Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

William D. Hardie1, Alyssa Piljan-Gentle1, Michelle R. Dunlavy1, Machiko Ikegami2, and Thomas R. Korfhagen2

Divisions of 1 Pulmonary Medicine and 2 Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha ) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 ± 40 pg/ml in the lowest expressing line to 1,247 ± 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.

pulmonary fibrosis; emphysema; epidermal growth factor receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR-alpha (TGF-alpha ) is a polypeptide member of the epidermal growth factor (EGF) family, which includes the EGF, amphiregulin and heparin-binding EGF. Synthesized as a 160-amino acid precursor polypeptide, mature 50-amino acid TGF-alpha peptide is released through proteolytic cleavage by specific elastase-like enzymes (3). TGF-alpha mRNA is present throughout prenatal lung development (12). In human fetal and postnatal lungs, TGF-alpha immunolocalizes to airway and alveolar epithelial cells and vascular smooth muscle (14). Although TGF-alpha is clearly produced in many cells in the developing lung, the precise physiological role for TGF-alpha is not completely understood. In vitro, TGF-alpha increases the proliferation of rabbit type II alveolar and cultured lung fibroblasts (2, 13), which suggests that TGF-alpha is involved in the propagation of cell populations in the lung during development.

Although TGF-alpha appears to be active in lung development, there is evidence that TGF-alpha also participates in diseases where there is significant lung remodeling. Strandjord and colleagues (14) demonstrated increased immunostaining for TGF-alpha in pulmonary epithelial cells of infants dying from respiratory distress syndrome or bronchopulmonary dysplasia (BPD). Lung biopsies from patients with idiopathic pulmonary fibrosis demonstrate increased TGF-alpha staining in the vascular endothelium, type II pneumocytes, and fibroblasts compared with controls (1). Lungs from patients with end-stage cystic fibrosis (CF) contain increased immunostaining for TGF-alpha in alveolar macrophages, airway epithelial cells, and diseased submucosal areas compared with healthy controls (5). Increased immunostaining with the proliferation marker Ki67 was detected in submucosal regions of the CF lungs, which suggests that TGF-alpha may be partially responsible for lung remodeling via increased proliferation of interstitial cells. The orderly production of TGF-alpha during lung development may be reactivated during disease processes that lead to lung remodeling.

Transgenic mice overexpressing high levels of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter demonstrate enlarged alveolar airspaces with pleural and peribronchial fibrosis (8) due to abnormal development during postnatal alveolarization (6). To determine the levels of TGF-alpha that induce alveolar emphysema and fibrosis and to assess mechanisms by which TGF-alpha causes lung remodeling, four lines of SP-C transgenic mice with distinct levels of transgene expression were compared for the extent of cellular proliferation, airspace enlargement, pulmonary fibrosis, and pulmonary function. Increased levels of transgenic TGF-alpha were associated with increasingly severe morphological and physiological alterations in the lung.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic mice and Southern blot analysis. Methods for generating transgenic mice that express human TGF-alpha under control of the human SP-C 3.7-kb promoter-enhancer sequence were previously described (8). Four founder lines (lines 6108, 4, 2, and 28) on the FVB/N background strain were evaluated compared with an FVB/N nontransgenic control. All mice studied were transgene heterozygotes. Transgenic mice were identified by a diagnostic 1.4-kb band on genomic Southern blots of PstI-digested genomic tail DNA as previously described (8). Studies were approved by the Institutional Animal Care and Use Committee of the Children's Hospital Research Foundation and the University of Cincinnati Medical Center (Cincinnati, OH).

Quantitation of TGF-alpha expression. The lungs from 2-mo-old mice from each transgenic line and the nontransgenic control were removed and the mice were euthanized. Lung tissue was homogenized in 2 ml of PBS (pH 7.2) and centrifuged at 1,500 g, and the supernatant was stored at -70°C. Human TGF-alpha levels were determined using a quantitative murine sandwich enzyme-linked immunosorbent assay (ELISA) kit (Oncogene Research Products, Cambridge, MA) 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). Only assays having standard curves with a calculated regression line value >0.95 were accepted for analysis.

To correct for possible endogenous mouse TGF-alpha cross- reaction with the anti-human TGF-alpha antibody, data were normalized to the average nontransgenic TGF-alpha concentration.

Pulmonary histology and immunohistochemistry. Two-month-old mice from each group were euthanized with intraperitoneal pentobarbital sodium. The tracheae were cannulated and the lungs were inflated (1 min at 25 cmH2O) with 4% paraformaldehyde in PBS (pH 7.2). After 24 h of immersion, the lungs were washed in PBS, dehydrated in graded alcohol and xylene, and embedded in paraffin (60°C). Paraffin-embedded lungs were sectioned (5 µm) and placed on Polysine glass slides (Erie Scientific, Portsmouth, NH). The slides were deparaffinized with xylene and rehydrated with graded alcohol washes and PBS. The sections were stained with hematoxylin and eosin and Gomori trichrome stain.

For immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU), 2-wk-old animals from each line were injected with BrdU-labeling reagent (0.1 ml/100 g body wt; Zymed Laboratories, San Francisco, CA) 2 h before death. BrdU incorporated into DNA was detected using anti-BrdU monoclonal antibody and a BrdU staining kit (Zymed Laboratories). BrdU staining for each animal was performed on three randomly selected fields from serial sections at ~2.0-mm intervals. Staining was analyzed only in the distal lung in the alveolar region, and the proliferation index was determined by counting the total number of BrdU-stained nuclei and dividing by the total number of nuclei in each field. Antibodies and procedures for immunostaining to detect proSP-C have been described previously (17). The total number of type II cells per field and the percentage of type II cells per nuclei were determined from random fields in three serial sections. As with BrdU detection, staining and analysis were performed only in the distal lung in the alveolar region.

To further identify subpopulations of proliferating cells, lung tissue sections were double-stained with proliferating cell nuclear antigen (PCNA) and proSP-C. Briefly, tissue sections were dehydrated and then stained first for proSP-C using methods briefly described (17). To distinguish PCNA nuclear staining from proSP-C, development of SP-C was modified by using alkaline phosphatase substrate reagent and counterstaining with vector red. This adaptation caused proSP-C to stain red in the cell cytoplasm. Tissue was then fixed with 4% paraformaldehyde. PCNA staining was performed using a staining kit with a biotinylated monoclonal antibody as per the manufacturer's recommendations. PCNA cells were identified with a dark-brown nuclear stain.

Morphometry. Mice were killed at 2 mo of age for morphometric studies. Lung tissue sections stained with hematoxylin and eosin were prepared as described (see Pulmonary histology and immunohistochemistry). Two serial sections at ~2.0-mm intervals were studied for each animal. Three representative fields of lung were studied per slide. The studied section was visually scanned, and fields were selected to contain terminal airspaces. Fields with large conducting airways, longitudinal sections of alveolar ducts, or blood vessels were not selected.

Morphometric measurements for terminal airspace area were collected using Image-1/Metamorph Imaging System version 2 for Microsoft Windows (Universal Imaging) and using methods previously described (6). Briefly, calibrations for ×4, ×10, and ×20 images were determined by acquiring images of a ruler (in micrometers) at these powers, which allows the computer to calculate the number of pixels per micrometer at each power. Images were corrected for tears in airspace walls. The airspaces were distinguished from tissue based on intensity, and the computer measured the number of pixels in each airspace. These measurements were then converted and recorded in square micrometers.

Pressure-volume curve. Mice were sedated with pentobarbital sodium (100 mg/kg ip) and placed in a box containing 100% oxygen to ensure complete collapse of the alveoli by oxygen absorption after spontaneous breathing ceased. The mice were euthanized by exsanguination. The trachea was cannulated and connected by a syringe to a pressure sensor (mouse pulmonary testing system) via a three-way connector. After the diaphragm was opened, the lungs were inflated in 75-µl increments every 10 s to a maximum pressure and were similarly deflated (15). Lung volume is expressed as milliliters per kilogram of body weight. Hysteresis was measured by counting the intersections of the squares and multiplying by the area of the square.

Bronchoalveolar lavage fluid cell count, differential count, and lung homogenate proinflammatory cytokines. After the mice were euthanized, the tracheae were cannulated and the lungs were lavaged three times with 1 ml of Hanks' balanced salt solution [containing (in mM) 137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.34 Na2HPO4, 4.2 NaHCO3, and 5.6 glucose]. Bronchoalveolar lavage (BAL) fluid was pooled and immediately cooled to 4°C. Differential cell counts were performed on Diff-Quik-stained (Baxter Diagnostics, McGaw Park, IL) cytospin (Cytospin 3, Shandon Scientific) slides of cells from 200 µl of BAL fluid; 200 cells/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 of cells were mixed with 10 µl of 0.4% trypan blue and placed on a hemocytometer. White blood cells were counted on a grid and normalized to cells per milliliter.

Tumor necrosis factor-alpha (TNF-alpha ), interleukin-1beta (IL-1beta ), and macrophage inflammatory protein-2 (MIP-2) were determined using quantitative murine sandwich ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's directions. All plates were read on a microplate reader (Molecular Devices) and analyzed with the use of a computer-assisted analysis program (Softmax). Only assays having standard curves with a calculated regression line value >0.95 were accepted for analysis.

Statistical analysis. Pressure-volume curve measurements were compared using ANOVA and subsequent Student-Newman-Keuls test. Differences in TGF-alpha lung homogenate levels, cell counts, BrdU, and type II cell indexes were compared using ANOVA. Differences in terminal airspace areas between strains of mice were determined using an ANOVA conducted with an alpha -level of 0.05. Scores from the individual mice were nested within groups to reduce extraneous variance. The analysis was performed with the SAS 6.12 statistical software (SAS Institute, Cary, NC) general linear model according to the manufacturer's recommended procedure.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Concentration of human TGF-alpha in lung homogenates. Human TGF-alpha levels were different between each transgenic line, with a threefold increase in the amount of TGF-alpha produced between the lowest expressing line 6108 and the highest expressing line 28 (Table 1). All values were also normalized to mean nontransgenic levels to control for endogenous mouse TGF-alpha .

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Mean lung homogenate TGF-alpha level

Histological and morphometric changes related to TGF-alpha expression. Lines 6108 and 4 demonstrate minimal alveolar airspace enlargement compared with nontransgenic mice (Fig. 1); the mean airspace areas were almost identical between line 6108 (510 ± 10 µm2) and line 4 (509 ± 11 µm2; Fig. 2). Although airspace area in lines 6108 and 4 was increased compared with that in nontransgenic control mice (417 ± 8 µm2), these differences did not reach statistical significance. For line 2 mice, the alveoli appeared emphysematous compared with nontransgenic, line 6108, and line 4 mice (Fig. 1). The mean airspace area was significantly increased in line 2 mice (1,447 ± 77 µm2) compared with nontransgenic control, line 6108, and line 4 mice. Line 28 lungs appeared most remodeled, and the pleural and septal thickening identified in these mice were not detected in the other transgenic lines (Fig. 1). In addition to the thickened pleura, there were occasional scattered areas of peribronchial and perivascular fibrosis (data not presented). Airspace area for line 28 mice (5,843 ± 319 µm2) was significantly greater than for the other transgenic and nontransgenic mice.


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 1.   Lung histology of surfactant protein C (SP-C) transforming growth factor-alpha (TGF-alpha ) mouse lines and nontransgenic control. Lungs from 2-mo-old mice were inflation fixed and stained with hematoxylin and eosin. Sections are representative of 4-6 mice for each line. Original magnification, approximately ×26.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Mean terminal airspace area for 2-mo-old SP-C TGF-alpha mouse lines and nontransgenic control. Four mice from each line were studied. Number of airspaces measured per line were 1,754 for control, 1,899 for line 6108, 1,421 for line 4, 1,503 for line 2, and 1,083 for line 28. Data are expressed as means ± SE; some of the error bars fall within the symbols. *P < 0.001 compared with nontransgenic controls.

Altered pressure-volume curves. As shown in Fig. 3, there were no differences in opening or maximal pressures between nontransgenic, line 6108, and line 4 mice. These three lines of mice had normal opening pressures of ~15 cmH2O. In line 2 and line 28 mice, the lung volume increased as the pressure increased, whereas these mice did not show obvious increases in opening pressure. Line 2 mice had significantly higher lung volumes at maximum pressure than other groups. The volume at 15 cmH2O on the inflation limb of line 2 and line 28 mice pressure-volume curves was more than twofold higher than that in control, line 6108, and line 4 mice. Hysteresis was significantly reduced in line 28 mice compared with all other transgenic lines or control mice (Fig. 3).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Average pressure-volume curves and relative hysteresis for 2-mo-old SP-C TGF-alpha mice; 4 or 5 mice from each line were studied. A: nontransgenic controls. B: transgenic line 6108. C: transgenic line 4. D: transgenic line 2. E: transgenic line 28. F: relative hysteresis. No differences in the pressure-volume curves or opening pressures were detected between nontransgenic controls and lines 6108 and 4. In lines 2 and 28 mice, the lung volume was greater as the pressure increased, and the volume at 15 cmH2O on the inflation limb of the line 2 and 28 mice pressure-volume curves were over twofold higher than control, line 6108, and line 4 mice. Line 28 mice demonstrated significantly less hysteresis than all other transgenic lines or nontransgenic control mice. Data are expressed as means ± SE; some of the error bars fall within the symbols. *P < 0.05 compared with nontransgenic controls.

TGF-alpha enhanced BrdU labeling and type II cell staining. Incorporation of BrdU into DNA was used to estimate cell proliferation in the different lines of transgenic mice. Significantly increased BrdU was detected only in line 28 mice, which demonstrated a threefold increase compared with control or the other transgenic lines (Table 2). BrdU labeling among line 28 mice was detected primarily in respiratory epithelial cells in the lung periphery, although proliferating cells were detected in fibrosing areas in the peribronchial, perivascular, and lung pleura (Fig. 4). Double staining with PCNA and proSP-C in line 28 sections confirmed that many of the proliferating cells were type II cells (Fig. 4).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Proportion of BrdU- and pro-SP-C-immunostained cells



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 4.   A: trichrome staining from line 28 mouse of an area of pleural fibrosis bridging 2 adjacent lobes of the lung. Arrow, area of mature fibrosis. Blue stain identifies areas of collagen deposition. B: serial section from line 28 mouse of area of pleural fibrosis (arrow) stained with 5-bromo-2'-deoxyuridine (BrdU) antibody detecting proliferating cells. BrdU staining includes cells that appear to be type II cells. A number of proliferating cells are not type II cells and appear to be either interstitial cells or inflammatory cells. C: proliferating cell nuclear antigen (PCNA) and proSP-C double staining in line 28 mice. proSP-C stains red in the cytoplasm, and PCNA stains the nucleus dark brown. Large arrow, representative cell staining for PCNA only; arrowheads, representative cells staining only for proSP-C; small arrows, representative cells staining for both PCNA and proSP-C. Original magnification, approximately ×65 for A and B and ×130 for C.

Although the total numbers of type II cells per field examined were decreased in line 2 and line 28 mice (data not shown), there were no differences in the percentage of type II cells per total number of cells in the examined alveolar regions among all transgenic lines (Table 2).

TGF-alpha -induced alterations are not associated with pulmonary inflammation. To determine whether TGF-alpha -induced lung remodeling was associated with inflammation, BAL fluid cell counts and lung homogenate proinflammatory cytokines were measured. There were no significant differences noted in the total cell counts or the lung differential counts among transgenic lines compared with nontransgenic mice (Table 3). Proinflammatory cytokine lung homogenate levels were measured in five nontransgenic control and five line 28 mice. No significant differences were detected for each cytokine between nontransgenic and line 28 mice. Measured levels for nontransgenic and line 28 mice were, respectively, as follows: TNF-alpha , 16.4 ± 3 and 12.8 ± 1 pg/ml; IL-1beta , 52.7 ± 10 and 31.1 ± 6 pg/ml; and MIP-2, 4.6 ± 1 and 3.7 ± 2 pg/ml.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   BAL cell counts


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that increased TGF-alpha expression in the lung produces incremental histological changes related to the level of transgenic human TGF-alpha expression. The two lower expressing TGF-alpha lines (lines 6108 and 4) demonstrated mild alveolar airspace enlargement, with the alveolar airspace area ~25% larger than the nontransgenic alveolar airspace; however, these values were not statistically significant. With the higher levels of TGF-alpha expression produced by line 2 mice, the alveoli were uniformly enlarged, and the airspace areas were three times larger than for nontransgenic mice. The highest expressing line, line 28, demonstrated significant lung remodeling with markedly enlarged alveolar airspace area as previously reported (6). Airspace areas were 14 times larger than control areas. Line 28 mice were the only transgenic line to demonstrate fibrosis, which was apparent on the pleural surface, alveolar septa, and peribronchial and perivascular regions. The degree of airspace remodeling was directly related to the level of human TGF-alpha expression, and the highest levels also induced pulmonary fibrosis.

Alterations in opening pressures and maximal volumes corresponded to the severity of lung remodeling. There were no differences detected in the pressure-volume curves in line 6108 and line 4 mice compared with controls, and hence there were no detectable physiological effects of the mild emphysema in these lines. Line 2 mice did not have higher opening pressure but did have higher lung volumes at maximum pressure than all the other groups. It is likely that the alveoli in line 2 mice are more distensible, but the molecular basis for these changes is not known. Lungs of high-level-expressing lines have altered elastin structure (8), and it is possible that a subtle alteration in elastin could also be present in other TGF-alpha transgenic lines. For line 28 mice, the lung volumes were slightly larger than in control, line 6108, and line 4 mice but not as large as for line 2 mice. The most likely explanation for lower lung volumes in line 28 mice compared with line 2 mice is that the pleural fibrosis in line 28 reduced lung expansion at the higher inflation volumes. Line 28 mice were the only line of mice to demonstrate significantly increased hysteresis that most likely reflects the more distensible emphysematous alveoli.

TGF-alpha is a known mitogen for a variety of cells in the lung including type II epithelial cells and fibroblasts (2, 13). To begin investigating the mechanisms by which TGF-alpha expression induces lung remodeling and the subsequent physiological effects, we examined the proliferative index in the distal alveolar region where TGF-alpha is expressed. We observed a mild yet statistically insignificant increase in BrdU-labeled cells in the lower-expressing lines, whereas for line 28 mice, BrdU-labeled cells were more abundant than in the other transgenic lines and three times greater than in nontransgenic mice. Cells demonstrating proliferation in line 28 mice included type II cells and interstitial cells. We did not specifically identify these other proliferating cells, but they appear to include fibroblasts and occasional macrophages. Because line 28 is the only transgenic line to develop histological evidence of fibrosis and significant increases in proliferating subsets of cells, these findings suggest that there is a threshold level of TGF-alpha expression in the lung, after which there is increased cellular proliferation. Increased numbers of interstitial cells could lead to increased collagen deposition in the lung. The precise molecular signaling mechanism whereby TGF-alpha expression induces fibrosis in the transgenic mice is not understood. We have previously generated transgenic mice where EGF receptor signaling was disrupted in type II cells. These transgenic mice expressed a mutated EGF receptor sequence that lacked a portion of the intracytoplasmic domain but contained the transmembrane and extracellular ligand-binding domain under control of the SP-C promoter (a mutated EGF receptor) (7). When bitransgenic mice were generated with the line 28 TGF-alpha transgene and the SP-C mutated EGF receptor transgene, there was no histological evidence of fibrosis. Because the mutated EGF receptor was expressed in type II cells, these findings suggest that line 28-induced fibrosis develops not through a direct paracrine effect from TGF-alpha acting on interstitial cells but rather through an undefined signaling pathway between type II cells and interstitial cells.

The mechanism for emphysema in the transgenic lines is also unclear. We have previously demonstrated in line 28 mice that emphysema develops in the immediate postnatal period when saccules are subdividing into alveoli (6). There are also histological abnormalities of elastin (8) that may disrupt the orderly secondary septation of saccules, thus resulting in more disorganized, emphysematous alveoli. However, two lines of evidence suggest that TGF-alpha -induced emphysema is not directly a result of increased proliferation. First, we were unable to demonstrate increased proliferation in line 2 mice despite significant histological and morphometric emphysema. Second, if the emphysema was secondary to increased proliferation of alveolar cells, we hypothesized that we would detect an increase in the type II cell population of the lung relative to other cells. Our analysis of type II cells demonstrated no changes in the percentage of type II cells as a proportion of total cell nuclei among all transgenic lines compared with nontransgenic controls. Ganser and colleagues (4) demonstrated that mouse lung explants produced a marked dilation of tubular end buds and reduced branching when treated with TGF-alpha . Increased activity of a type IV collagenase-gelatinase (possibly matrix metalloproteinase-2) was detected, which suggests that TGF-alpha induces proteinase-elastase activity. We have previously reported that in TGF-alpha transgenic mice, elastin fibers appear to be less abundant and blunted in the bronchiolar regions and alveolar septa compared with normal nontransgenic lungs (8). TGF-alpha produced in the transgenic mouse lung may induce or activate matrix-degrading enzymes that subsequently disrupt or degrade the elastin network and inhibit the formation of normal alveoli during postnatal alveologenesis.

Previous experimental data indicate that TGF-alpha is induced after various forms of lung injury. TGF-alpha is released from alveolar macrophages stimulated by endotoxin in vitro (11). Rodent lungs injured with intratracheal administration of asbestos, naphthalene, or bleomycin produce increased TGF-alpha mRNA transcripts or protein in the airway and alveolar epithelium as well as the interstitium (9, 10, 16). In the transgenic lines, lung remodeling was not related to or induced by lung inflammation. There were no differences in the total white cell counts or differential counts in the BAL fluid cells between any of the transgenic and control mice, nor were there any differences in selected proinflammatory lung homogenate cytokines between nontransgenic and line 28 mice. TGF-alpha expression in the lung does not induce further inflammation but rather induces direct changes in the lung architecture that are independent of inflammation.

The postnatal lung remodeling of TGF-alpha transgenic mice may have clinical relevance with regard to lung diseases of infancy and childhood, specifically BPD and CF. Premature lungs are injured after birth from a variety of causes including barotrauma, reduced surfactant, and infection. These premature lungs develop inflammation and significant lung remodeling during the chronic phases of repair. Some of the pathological features of BPD resemble the lung remodeling seen in transgenic TGF-alpha mice, including emphysematous alveoli and fibrosis of the pleural surface. Immunohistochemistry of infants who died from BPD demonstrates a prominence of EGF, TGF-alpha , and EGF receptor in the diseased areas of the lung (14). Analysis of the lungs of patients with end-stage CF demonstrated marked TGF-alpha staining in inflamed airway epithelium and fibrosing submucosal areas (5). Hence the findings of increased TGF-alpha and EGF and the similarities in some features of lung remodeling seen in BPD and CF and in the TGF-alpha transgenic mice suggest that injury-induced TGF-alpha expression leads to morphological and functional abnormalities of the lung.

In summary, this study demonstrates that TGF-alpha expression in the lung can induce changes in the lung architecture in a dose-response fashion that are independent of lung inflammation. Because TGF-alpha is known to be released in disease states where there is subsequent lung remodeling, it is likely that TGF-alpha is a component of the complex cascade of growth factors and cytokines that cause postnatal lung remodeling.


    ACKNOWLEDGEMENTS

We thank Judy Bean for assistance with statistics, Marie Chappel for manuscript preparation, Sherri Profitt and Susan Wert for morphology and histology, and Jeff Whitsett for helpful advice and critical reading of the manuscript.


    FOOTNOTES

This work was sponsored by National Heart, Lung, and Blood Institute Grant KO8-HL-04172.

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

Received 22 March 2001; accepted in final form 18 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baughman, RP, Lower EE, Miller MM, Bejarano 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.   Derynck, R. Transforming growth factor-alpha . Cell 54: 593-595, 1988[ISI][Medline].

3.  Derynck R, Roberts AB, Winkler ME, Chen EY, and Goeddel DV. Human transforming growth factor-alpha : precursor, structure, and expression in E. coli. Cell 38: 287-297.

4.   Ganser, GL, Stricklin GP, and Matrisian L. EGF and TGF-alpha influence in vitro lung development by the induction of matrix-degrading metalloproteinases. Int J Dev Biol 35: 453-461, 1991[Medline].

5.   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[ISI][Medline].

6.   Hardie, WD, Huelsman K, Bruno M, Iwamoto H, Carrigan P, Leikauf G, and Korfhagen T. Effects of TGF-alpha during postnatal alveolarization in transgenic mice. Am J Pathol 151: 1075-1083, 1997[Abstract].

7.   Hardie, WD, Kerlakian CB, Bruno MD, Huelsman KM, Wert SE, Glasser SW, Whitsett JA, and Korfhagen TR. Reversal of lung lesions in transgenic TGF-alpha mice by expression of mutant EGF-R. Am J Respir Cell Mol Biol 15: 499-508, 1996[Abstract].

8.   Korfhagen, TR, Swantz RJ, Wert SE, McCarty JM, Kerlakian 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].

9.   Liu, J, Morris G, Lei W, Corti M, and Brody A. Up-regulated expression of transforming growth factor-alpha in the bronchiolar-alveolar duct regions of asbestos-exposed rats. Am J Pathol 149: 205-217, 1996[Abstract].

10.   Madtes, DK, Busby HK, Strandjord TP, and Clark JG. Expression of transforming growth factor-alpha and epidermal growth factor receptor is increased following bleomycin-induced lung injury in rats. Am J Respir Cell Mol Biol 11: 540-551, 1994[Abstract].

11.   Madtes, DK, Raines EW, Sakariassen KS, Assoian RK, Sporn MB, Bell GI, and Ross R. Induction of transforming growth factor-alpha in activated human alveolar macrophages. Cell 53: 285-293, 1988[ISI][Medline].

12.   Ruocco, S, Lallemand A, Tournier JM, and Gaillard D. Expression and localization of epidermal growth factor, transforming growth factor-alpha , and localization of their common receptor in fetal human lung development. Pediatr Res 39: 448-455, 1996[Abstract].

13.   Ryan, RM, Mineo-Kuhn MM, Kramer CM, and Finkelstein JN. Growth factors alter neonatal type II alveolar epithelial cell proliferation. Am J Physiol Lung Cell Mol Physiol 266: L17-L22, 1994[Abstract/Free Full Text].

14.   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].

15.   Tokieda, K, Ikegami M, Wert SE, Baatz JE, Zou Y, and Whitsett JA. Surfactant protein B corrects oxygen-induced pulmonary dysfunction in heterozygous surfactant protein B-deficient mice. Pediatr Res 46: 708-714, 1999[Abstract].

16.   Van Winkle, LS, Isaac JM, and Plopper CG. Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse. Am J Pathol 151: 443-459, 1997[Abstract].

17.   Zhou, L, Lim L, Costa RH, and Whitsett JA. Thyroid transcription factor-1, hepatocyte nuclear factor-3beta , surfactant protein B, C, and Clara cell secretory protein in developing mouse lung. J Histochem Cytochem 44: 1183-1193, 1996[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 281(5):L1088-L1094
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society