Attenuation of acute lung injury in transgenic mice expressing human transforming growth factor-alpha

William D. Hardie1, Daniel R. Prows2, George D. Leikauf2, and Thomas R. Korfhagen1

1 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati 45229-3039; and 2 Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267-0056


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-alpha (TGF-alpha ) is produced in the lung in experimental and human lung diseases; however, its physiological actions after lung injury are not understood. To determine the influence of TGF-alpha on acute lung injury, transgenic mouse lines expressing differing levels of human TGF-alpha in distal pulmonary epithelial cells under control of the surfactant protein C gene promoter were generated. TGF-alpha transgenic and nontransgenic control mice were exposed to polytetrafluoroethylene (PTFE; Teflon) fumes to induce acute lung injury. Length of survival of four separate TGF-alpha transgenic mouse lines was significantly longer than that of nontransgenic control mice, and survival correlated with the levels of TGF-alpha expression in the lung. The transgenic line expressing the highest level of TGF-alpha (line 28) and nontransgenic control mice were then compared at time intervals of 2, 4, and 6 h of PTFE exposure for differences in pulmonary function, lung histology, bronchoalveolar lavage fluid protein and cell differential, and lung homogenate proinflammatory cytokines. Line 28 TGF-alpha transgenic mice demonstrated reduced histological changes, decreased bronchoalveolar lavage fluid total protein and neutrophils, and delayed alterations in pulmonary function measures of airway obstruction compared with those in nontransgenic control mice. Both line 28 and nontransgenic control mice had similar increases in interleukin-1beta protein levels in lung homogenates. In contrast, interleukin-6 and macrophage inflammatory protein-2 levels were significantly reduced in line 28 transgenic mice compared with those in nontransgenic control mice. In the transgenic mouse model, TGF-alpha protects against PTFE-induced acute lung injury, at least in part, by attenuating the inflammatory response.

Teflon; ultrafine particulates; macrophage inflammatory protein-2; interleukin-6


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR-alpha (TGF-alpha ) is a polypeptide member of the epidermal growth factor (EGF) family that binds and activates the EGF receptor (6). TGF-alpha is produced by most epithelial cells and is mitogenic for both epithelial cells and fibroblasts in many organs (2, 3, 23, 24, 29).

In response to acute injury, TGF-alpha and EGF are produced in the lung. Rodent lungs injured with intratracheal administration of asbestos, naphthalene, or bleomycin produce increased TGF-alpha mRNA transcripts or protein in the airway and alveolar epithelia as well as in the interstitium (19, 21, 32). In humans, elevated TGF-alpha protein levels are found in acute and chronic lung injury. TGF-alpha levels from bronchoalveolar lavage (BAL) fluid increase in patients with acute respiratory distress syndrome (22). Infants dying from bronchopulmonary dysplasia have increased immunohistochemical staining for EGF and TGF-alpha in alveolar and airway epithelial cells and macrophages (30, 31). Lung biopsies from patients with idiopathic pulmonary fibrosis and cystic fibrosis have increased immunohistochemical TGF-alpha staining in airway epithelial cells, peribronchial submucosal cells, and macrophages compared with that in nondiseased control subjects (4, 9).

The precise physiological actions of TGF-alpha after lung injury is not completely understood. In the lungs of rodents exposed to asbestos or naphthalene, increased levels of TGF-alpha protein were detected in foci of cellular proliferation (19, 32). An increased number of cells expressing the nuclear proliferation antigen Ki67 and TGF-alpha was detected in diseased submucosal areas of cystic fibrosis lungs, suggesting that TGF-alpha may promote proliferation of various cells in response to injury (9). In addition, TGF-alpha promotes wound healing in the skin, gut, and lungs by augmenting cell motility and spreading (8, 13-16, 28). Finally, a recent study (1) supports a role for TGF-alpha in inducing cellular resistance to the antiproliferative activity of tumor necrosis factor-alpha (TNF-alpha ), suggesting that TGF-alpha may alter cellular signaling in inflammation.

In the present study, we examined the effects of TGF-alpha expression in acute lung injury produced by the inhalation of polytetrafluoroethylene (PTFE; Teflon) fumes. Lung injury from these fumes is caused primarily by ultrafine particulates generated from the heating of PTFE powder (25). We chose to expose mice to PTFE because this is a well-characterized model of acute lung injury in rodents (12). A previous study in rats (12) demonstrated that inhalation of PTFE caused acute pulmonary hemorrhage and inflammation. In this model, acute injury was associated with the recruitment of neutrophils and the production of proinflammatory cytokines [TNF-alpha , interleukin (IL)-6, and IL-1beta ], chemokines [macrophage inflammatory protein-2 (MIP-2)], and antioxidants. To study the in vivo effects of TGF-alpha on the pulmonary toxicity of PTFE, we used previously generated transgenic mice expressing human TGF-alpha (hTGF-alpha ) in distal respiratory epithelial cells under control of the human surfactant protein C (SP-C) promoter (17). We exposed four separate transgenic lines expressing different levels of TGF-alpha and their nontransgenic controls to PTFE fumes and monitored survival time. Survival was increased in all transgenic lines compared with the age- and strain-matched nontransgenic control lines. Alterations in lung morphology, BAL fluid protein and neutrophils, pulmonary function, and proinflammatory cytokines and chemokines were markedly less in the highest-expressing TGF-alpha transgenic mice (line 28) compared with those in nontransgenic control mice. Taken together, these data suggest that TGF-alpha reduces the extent of PTFE-induced acute lung injury by modifying the inflammatory response.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental design. To induce acute lung injury, mice were exposed to PTFE with the method described by Oberdorster et al. (25). To determine whether TGF-alpha protects against acute lung injury, four transgenic and one nontransgenic mouse lines were continuously exposed to PTFE for up to 50 h, and survival time was recorded. The mouse lines were selected because each expresses different levels of TGF-alpha in the lung. To examine PTFE-induced lung injury, pulmonary histology, physiology, and BAL fluid protein concentrations were compared between mice with the highest TGF-alpha expression (line 28) and age- and strain-matched (FVB/N) nontransgenic mice at time 0 and 2, 4, and 6 h of exposure. To assess lung inflammation, BAL fluid cell differentials and lung homogenate cytokine levels (TNF-alpha , IL-1beta , IL-4, IL-6, and MIP-2) were also determined in line 28 and nontransgenic mice.

Transgenic mice and Southern blot analysis. Methods for generating transgenic mice expressing hTGF-alpha under control of the human SP-C 3.7-kb promoter/enhancer sequences were previously described (17). Mice from four founder lines were studied. Line 28 mice express hTGF-alpha at high levels, with average lung homogenate concentrations of 570 pg/ml by ELISA. Line 28 mice develop a consistent pattern of lung pathology, with emphysema and pleural and peribronchial fibrosis, but have no differences in life span compared with nontransgenic littermate control mice (10, 17). Line 2 mice express 540 pg/ml of hTGF-alpha (95% of line 28) and have less extensive emphysema with no fibrosis. Line 4 mice express 230 pg/ml of hTGF-alpha (40% of line 28) and have minimal air space enlargement and no fibrosis. Transgenic line 6108 mice express 120 pg/ml of hTGF-alpha (21% of line 28) and have no gross pathological abnormalities. Mice were maintained in a virus-free environment and handled in accordance with the Institutional Animal Care and Use Committee of the Children's Hospital Research Foundation and the University of Cincinnati Medical Center (Cincinnati, OH). Transgenic mice were identified by a diagnostic 1.4-kb band on genomic Southern blots of Pst I-digested genomic tail DNA as previously described (17).

Aerosol generation. Air-generated PTFE (Fluon, ICI Chemical) exposures were conducted with the method described by Oberdorster et al. (25). Briefly, PTFE (0.75 g) was heated (420-430°C; Lindberg Hevi-Duty Furnace, Sola Basics Industries, Watertown, WI) to produce a mixture of ultrafine particles (count median diameter 18 nm) and hydrogen fluorocarbon gases (flow rate 5 l/min) (26). This mixture was combined with air (30 l/min) and introduced into a 43-liter Plexiglas inhalation chamber. Before exposure, PTFE was heated for >20 h to achieve a particle concentration of 107 particles/cm3 (electric aerosol size analyzer model 3030, Thermo-Systems, St. Paul, MN). In rats, Johnston et al. (12) reported that this exposure produces pulmonary edema, inflammation, and increased transcript levels of inflammatory cytokine and antioxidant enzymes, consistent with oxidant-induced acute lung injury.

Pulmonary histology. To assess lung injury after PTFE exposure, mice were killed with intraperitoneal pentobarbital sodium, the tracheae were cannulated, and the lungs were inflated (1 min, 25 cmH2O) with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.2. 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.

Pulmonary function. Pulmonary function was assessed in conscious mice by whole body plethysmography (model PLY 3115, Buxco Electronics, Troy, NY) as previously described (10, 18). Chamber pressure was measured with a transducer (model TRD 5700) connected to preamplifier modules (model CHA 0150) and analyzed by the XA software of the system (model SFT 1810; all from Buxco Electronics). The chamber pressure was used as a measure of the difference between thoracic expansion (or contraction) and air volume removed from (or added to) the chamber during inspiration (or expiration) and was differentiated to produce a pseudoflow that was proportional to the difference between the rate of change of the thoracic volume and nasal flow. The mice were placed in each chamber until stable breathing patterns were observed (2-6 min). Continuous measurements including respiratory frequency, tidal volume, expiratory time (TE), inspiratory time, relaxation time (RT), peak expiratory flow (PEF), and peak inspiratory flow (PIF) were obtained. Airflow obstruction was assessed by measuring the enhanced pause (Penh) with the manufacturer's recommended formula: Penh = [(TE/0.3RT) - 1] × (2PEF/3PIF). This measurement was calculated from the mean data of repetitive samples obtained over 2 min.

BAL fluid cell differential and protein. After death, the tracheae were cannulated, and the lungs were 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 µl of BAL fluid. Two hundred cells per slide were counted. Total BAL protein was measured with the bicinchoninic acid method as previously described (20), with bovine serum albumin for the standard.

Cytokine production. After death, the lungs were homogenized in 2 ml of PBS, pH 7.2 and centrifuged at 1,500 g, and the supernatant was stored at -70°C. TNF-alpha , IL-1beta , IL-6, IL-4, and MIP-2 were determined with a quantitative murine sandwich enzyme-linked immunosorbent assay kit (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). Only assays having standard curves with a calculated regression line value >0.95 were accepted for analysis.

Statistics. Statistics for survival time were based on Kaplan-Meier product limit survival curves, and differences in survival time between groups was tested by log rank test. For all other parameters, differences were compared with paired and unpaired t-tests. A P value < 0.05 was used as the level of significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Survival. Average survival time for the four transgenic lines and the wild-type control mice are presented in Fig. 1. All TGF-alpha transgenic mice survived significantly longer than strain-matched nontransgenic mice. Mouse lines with >500 pg/ml of transgenic TGF-alpha survived the longest, with all line 2 and five of nine line 28 mice alive at 50 h of exposure.


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Fig. 1.   Survival time of nontransgenic and transforming growth factor (TGF)-alpha transgenic mice exposed to polytetrafluoroethylene (PTFE; Teflon) ultrafine particles. Values are means ± SE; n = 13 FVB/N nontransgenic mice, 5 TGF-alpha line 6108 mice, 5 line 4 mice, 4 line 2 mice, and 9 line 28 mice. * P < 0.01 compared with FVB/N nontransgenic mice.

Histology. Two hours into PTFE exposure, all nontransgenic mice demonstrated infrequent areas of alveolar hemorrhage as well as evidence of early pulmonary edema signified by perivascular swelling (Fig. 2, arrow). By 4 h, extensive pulmonary edema was noted, with pronounced perivascular swelling and interstitial thickening (Fig. 2, arrow). Alveolar hemorrhage was extensive, and interstitial and alveolar macrophages and neutrophils were increased. At 6 h, pathological changes were similar to those at 4 h. Line 28 transgenic mice did not exhibit significant histological changes at any point during PTFE exposure compared with time 0 except for small, infrequent foci of alveolar hemorrhage seen at 4 and 6 h.


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Fig. 2.   Lung histology after PTFE exposure. Lungs from transgenic (line 28 TGF-alpha ) and age-matched nontransgenic (wild-type) mice were inflation fixed and stained with hematoxylin and eosin. Sections are representative of 3-6 individual mice killed at each time point. Arrows, perivascular edema in nontransgenic mouse lungs 2 and 4 h into PTFE exposure. A, airways; v, blood vessels. Original magnification, approximately ×26.

In line 2 mice, the histological changes were similar to those described in line 28 mice. Small foci of alveolar hemorrhage were occasionally detected after 4 or 6 h of exposure. However, at 6 h, mild perivascular swelling was detected in two of five mice.

Physiological comparisons between nontransgenic and transgenic mice. At time 0, no significant differences in pulmonary function were found between nontransgenic control and line 28 mice (Table 1). After PTFE exposure, PIF, PEF, and respiratory frequency decreased for both nontransgenic and line 28 mice. These changes were accompanied by increases in inspiratory time, TE, and Penh. In line 28 mice, all post-PTFE exposure changes in pulmonary function were attenuated compared with those in nontransgenic mice, with significant differences in respiratory frequency and TE at 2 and 4 h and Penh at 2 h. Data for pulmonary function were obtained over three separate experiments and were reproducible.

                              
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Table 1.   Pulmonary function

BAL fluid protein and cellular content. At time 0, there were no differences in total protein recovered in BAL fluid from nontransgenic and line 28 transgenic mice. Increased BAL fluid protein was observed in nontransgenic and line 28 mice after PTFE exposure; however, this increase was attenuated in transgenic mice compared with that in nontransgenic mice (Fig. 3).


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Fig. 3.   Bronchoalveolar lavage fluid protein levels after PTFE exposure. Open bars, nontransgenic mice; solid bars, line 28 transgenic mice. Values are means ± SE; n = 4-5 mice/group at each time point. * P < 0.05 compared with control mice at same time point (by t-test).

The percent neutrophils in BAL fluid in line 28 mice after PTFE exposure did not change during the experimental period, with a mean percent neutrophils of 2.8 ± 0.2% at time 0, increasing to 3.7 ± 1.8% at 6 h. In contrast, the percent neutrophils increased >50-fold in nontransgenic mice, from 0.3 ± 0.2% at time 0 to 16.1 ± 7.7% at 6 h.

Cytokines. After PTFE exposure, IL-1beta levels increased equally in both line 28 transgenic and nontransgenic mice (Fig. 4A). TNF-alpha and IL-4 levels at all time points increased only minimally from time 0 for both groups of mice (data not shown). IL-6 and MIP-2 levels increased significantly in both groups of mice from time 0 after PTFE exposure; however, the increases were significantly less in line 28 mice. IL-6 levels were significantly decreased in line 28 transgenic mice compared with those in nontransgenic mice at 4 and 6 h (Fig. 4B). MIP-2 levels were significantly decreased in line 28 transgenic mice compared with those in nontransgenic mice at each time point (Fig. 4C).


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Fig. 4.   Lung homogenate cytokine levels after PTFE exposure. A: interleukin-1beta . B: interleukin-6. C: macrophage inflammatory protein-2. Open bars, nontransgenic mice; solid bars, line 28 transgenic mice. Values are means ± SE. * P < 0.05 compared with control mice at same time point (by t-test).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present work demonstrates the differences between TGF-alpha transgenic and nontransgenic mice in the development of acute lung injury after PTFE exposure. All four TGF-alpha transgenic mouse lines survived significantly longer than the nontransgenic strain-matched mice. Inflammation and airway obstruction were attenuated in the higher TGF-alpha -expressing transgenic lines compared with those in nontransgenic mice. These findings suggest that hTGF-alpha expression protects the lungs from oxidant-induced injury by attenuating the inflammatory response.

Increased survival was associated with higher TGF-alpha -expressing transgenic lines and resembled a dose-response curve. Among the two highest expressing lines, line 2 mice survived longer than the higher-expressing line 28 mice. Previous characterizations (10, 17) of line 28 mice demonstrated significant lung remodeling, with emphysematous alveoli and pleural fibrosis associated with physiological abnormalities of increased compliance and airflow obstruction. In contrast, line 2 mice have less emphysema and no baseline pulmonary function abnormalities. The lower survival among line 28 mice may reflect their increased lung remodeling and physiological abnormalities. Thus these findings implicate increasing TGF-alpha gene dosage with increased survival.

The amount of PTFE particulate deposited into the lungs and alveoli of nontransgenic and transgenic mice was not quantified. We, therefore, cannot dismiss the possibility that lung remodeling of higher-expressing transgenic lines influenced particle deposition in the lungs. However, increased survival was demonstrated in transgenic lines where no lung remodeling was detected, and thus no change in PTFE distribution would be expected. In addition, previous studies (5, 11, 27) in animal models of emphysema have demonstrated that emphysematous lungs are generally no more or less susceptible to injury by gaseous and water-soluble irritants than normal lungs. Furthermore, because of the small size of the singlet ultrafine PTFE particle (10-20 nm), deposition would be more similar to an insoluble gas rather than to a longer diameter particle. Such particles may diffuse equally in emphysematous and nonemphysematous lungs.

The characterization of the inflammatory response in nontransgenic mice after PTFE exposure is similar to the response in rats described by Johnston et al. (12). In both Johnston et al.'s study and ours, an increase in BAL fluid protein and neutrophils as well as a marked increase in proinflammatory chemokines and cytokines, including MIP-2, IL-6, and IL-1beta , was present. In TGF-alpha transgenic mice, this inflammatory response was attenuated, suggesting that TGF-alpha may possess anti-inflammatory properties. A direct effect of TGF-alpha on chemokine and cytokine levels has not been described. However, TGF-alpha has been reported to decrease some of the biological activities of TNF-alpha . Aggarwal et al. (1) demonstrated that either transfection or exogenous addition of TGF-alpha to cells in vitro decreased the antiproliferative effects of TNF-alpha . The mechanism of this protection is unknown; however, the proliferative effects of TGF-alpha in cells are mediated through tyrosine kinase protein phosphorylation, whereas the antiproliferative effects of TNF-alpha are mediated through dephosphorylation (1). Thus TGF-alpha may inactivate the protein tyrosine phosphatase pathway, resulting in cellular resistance to the effects of TNF-alpha .

In the present study, levels of TNF-alpha and IL-4 were only minimally increased and no differences between transgenic and nontransgenic mice were found. However, IL-6 and MIP-2 were significantly lower in TGF-alpha transgenic mice compared with nontransgenic control mice after PTFE exposure. MIP-2 is a known chemoattractant for neutrophils (7). In TGF-alpha mice, neutrophil counts were unchanged, in association with only modest increases in MIP-2 levels after exposure. In contrast, in nontransgenic control mice, neutrophil counts increased 50-fold 6 h into PTFE exposure in association with marked increases in MIP-2 levels. Thus TGF-alpha may inhibit MIP-2 release after PTFE exposure, thereby reducing neutrophil migration to the lung and preventing neutrophil-mediated lung injury. The effects of TGF-alpha on the levels of inflammatory mediators were specific to MIP-2 and IL-6 because IL-1beta levels were equally increased in both transgenic and nontransgenic mice. TGF-alpha -mediated protection of the lung may be due to selective inhibition of inflammatory cytokines and chemokines.

In summary, transgenic mice overexpressing TGF-alpha demonstrated increased survival and reduced inflammation after acute lung injury caused by exposure to PTFE. Transgenic mice had reduced BAL fluid neutrophils and little evidence of lung injury at the same study points where nontransgenic mice demonstrated hemorrhagic inflammation with increased neutrophil influx. There was a marked difference in IL-6 and MIP-2 levels in lung homogenates between transgenic and nontransgenic mice, whereas TNF-alpha , IL-4, and IL-1beta levels were similar. These findings suggest that TGF-alpha protects against acute lung injury by altering the extent of pulmonary inflammation.


    ACKNOWLEDGEMENTS

We thank Kim Kurak and Jaymi Semona for assistance with mouse husbandry, Patricia Carrigan for assistance with pulmonary function testing and graphs, and Jennifer Westrich and Ann Maher for assistance with manuscript preparation.


    FOOTNOTES

This work was supported in part by a Trustee's Grant from the Children's Hospital Research Foundation (Cincinnati, OH); grants from the Center for Environmental Genetics (Cincinnati, OH); National Institute of Environmental Health Sciences Grant ES-06096; National Heart, Lung, and Blood Institute Grant HL-58275; and the Health Effects Institute (Cambridge, MA).

This work was presented in part at the American Thoracic Society meeting in Chicago, IL, in April 1998.

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. §1734 solely to indicate this fact.

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

Received 29 January 1999; accepted in final form 1 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aggarwal, B. B., E. Pocsik, F. Ali-Osman, and K. Totpal. Transfection of cells with transforming growth factor-alpha leads to cellular resistance to the antiproliferative effects of tumor necrosis factor. FEBS Lett. 354: 12-16, 1994[Medline].

2.   Bade, E. G., and S. Feindler. Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor-alpha is associated with changes in the gene expression of secreted proteins in vitro. In Vitro Cell. Dev. Biol. 24: 149-154, 1988[Medline].

3.   Basson, M. D., I. M. Modlin, and J. A. Madri. Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J. Clin. Invest. 90: 15-23, 1992[Medline].

4.   Baughman, R. P., E. E. Lower, M. A. Miller, P. A. Bejarano, and S. C. Heffelfinger. Overexpression of transforming growth factor-alpha and epidermal growth factor receptor in idiopathic pulmonary fibrosis. Sarcoidosis Vasc. Diffuse Lung Dis. 16: 57-61, 1999[Medline].

5.   Busch, R. H., R. L. Buschbom, and W. C. Cannon. Effects of ammonium sulfate aerosol exposure on lung structure of normal and elastase-impaired rats and guinea pigs. Environ. Res. 33: 454-472, 1984[Medline].

6.   Derynck, R. Transforming growth factor-alpha . Cell 54: 593-595, 1988[Medline].

7.   Driscoll, K. E. Macrophage inflammatory proteins: biology and role in pulmonary inflammation. Exp. Lung Res. 20: 473-490, 1994[Medline].

8.   Egger, B., F. Procaccino, J. Lakshmanan, M. Reinshagen, P. Hoffmann, A. Patel, W. Reuben, S. Gnanakkan, L. Liu, L. Barajas, and V. E. Eysselein. Mice lacking transforming growth factor-alpha have an increased susceptibility to dextran sulfate-induced colitis. Gastroenterology 113: 825-832, 1997[Medline].

9.   Hardie, W. D., P. A. Bejarano, M. A. Miller, J. R. Yankaskas, J. H. Ritter, J. A. Whitsett, and T. R. Korfhagen. 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.[Medline]

10.   Hardie, W. D., M. D. Bruno, K. M. Huelsman, H. S. Iwamoto, P. E. Carrigan, G. D. Leikauf, J. A. Whitsett, and T. R. Korfhagen. Postnatal lung function and morphology in transgenic mice expressing transforming growth factor-alpha . Am. J. Pathol. 151: 1075-1083, 1997[Abstract].

11.   Harkema, J. R., J. L. Mauderly, and F. F. Hahn. The effects of emphysema on oxygen toxicity in rats. Am. Rev. Respir. Dis. 126: 1058-1065, 1982[Medline].

12.   Johnston, C. J., J. N. Finkelstein, R. Gelein, R. Baggs, and G. Oberdorster. Characterization of the early pulmonary inflammatory response associated with PTFE fume exposure. Toxicol. Appl. Pharmacol. 140: 154-163, 1996[Medline].

13.   Kheradmand, F., H. G. Folkesson, L. Shum, R. Derynk, R. Pytela, and M. A. Matthay. Transforming growth factor enhances alveolar epithelial cell repair in a new in vitro model. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L728-L738, 1994[Abstract/Free Full Text].

14.   Konturek, S. J. Role of growth factors in gastroduodenal protection and healing of peptic ulcer. Gastroenterol. Clin. North Am. 19: 41-65, 1990[Medline].

15.   Konturek, S. J., T. Brzozowski, J. Majka, A. Dembinski, A. Slomiany, and B. Slomiany. Transforming growth factor-alpha and epidermal growth factor in protection and healing of gastric mucosal injury. Scand. J. Gastroenterol. 27: 649-655, 1992[Medline].

16.   Konturek, S. J., M. Cieszkowski, J. Jaworek, T. Brzozowski, and H. Gregory. Effects of epidermal growth factor on gastrointestinal secretion. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G580-G586, 1984[Abstract/Free Full Text].

17.   Korfhagen, T. R., R. J. Swantz, S. E. Wert, J. M. McCarty, C. B. Kerlakian, S. W. Glasser, and J. A. Whitsett. Respiratory epithelial cell expression of human transforming growth factor-alpha induced lung fibrosis in transgenic mice. J. Clin. Invest. 93: 1691-1699, 1994[Medline].

18.   Lee, J. J., M. P. McGarry, S. C. Farmer, K. L. Denzler, K. A. Larson, P. E. Carrigan, J. F. Brennelsen, M. A. Horton, E. W. Gelfand, G. D. Leikauf, and N. A. Lee. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J. Exp. Med. 185: 2143-2156, 1997[Abstract/Free Full Text].

19.   Liu, J., G. Morris, W. Lei, M. Corti, and A. Brody. 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].

20.   Lowry, O. H., M. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

21.   Madtes, D. K., H. K. Busby, T. P. Strandjord, and J. G. Clark. 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].

22.   Madtes, D. K., G. Rubenfeld, L. D. Klima, J. A. Milberg, K. P. Steinberg, T. R. Martin, G. Raghu, L. D. Hudson, and J. G. Clark. 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.   Mawatari, M., K. Okamura, T. Matsuda, R. Hamanaka, H. Mizoguchi, K. Higashio, K. Kohno, and M. Kuwano. Tumor necrosis factor and epidermal growth factor modulate migration of human microvascular endothelial cells and production of tissue-type plasminogen activator and its inhibitor. Exp. Cell Res. 192: 574-580, 1991[Medline].

24.   Nickoloff, B. J., R. S. Mitra, B. L. Riser, V. M. Dixit, and J. Varani. Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferative factors. Am. J. Pathol. 132: 543-551, 1988[Abstract].

25.   Oberdorster, G., R. Gelein, J. Ferin, and B. Weiss. Association of particle air pollution and acute mortality: involvement of ultrafine particles? Inhal. Toxicol. 7: 111-124, 1995.[Medline]

26.   Oberdorster, G., R. Gelein, C. Johnston, P. Mercer, N. Corson, and J. N. Finkelstein. Ambient ultrafine particles: inducers of acute lung injury? In: Relationships Between Respiratory Disease and Exposure to Air Pollution, edited by D. L. Dungworth, J. D. Brain, K. E. Driscoll, R. C. Grafstrum, and C. C. Harris. Washington, DC: Int. Life Sci. Inst., 1998, p. 216-229.

27.   Raub, J. A., F. J. Miller, J. A. Graham, D. E. Gardner, and J. J. O'Neil. Pulmonary function in normal and elastase-treated hamsters exposed to a complex mixture of olefin-ozone-sulfur dioxide reaction products. Environ. Res. 31: 302-310, 1983[Medline].

28.   Schultz, G. S., M. White, R. Mitchel, G. Brown, J. Lynch, D. R. Twardzik, and G. J. Todaro. Epithelial wound healing enhanced by transforming growth factor-alpha and vaccinia growth factor. Science 235: 350-352, 1987[Medline].

29.   Scott-Burden, T., T. J. Resnik, and F. R. Buhler. Growth regulation in smooth muscle cells from normal and hypertensive rats. J. Cardiovasc. Pharmacol. 12, Suppl. 5: S124-S127, 1988[Medline].

30.   Stahlman, M. T., D. N. Orth, and M. E. Gray. Immunocytochemical localization of epidermal growth factor in the developing human respiratory system and in acute and chronic lung disease in the neonate. Lab. Invest. 60: 539-547, 1989[Medline].

31.   Strandjord, T. P., J. G. Clark, D. E. Guralnick, and D. K. Madtes. Immunolocalization of transforming growth factor-alpha , epidermal growth factor (EGF), and EGF-receptor in normal and inured developing human lung. Pediatr. Res. 38: 851-856, 1995[Abstract].

32.   Van Winkle, L. S., J. M. Isaac, and C. G. Plopper. 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].


Am J Physiol Lung Cell Mol Physiol 277(5):L1045-L1050
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society