pentachrome (8) for detection of collagen and extracellular matrix deposition. For immunohistochemical detection of bromodeoxyuridine (BrdU), animals were injected with 0.1 ml of BrdU labeling reagent (Zymed Laboratories) per 100 g body wt 2 h before death. BrdU incorporated into DNA was detected using anti-BrdU monoclonal antibody and a BrdU staining kit (Zymed Laboratories) on paraffin-embedded sections of lung tissue. The total number of BrdU-staining nuclei was counted as well as the total number of nuclei in 10 randomly selected uniform fields (26.2 mm2) that encompassed alveolar, pleural, and adventitial regions of the lung. We determined the proliferation index by counting the total number of BrdU-staining nuclei and dividing by the total number of nuclei in each field.

For immunohistochemical detection of human TGF-{alpha}, lung sections were pretreated with antigen retrieval using microwave heat. Sections were treated using mouse on mouse staining kits (Vector Laboratories) before application of anti-human TGF-{alpha} antibody (Oncogene Research Products, Cambridge, MA) diluted 1:40.

For immunohistochemical detection of TGF-{beta}1, paraffin-embedded sections were processed as previously described (26). We performed antigen retrieval by treating sections with hyaluronidase (1 mg/ml in 0.1 M sodium acetate pH 5.5, 0.85% NaCl) for 30 min at 37°C. The sections were incubated with TGF-{beta}1 primary antibody (5 µg/ml, generously provided by Dr. Leslie I. Gold, New York University Medical Center) in blocking serum overnight at 4°C. Control slides were incubated without the primary antibodies.

Hydroxyproline assay. A hydroxyproline assay was performed as previously described (7) with minor modifications. Briefly, lung tissue was lyphophilized overnight, and 10 mg of dried tissue were incubated overnight in 500 µl of 6 N HCl. Five microliters of the samples and standards were applied to an ELISA plate. Fifty microliters of citric-acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% NaOH, and 1.2% glacial acetic acid, pH 6.0) and 100 µl of chloramines T solution (564 mg of chloramines T, 4 ml of H2O, 4 ml of n-propanol, and 32 ml of citrate-acetate buffer) were added and incubated for 20 min at room temperature. Then 100 µl of Ehrlich's solution (4.5 g 4-dimethylaminobenzaldehyde, 18.6 ml n-propanol, and 7.8 ml sulfuric acid) were added and incubated for 15 min at 100°C. Reaction product was read at optical density of 525 nm. Hydroxyproline (Sigma, St. Louis, MO) standard solutions of 0–800 µg/ml were used to construct the standard curve.

Protein studies. After euthanization, lungs were removed and homogenized in 2 ml of PBS, pH 7.2, and centrifuged at 1,500 g, and the supernatant was stored at -70°C. TGF-{alpha} levels were determined with an ELISA kit (Oncogene Research Products) according to the manufacturer's directions. Lung homogenate tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) were determined with ELISA kits (R & D Systems, Minneapolis, MN) according to the manufacturer's directions. All plates were read on a microplate reader (Molecular Devices, Menlo Park, CA) and analyzed with the use of a computer-assisted analysis program (Softmax, Molecular Devices).

BAL cell counts. After euthanization, the tracheae of mice were cannulated, and the lungs lavaged three times with 1 ml of Hanks' balanced salt solution (137 mM NaCl, 5.4 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 4.2 mM NaHCO3, and 5.6 mM glucose). BAL fluid was pooled and immediately cooled to 4°C. Differential cell counts were performed on Diff-Quick-stained (Baxter Diagnostics, McGaw Park, IL) cytospin (Cytospin 3, Shandon Scientific) slides of cells from 200 µlofBAL fluid. Two hundred cells per slide were counted. The remaining BAL fluid was then centrifuged at 1,300 rpm for 10 min. Cells were resuspended in PBS, and 10 µl were mixed with 10 µl of trypan blue (0.4%) and counted on a hemocytometer.

Mink lung epithelial assay. Measurement of active TGF-{beta} in whole lung homogenate was performed using a mink lung epithelial (MLE) assay as previously described (25). Briefly, MLE cells were subcultured to ~50% density then incubated with increasing concentrations of recombinant human TGF-{beta}1 (R&D Systems) that had been activated by incubation with 4 mM HCl. Cultures were incubated for 72 h, and then proliferation was assessed by measuring [3H]thymidine incorporation with a scintillation counter to establish a reference standard. Whole lung homogenate from bitransgenic mice with and without Dox was acidified with 4 mM HCl to activate latent TGF-{beta}, then added at increasing volumes to the media of MLE cells, and incubated for 72 h, and then [3H]thymidine incorporation was measured.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of TGF-{alpha} expression in the postnatal lung. Two separate founder mouse lines (lines 22.1 and 23.5) bearing the target construct were identified by Southern blot analysis. Transgenic mice bearing either the CCSP-rtTA or (TetO)7-cmv TGF-{alpha} transgenes singly did not have pathologic alterations detectable by histological examination of the lung and survived normally in the vivarium. (TetO)7-cmv TGF-{alpha} target mice were bred to CCSP activator mice to obtain transgenic mice in which TGF-{alpha} expression was regulated by administration of Dox. After 4 days of Dox induction, lungs of 2-mo-old bitransgenic progeny contained significant increases in TGF-{alpha} with values ranging from 1,506 to 1,991 pg/ml, which is 13–18 times above nontransgenic mice (Table 1). Bitransgenic 22.1 mice not treated with Dox had TGF-{alpha} levels similar to those of nontransgenic controls, indicating no significant promoter leak in bitransgenic mice in the absence of Dox.


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Table 1. Mean lung homogenate TGF-{alpha} levels

 

Histological changes following TGF-{alpha} expression. To determine the effects of TGF-{alpha} in the adult lung, we examined lung histology of bitransgenic lines 22.1 and 23.5 following 3 wk of Dox treatment. Fibrosis was detected in peribronchial and perivascular adventitial regions (Fig. 1) as well as the lung pleura in both lines. Inflammatory cell infiltration was not observed in any of the fibrotic regions or other areas of the lung. Bitransgenic mice not administered Dox did not have detectable fibrosis.



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Fig. 1. Adventitial and peribronchial fibrosis. Representative hematoxylin and eosin (H&E) stain of lung section in 2-mo-old line 23.5 and 22.1 transforming growth factor (TGF)-{alpha} inducible mice following 3 wk of continuous doxycycline (Dox). Adventitial and peribronchial fibrosis is evident in both lines of bitransgenic mice following Dox. Inflammatory cell infiltration is not detected in the fibrotic regions of the lung. Photomicrographs are representative of 2–6 mice after 3 wk of Dox treatment. All photomicrographs were taken at the same magnification.

 

Temporal progression of fibrosis. There were no significant differences in total body weight between line 22.1 mice and nontransgenic mice at 2 mo of age before Dox was administered (23.4 ± 1 g for line 22.1 vs. 24.7 ± 1 g for nontransgenic mice). After 2 wk of continuous Dox administration, line 22.1 mice began to progressively lose weight and lost on average 15% of preinduction body weight following 6 wk of Dox (range +1% to -37%, Fig. 2). Females tended to lose more weight than males (-20% females, -12% males; P = 0.20). In contrast, nontransgenic mice progressively gained weight during this time interval with a 8% increase in body weight 6 wk into treatment.



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Fig. 2. Whole body weight changes following Dox. Whole body weights of 2-mo-old line 22.1 and nontransgenic mice were measured before we administered Dox and at weekly intervals for 6 wk. There were no differences in weights between transgenic and nontransgenic mice before Dox was initiated. Nontransgenic mice demonstrated progressive weight gain during the study interval, whereas line 22.1 mice developed progressive weight loss after 2 wk of Dox. Solid lines, line 22.1 mice; dashed lines, nontransgenic mice. *P < 0.05 between line 22.1 and nontransgenic mice; n = 13 mice in each group (8 males and 5 females).

 

To characterize the temporal effects of chronic TGF-{alpha} expression on the adult lung, we studied line 22.1 mice. After 4 days of Dox treatment, there was thickening in the adventitial regions of medium-sized vessels and to a lesser extent in the peribronchial region (Fig. 3). After 1–2 wk of Dox, fibrosis became more organized in the perivascular adventitia and began to further surround the peribronchial areas. Areas of pleural thickening were first detected between 2 and 3 wk of Dox treatment, although pleural thickening was not uniform (Figs. 4 and 7). By 3–4 wk of Dox, fibrosis encompassed the entire perivascular adventitia and peribronchial area and began to extend into the interstitium. By 6 wk, fibrosis progressed further from the adventitia and peribronchial area into the interstitium and pleural fibrosis was extensive and uniform (Fig. 4). At all time points, there was no histological evidence of inflammatory cell infiltration. Enlarged distal air spaces were primarily detected adjacent to areas of severe adventitial, peribronchial, or pleural fibrosis (Figs. 3 and 4).



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Fig. 3. Progressive adventitial and peribronchial fibrosis. Representative H&E stains of the adventitia in 2- to 3-mo-old line 22.1 TGF-{alpha} inducible mice without Dox (A) and selected intervals of continuous Dox. No fibrosis was detected in bitransgenic mice not administered Dox; after 4 days of Dox treatment, adventitial (arrow, B) and minimal peribronchial fibrosis was detected. By 1 wk, adventitial fibrosis expands (arrow, C) and by 2 wk begins to extend further along the peribronchial region (arrow, D). By 3 wk of Dox treatment fibrosis progressed to involve the entire adventitial and peribronchial region and began to extend into the interstitium (E), and after 6 wk of Dox there was extensive interstitial fibrosis effacing normal alveolar architecture (F). Enlarged air spaces were seen only 6 wk after Dox and only adjacent to areas of pronounced fibrosis. Photomicrographs are representative of 2–6 mice per time point. A, airway. All photomicrographs were taken at the same magnification.

 


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Fig. 4. Pleural fibrosis. Representative H&E stains of the pleural surface in 2- to 3-mo-old line 22.1 TGF-{alpha} inducible mice without Dox and selected intervals of continuous Dox. Pleural fibrosis was not detected in mice not administered Dox; pleural fibrosis (arrow) was detected between 2 and 3 wk of induction but was not uniform in distribution. By 6 wk of Dox pleural fibrosis was extensive and uniform. A: 1 wk of Dox; B: 3 wk of Dox; C: 6 wk of Dox. Photomicrographs are representative of 6 mice per time point. All photomicrographs were taken at the same magnification.

 


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Fig. 7. Cellular sites of proliferation. BrdU incorporation (black staining cells) was detected in epithelial and mesenchymal cells in bitransgenic mice not receiving Dox (A). By 4 days of Dox treatment there was marked increase in the number of proliferating cells in the adventitia and peribronchial regions at sites of developing fibrosis (B). By 3 wk of Dox treatment proliferating cells are noted within fibrotic regions as well as along the leading edge of fibrotic regions extending from the pleural surface (arrow) and adventitia (not shown) into the interstitium (C). By 6 wk of Dox BrdU staining was infrequent in the more expansive fibrotic regions, whereas BrdU staining remained prominent at the leading edge of the fibrosis extending into the interstitium (arrow, D). Each photomicrograph is representative of 4 mice per time point. Note higher magnification for 3- and 6-wk time points.

 

Collagen and extracellular matrix deposition. Total collagen and extracellular matrix deposition were increased following Dox induction. Increased trichrome staining consistent with collagen deposition was detected at all time points following Dox and was most prominent 6 wk after induction in the pleural, adventitial, peribronchial, and interstitial areas (Fig. 5). Hydroxyproline was increased over 70% between line 22.1 mice not treated with Dox versus mice following 6 wk of Dox (11.7 ± 0.8 vs. 20.0 ± 2 µg hydroxyproline per mg dry lung, respectively; P < 0.01). With pentachrome stain the pattern of extracellular matrix and collagen deposition demonstrated a leading edge of less mature, organizing collagen and extracellular matrix extending from the adventitia and peribronchium into the interstitium (Fig. 6).



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Fig. 5. Trichrome staining. Gomori's trichrome stain demonstrating increased deposition of total collagen (dark blue stain) around the adventitia, peribronchial region, and pleural surface. Photomicrograph is representative of 6 line 22.1 mice after 6 wk of Dox treatment. Photomicrographs were taken at the same magnification.

 


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Fig. 6. Pentachrome stain. Pentachrome stain of 22.1 transgenic mice following 6 wk of Dox treatment. Mature collagen and reticulum fibers (yellow stain) immediately surround vascular and airway adventitia, whereas immature ground substance (blue stain) extends from the adventitia and peribronchial region into the interstitium (arrow). Photomicrograph is representative of 6 mice.

 

Fibrosis is associated with increased cellular proliferation. With BrdU labeling total cellular proliferation per lung field was significantly increased at all time points, with the percentage of proliferating cells/field increasing 4.2-, 15-, and 11.2-fold following 4 days and 3 and 6 wk of Dox, respectively, compared with mice not receiving Dox (Table 2). In association with the increased proliferation, the total number of nuclei also increased, with a 30% increase after 4 days of Dox, a doubling of the total number of cells 3 wk after Dox, and a 150% increase 6 wk after Dox. Although the total cells per lung field were highest 6 wk after Dox, the number of proliferating cells/field and the proliferation index decreased between 3 and 6 wk of Dox. BrdU-labeled cells were usually detected in areas of developing fibrosis and were infrequently detected in the mature fibrotic regions in the lung (Fig. 7).


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Table 2. TGF-{alpha} expression induces proliferation

 

Immunolocalization of TGF-{alpha}. After Dox induction increased TGF-{alpha} immunostaining was detected in airway and alveolar epithelial cells at early and late time points compared with bitransgenic mice treated with water. In the peribronchial region, developing fibrosis was seen immediately adjacent to airway and alveolar epithelial cells with TGF-{alpha} immunostaining (Fig. 8A). TGF-{alpha} immunostaining was not detected in the vascular endothelium. In the pleural regions, areas of fibrosis were associated with alveolar sites of TGF-{alpha} immunostaining (Fig. 8B).



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Fig. 8. TGF-{alpha} immunostaining. TGF-{alpha} was detected by immunohistochemistry as described in METHODS (black stain). TGF-{alpha} was detected in airway (arrows, A) and alveolar epithelial cells proximal to developing adventitial fibrosis following 1 wk of Dox. TGF-{alpha} was also detected in airway epithelial cells immediately adjacent to pleural fibrosis in mice following 6 wk of Dox (arrows, B). Each photomicrograph is representative of 4 mice per time point. Both photomicrographs were taken at the same magnification.

 

TGF-{alpha}-induced fibrosis progresses in the absence of pulmonary inflammation. BAL total and differential cell counts did not differ at early and late time points following Dox compared with bitransgenic mice not receiving Dox (Table 3). Proinflammatory cytokines (TNF-{alpha} or IL-6) were not increased in Dox-induced transgenic mice during the progression of fibrosis (Table 3).


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Table 3. BAL cell counts and mean lung homogenate cytokines

 

TGF-{alpha}-induced fibrosis initiates in the absence of TGF-{beta} activation. To determine whether TGF-{beta} was increased following TGF-{alpha} induction, we used the MLE cell assay. Activated TGF-{beta}1 caused a concentration-dependent decrease in MLE proliferation (Fig. 9A). There were no differences in MLE proliferation in response to the addition of TGF-{alpha} in the dose range of 0.1–100 ng/ml without activated TGF-{beta}1 (data not shown), demonstrating that lung homogenate TGF-{alpha} did not influence MLE proliferation. MLE proliferation was not altered at increasing volumes of lung homogenate from line 22.1 inducible mice not administered Dox. Similarly there were no changes in MLE proliferation at increasing volumes of lung homogenate for mice after 1 and 4 days of Dox (Fig. 9B).



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Fig. 9. Total TGF-{beta} activity. Active TGF-{beta} was measured using the mink lung epithelial (MLE) cell proliferation assay as described in METHODS. Activated TGF-{beta}1 added to lung homogenates remained active, indicating that TGF-{beta} is not inhibited by lung homogenate preparation (A). To determine whether active TGF-{beta} is present in lung homogenate from mice following TGF-{alpha} induction, increasing volumes of lung homogenate were added to the media of MLE cells, and 3H incorporation was measured. There were no differences in active TGF-{beta} in transgenic mice not receiving Dox compared with mice receiving Dox for 1 and 4 days. The x-axis depicts 3H counts; the y-axis depicts increasing lung homogenate volumes added to MLE cells (B); n = 3 each time point.

 

Although TGF-{beta}1 was detected by immunostaining in epithelial cells, changes in the intensity of staining in the airway and peribronchial regions of fibrosis were not detected following TGF-{alpha} induction (Fig. 10). Increased immunostaining was detected only at the peripheral pleural fibrotic regions after 6 wk of Dox.



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Fig. 10. TGF-{beta}1 immunostaining. TGF-{beta}1 immunostaining was detected in bronchial epithelium but was barely detectable in developing peribronchial and perivascular fibrotic regions at all time points. TGF-{beta}1 immunostaining was most prominent only at the pleural surface following 6 wk of Dox (arrow).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study demonstrates that chronic conditional expression of TGF-{alpha} in the pulmonary epithelium of the adult lung induces extensive and progressive pulmonary fibrosis independently of detectable markers of inflammation or activation of TGF-{beta}. Fibrosis was first detected in the vascular adventitia and peribronchial regions 4 days after TGF-{alpha} expression was induced, and it expanded to encompass the entire adventitia and peribronchium after 3 wk. Between 3 and 6 wk, fibrosis extended into the lung interstitium, effacing normal alveolar architecture. In addition, TGF-{alpha} caused progressive fibrosis on the lung pleura. Pleural thickening was detected after 2–3 wk of TGF-{alpha} induction and by 6 wk was marked with uniform pleural thickening.

The areas of airway, vascular, and pleural fibrosis in this study were juxtaposed to epithelial sites of TGF-{alpha} expression. In the constitutive SP-C TGF-{alpha} transgenic mice, fibrosis also correlated with sites of TGF-{alpha} expression with smaller foci of fibrosis seen in the lung pleura, distal airways, and vessels, but fibrosis was not seen around larger airways and vasculature (13, 14, 18). Differences in sites of fibrosis between both lines likely represent the unique expression patterns due to different promoters. In the conditional model, enlargement of distal air spaces was most prominent in air spaces immediately adjacent to areas of extensive interstitial or pleural fibrosis. Enlargement of these distal air spaces may be due to traction effects of the fibrosis on adjacent alveoli or to early emphysematous changes. In the constitutive SP-C TGF-{alpha} mouse, enlargement of distal air spaces is detected within 1 wk of age and progresses throughout the rest of lung development (13). These findings demonstrate that TGF-{alpha} induces air space enlargement in the SP-C TGF-{alpha} mouse by disrupting postnatal alveolar development, and TGF-{alpha} causes primarily pulmonary fibrosis in the adult lung.

Cellular proliferation was increased at all time points following TGF-{alpha} induction, and proliferating cells were seen in the areas of extracellular matrix and collagen deposition. Increased cellular proliferation and fibrosis were also reported in transgenic mice overexpressing TGF-{alpha} in other organs including the pancreas, skin, liver, and mammary glands (15, 30). Collectively, these findings indicate that overexpression of TGF-{alpha} in many organs can lead to both increased proliferation and matrix protein deposition.

BAL cell counts and measurements of proinflammatory cytokines showed that the induction of fibrosis in the conditional TGF-{alpha} mice developed in the absence of detectable inflammation. In contrast, bleomycin installation, expression of TNF-{alpha} and IL-13 in transgenic mice, expression of TGF-{beta}1 and IL-1{beta} with adenoviral vectors in mice, and fibrosis in SP-C knockout mice were all associated with increased inflammatory cell infiltration and cytokine release (7, 10, 17, 19, 31). A number of studies support a role for activation of the EGFR signaling pathway in epithelial cells by inflammatory mediators, including bacterial lipopolysaccharides, oxidative stress, and proinflammatory cytokines TNF-{alpha}, IL-4, and IL-13 (2, 5, 16, 20, 38). The absence of inflammation following chronic expression of TGF-{alpha} supports the concept that the EGFR pathway may be downstream of the inflammatory cascade following lung injury and may contribute to the development of pulmonary fibrosis following prolonged inflammation.

Although pulmonary fibrosis has been traditionally viewed as a consequence of inflammation, in diseases such as IPF, clinical measures of inflammation do not correlate with disease progression, and anti-inflammatory drugs, such as corticosteroids, often do not significantly improve clinical outcome (6, 24). In vitro, corticosteroids have no effect on bronchial epithelial EGFR expression levels following injury, suggesting that the EGFR pathway can continue to be active following anti-inflammatory therapy with steroids (28). Thus considering that the EGFR pathway is increased in human diseases of pulmonary fibrosis, that chronic expression of TGF-{alpha} causes fibrosis independently of inflammation, and that the EGFR pathway is not responsive to corticosteroids, these findings support further exploring the EGFR pathway as a molecular target to disrupt fibrogenesis, especially in diseases where fibrosis progresses despite anti-inflammatory therapy.

Several lines of evidence implicate activated TGF-{beta}1 as a key cytokine in the pathogenesis of fibrosis in a number of organs including the lung (3, 17, 36, 37). In our study, total TGF-{beta} activity measured with the MLE assay did not demonstrate active TGF-{beta} 1 or 4 days following TGF-{alpha} induction when fibrosis is initiated. Furthermore, increased immunohistochemistry for TGF-{beta}1 was not detected in areas of developing peribronchial or perivascular fibrosis at early and late time points. Increased immunostaining for TGF-{beta}1 was detected only at the leading edge of pleural fibrosis 6 wk into Dox induction. Interestingly, previous studies with TGF-{alpha} transgenic mice driven by the keratin 14 promoter demonstrate reduced TGF-{beta} signaling in the olfactory epithelium, suggesting that TGF-{alpha} may downregulate TGF-{beta} signaling (9). Together, these findings suggest TGF-{alpha}-induced fibrosis is independent of TGF-{beta} during the progression of fibrosis and suggests an indirect role for TGF-{beta} only in the later period of pleural fibrosis.

The levels of TGF-{alpha} in the transgenic mice are comparable to levels detected in samples from patients with lung disease. Adult patients with pulmonary edema from acute lung injury had TGF-{alpha} levels in the pulmonary edema fluid in a range of 35–2,570 pg/ml (4). TGF-{alpha} levels produced in SP-C TGF-{alpha} constitutive (1,247 pg/ml) and the CCSP conditional transgenic mice (1,991 pg/ml) are similar to levels of TGF-{alpha} that have been described in human disease and are therefore biologically relevant for understanding the effects of increased TGF-{alpha} in the lung.

In summary, conditional overexpression of TGF-{alpha} in the adult lung epithelium caused progressive vascular adventitial, peribronchial, interstitial, and pleural fibrosis independently of developmental influences. Fibrosis was tangential to epithelial sites of TGF-{alpha} expression and was associated with increased collagen and extracellular matrix protein deposition, as well as increased cellular proliferation. Fibrosis occurred in the absence of inflammatory cell influx, increased proinflammatory cytokines, or direct activation of TGF-{beta}. These findings further support the EGFR as a significant pathway in the pathogenesis of pulmonary fibrosis in the adult lung.


    ACKNOWLEDGMENTS
 
We thank Dr. Jeff Whitsett for helpful critiques, Dr. Thomas Doetschman and Moying Yin for assistance with TGF-{beta} immunohistochemistry and MLE assays, and Adrienne Prestridge and Crystal Lee for technical assistance.

GRANTS

This work was sponsored by National Heart, Lung, and Blood Institute Grant KO8-04172 (W. D. Hardie), the Translational Research Initiative of Cincinnati Children's Hospital (W. D. Hardie), and The University of Cincinnati Center for Environmental Genetics (ES-06096) (W. D. Hardie).


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. D. Hardie, Div. of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: william.hardie{at}cchmc.org).

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


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baughman RP, Lower EE, Miller MA, Bejarano Publo PA, and Heffelfinger SC. Overexpression of transforming growth factor-{alpha} and epidermal growth factor-receptor in idiopathic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis 16: 57-61, 1999.[ISI][Medline]
  2. Booth BW, Adler KB, Bonner JC, Tournier F, and Martin LD. Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-{alpha}. Am J Respir Cell Mol Biol 25: 739-743, 2001.[Abstract/Free Full Text]
  3. Border WA and Noble NA. Transforming growth factor {beta} in tissue fibrosis. N Engl J Med 331: 1286-1292, 1994.[Free Full Text]
  4. Chesnutt A, Kheradmand F, Folkesson H, Alberts M, and Matthay M. Soluble transforming growth factor-{alpha} is present in the pulmonary edema fluid of patients with acute lung injury. Chest 111: 652-656, 1997.[Abstract/Free Full Text]
  5. Churg A, Gilks B, and Dai J. Induction of fibrogenic mediators by fine and ultrafine titanium dioxide in rat tracheal explants. Am J Physiol Lung Cell Mol Physiol 277: L975-L982, 1999.[Abstract/Free Full Text]
  6. Cook DN, Brass DM, and Schwartz DA. A matrix for new ideas in pulmonary fibrosis. Am J Respir Cell Mol Biol 27: 122-124, 2002.[Free Full Text]
  7. Fujita M, Shannon J, Irvin C, Fagan K, Cool C, Augustin A, and Mason R. Overexpression of tumor necrosis factor-{alpha} produces an increase in lung volumes and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 280: L39-L49, 2001.[Abstract/Free Full Text]
  8. Garvey W, Fathi A, Bigelow F, Carpenter B, and Jimenez C. Improved Movat pentachrome stain. Stain Technol 61: 60-62, 1986.[ISI][Medline]
  9. Getchell ML, Boggess MA, Pruden SJ, Little SS, Buch S, and Getchell TV. Expression of TGF-beta type II receptors in the olfactory epithelium and their regulation in TGF-alpha transgenic mice. Brain Res 945: 232-241, 2002.[CrossRef][ISI][Medline]
  10. Glasser SW, Detmer EA, Ikegami M, Na CL, Stahlman MT, and Whitsett JA. Pneumonitis and emphysema in SP-C gene targeted mice. J Biol Chem 278: 14291-14298, 2003.[Abstract/Free Full Text]
  11. Gossen M and Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547-5551, 1992.[Abstract]
  12. Hardie WD, Bejarano PA, Miller MA, Yankaskas JR, Ritter JH, Whitsett JA, and Korfhagen TR. Immunolocalization of transforming growth factor {alpha} and epidermal growth factor receptor in lungs of patients with cystic fibrosis. Pediatr Dev Pathol 2: 415-423, 1999.[CrossRef][ISI][Medline]
  13. Hardie WD, Huelsman K, Bruno M, Iwamoto H, Carrigan P, Leikauf G, and Korfhagen T. Postnatal lung function and morphology in transgenic mice expressing transforming growth factor-alpha. Am J Pathol 151: 1075-1083, 1997.[Abstract]
  14. Hardie WD, Piljan-Gentle A, Dunlavy MR, Ikegami M, and Korfhagen TR. Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-{alpha}. Am J Physiol Lung Cell Mol Physiol 281: L1088-L1094, 2001.[Abstract/Free Full Text]
  15. Jhappen C, Stahle C, Harkins RN, Fausto N, Smith GH, and Merlino GT. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61: 1137-1146, 1990.[ISI][Medline]
  16. Kang JL, Lee HW, Lee HS, Pack IS, Chong Y, Castranova V, and Koh Y. Genistein prevents nuclear factor-kappa B activation and acute lung injury induced by lipopolysaccharide. Am J Respir Crit Care Med 164: 2206-2212, 2001.[Abstract/Free Full Text]
  17. Kolb M, Margetts P, Anthony D, Pitossi F, and Gauldie J. Transient expression of IL-1{beta} induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest 107: 1529-1536, 2001.[Abstract/Free Full Text]
  18. Korfhagen TR, Swantz RJ, Wert SE, McCarty JM, Merlakian CB, Glasser SW, and Whitsett JA. Respiratory epithelial cell expression of human transforming growth factor-{alpha} induces lung fibrosis in transgenic mice. J Clin Invest 93: 1691-1699, 1994.[ISI][Medline]
  19. Lanone S, Zheng T, Zhu Z, Liu W, Lee CG, Ma B, Chen Q, Homer R, Wang J, Rabach L, Rabach M, Shipley J, Shapiro S, Senior R, and Elias J. Overlapping and enzyme-specific contributions of matrix metalloporoteinases-9 and -12 in IL-13-induced inflammation and remodeling. J Clin Invest 110: 463-474, 2002.[Abstract/Free Full Text]
  20. Lordan JL, Bucchieri F, Richter A, Konstantinidis A, Holloway JW, Thornber M, Puddicombe SM, Buchanan D, Wilson SJ, Djukanovic R, Holgate ST, and Davies DE. Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells. J Immunol 169: 407-414, 2002.[Abstract/Free Full Text]
  21. Madtes DK, Elston AL, Hackman RC, Dunn AR, and Clark JG. Transforming growth factor-{alpha} deficiency reduces pulmonary fibrosis in transgenic mice. Am J Respir Cell Mol Biol 20: 924-934, 1999.[Abstract/Free Full Text]
  22. Madtes DK, Rubenfeld G, Klima LD, Milberg JA, Steinberg KP, Martin TR, Raghu G, Hudson LD, and Clark JG. Elevated transforming growth factor-{alpha} levels in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 158: 424-430, 1998.[Abstract/Free Full Text]
  23. Modi S and Seidman A. An update on epidermal growth factor receptor inhibitors. Curr Oncol Rep 4: 47-55, 2002.[Medline]
  24. National Heart, Lung, and Blood Institute Working Group. Future research directions in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 166: 236-246, 2002.[Abstract/Free Full Text]
  25. Ogawa Y and Seyedin SM. Purification of transforming growth factors beta 1 and beta 2 from bovine bone and cell culture assays. Methods Enzymol 198: 317-327, 1991.[ISI][Medline]
  26. Pelton RW, Johnson MD, Perkett EA, Gold LI, and Moses HL. Expression of transforming growth factor-beta 1, beta 2 and -beta 3 mRNA and protein in the murine lung. Am J Respir Cell Mol Biol 5: 522-530, 1991.[ISI][Medline]
  27. Polosa R, Puddicombe SM, Krishna T, Tuck AB, Howarth PH, Holgate ST, and Davies DE. Expression of c-erbB receptors and ligands in the bronchial epithelium of asthmatic subjects. J Allergy Clin Immunol 109: 75-81, 2002.[CrossRef][ISI][Medline]
  28. Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST, and Davies DE. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J 14: 1362-1374, 2000.[Abstract/Free Full Text]
  29. Rice AB, Moomaw CR, Morgan DL, and Bonner JC. Specific inhibitors of platelet-derived growth factor of epidermal growth factor receptor tyrosine kinase reduce pulmonary fibrosis in rats. Am J Pathol 155: 213-221, 1999.[Abstract/Free Full Text]
  30. Sandgren EP, Leutteke NC, Palmiter RD, Brinster RL, and Lee DC. Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61: 1121-1135, 1990.[ISI][Medline]
  31. Sime PJ, Xing Z, Graham FL, Csaky KG, and Gauldie J. Adenovectormediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 100: 768-776, 1997.[Abstract/Free Full Text]
  32. Stahlman MT, Orth DN, and Gray ME. Immunocytochemical localization of epidermal growth factor in the developing human respiratory system and in acute and chronic lung disease in the neonate. Mod Pathol 60: 539-547, 1989.
  33. Strandjord TP, Clark JG, Guralnick DE, and Madtes DK. Immunolocalization of transforming growth factor-{alpha}, epidermal growth factor (EGF), and EGF-receptor in normal and injured developing human lung. Pediatr Res 38: 851-856, 1995.[Abstract]
  34. Takeyama K, Fahy JV, and Nadel JA. Relationship of epidermal growth factor receptors to goblet cell production in human bronchi. Am J Respir Crit Care Med 163: 511-516, 2001.[Abstract/Free Full Text]
  35. Tichelaar JW, Lu W, and Whitsett JA. Conditional expression of fibroblast growth factor-7 in the developing and mature lung. J Biol Chem 275: 11858-11864, 2000.[Abstract/Free Full Text]
  36. Ueberham E, Low R, Ueberham U, Schonig K, Bujard H, and Gebhardt R. Conditional tetracycline-regulated expression of TGF-{beta}1 in liver of transgenic mice leads to reversible intermediary fibrosis. Hepatology 37: 1067-1078, 2003.[CrossRef][ISI][Medline]
  37. Xu YD, Hua J, Mui A, O'Conner R, Grotendorst G, and Khalil N. Release of biologically active TGF-{beta}1 by alveolar epithelial cells results in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 285: L522-L526, 2003.[Free Full Text]
  38. Zhang L, Rice AB, Adler K, Sannes P, Martin L, Gladwell W, Koo JS, Gray TE, and Bonner JC. Vanadium stimulates human bronchial epithelial cells to produce heparin-binding epidermal growth factor-like growth factor: a mitogen for lung fibroblasts. Am J Respir Cell Mol Biol 24: 123-131, 2001.[Abstract/Free Full Text]