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
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ABSTRACT |
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collagen; proliferation; epidermal growth factor receptor; transforming growth factor-
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- (TGF-
), 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-
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-
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-
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-
, 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- under control of the human surfactant protein C (SP-C) promoter (18). The degree of remodeling was directly proportional to the levels of TGF-
expressed in different lines of SP-C/TGF-
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-
over-expression in the adult lung is sufficient to cause emphysema and pulmonary fibrosis. To determine whether TGF-
causes remodeling in the adult lung independently of developmental influences, we generated transgenic mice in which TGF-
was conditionally regulated with tetracycline-responsive promoters (11). We demonstrate that induction of TGF-
expression in the respiratory epithelium of adult transgenic mice produced progressive fibrosis in the absence of increased inflammatory cells, proinflammatory cytokines, or early TGF-
activation.
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METHODS |
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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- transgene with PCR with 5' primer in human TGF-
coding region 5'-CCT GTT CGC TCT GGG TAT TGT GTT and 3' primer in human TGF-
coding region 5'-CGT GGT CCG CTG ATT TCT TCT CTA. Amplification of PCR products for (TetO)7-cmv TGF-
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- mice. To induce TGF-
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-, 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-
antibody (Oncogene Research Products, Cambridge, MA) diluted 1:40.
For immunohistochemical detection of TGF-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-
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 0800 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- levels were determined with an ELISA kit (Oncogene Research Products) according to the manufacturer's directions. Lung homogenate tumor necrosis factor-
(TNF-
) 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- 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-
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-
, then added at increasing volumes to the media of MLE cells, and incubated for 72 h, and then [3H]thymidine incorporation was measured.
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RESULTS |
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Histological changes following TGF- expression. To determine the effects of TGF-
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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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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To characterize the temporal effects of chronic TGF- 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 12 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 34 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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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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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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Immunolocalization of TGF-. After Dox induction increased TGF-
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-
immunostaining (Fig. 8A). TGF-
immunostaining was not detected in the vascular endothelium. In the pleural regions, areas of fibrosis were associated with alveolar sites of TGF-
immunostaining (Fig. 8B).
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TGF--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-
or IL-6) were not increased in Dox-induced transgenic mice during the progression of fibrosis (Table 3).
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TGF--induced fibrosis initiates in the absence of TGF-
activation. To determine whether TGF-
was increased following TGF-
induction, we used the MLE cell assay. Activated TGF-
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-
in the dose range of 0.1100 ng/ml without activated TGF-
1 (data not shown), demonstrating that lung homogenate TGF-
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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Although TGF-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-
induction (Fig. 10). Increased immunostaining was detected only at the peripheral pleural fibrotic regions after 6 wk of Dox.
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DISCUSSION |
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The areas of airway, vascular, and pleural fibrosis in this study were juxtaposed to epithelial sites of TGF- expression. In the constitutive SP-C TGF-
transgenic mice, fibrosis also correlated with sites of TGF-
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-
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-
induces air space enlargement in the SP-C TGF-
mouse by disrupting postnatal alveolar development, and TGF-
causes primarily pulmonary fibrosis in the adult lung.
Cellular proliferation was increased at all time points following TGF- 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-
in other organs including the pancreas, skin, liver, and mammary glands (15, 30). Collectively, these findings indicate that overexpression of TGF-
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- mice developed in the absence of detectable inflammation. In contrast, bleomycin installation, expression of TNF-
and IL-13 in transgenic mice, expression of TGF-
1 and IL-1
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-
, IL-4, and IL-13 (2, 5, 16, 20, 38). The absence of inflammation following chronic expression of TGF-
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- 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-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-
activity measured with the MLE assay did not demonstrate active TGF-
1 or 4 days following TGF-
induction when fibrosis is initiated. Furthermore, increased immunohistochemistry for TGF-
1 was not detected in areas of developing peribronchial or perivascular fibrosis at early and late time points. Increased immunostaining for TGF-
1 was detected only at the leading edge of pleural fibrosis 6 wk into Dox induction. Interestingly, previous studies with TGF-
transgenic mice driven by the keratin 14 promoter demonstrate reduced TGF-
signaling in the olfactory epithelium, suggesting that TGF-
may downregulate TGF-
signaling (9). Together, these findings suggest TGF-
-induced fibrosis is independent of TGF-
during the progression of fibrosis and suggests an indirect role for TGF-
only in the later period of pleural fibrosis.
The levels of TGF- 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-
levels in the pulmonary edema fluid in a range of 352,570 pg/ml (4). TGF-
levels produced in SP-C TGF-
constitutive (1,247 pg/ml) and the CCSP conditional transgenic mice (1,991 pg/ml) are similar to levels of TGF-
that have been described in human disease and are therefore biologically relevant for understanding the effects of increased TGF-
in the lung.
In summary, conditional overexpression of TGF- 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-
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-
. These findings further support the EGFR as a significant pathway in the pathogenesis of pulmonary fibrosis in the adult lung.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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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.
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REFERENCES |
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