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Published ahead of print on June 19, 2003, doi:10.1165/rcmb.2002-0046OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 669-676, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0046OC

Overexpression of Tumor Necrosis Factor-{alpha} Diminishes Pulmonary Fibrosis Induced by Bleomycin or Transforming Growth Factor-ß

Masaki Fujita, John M. Shannon, Osamu Morikawa, Jack Gauldie, Nobuyuki Hara and Robert J. Mason

Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado; Department of Pathology, University of McMaster, Hamilton, Ontario, Canada; and Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Address correspondence to: Robert J. Mason, M.D., Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: masonb{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) is thought to be important in the development of pulmonary fibrosis. However, surfactant protein-C/TNF-{alpha} transgenic mice do not spontaneously develop pulmonary fibrosis but instead develop alveolar enlargement and loss of elastic recoil. We hypothesized that overexpression of TNF-{alpha} in the lung requires an additional insult to produce fibrosis. In this study we evaluated whether TNF-{alpha} overexpression altered the development of pulmonary fibrosis due to bleomycin or transforming growth factor-ß (TGF-ß). Either 0.2 U bleomycin or saline was administered into left lung of TNF-{alpha} transgenic mice and their transgene-negative littermates. To overexpress TGF-ß, an adenovirus vector containing either active TGF-ß (AdTGF-ß) or LacZ was administered at a dose of 3 x 108 plaque-forming units per mouse. Fibrosis was assessed histologically and by measurement of hydroxyproline. TNF-{alpha} transgenic mice tolerated bleomycin or AdTGF-ß, whereas the transgene-negative littermates demonstrated severe pulmonary fibrosis after either agent. An increase in prostaglandin E2 and downregulation of TNF receptor I expression were observed in the TNF-{alpha} transgenic mice. In addition, recombinant human TNF-{alpha} attenuated bleomycin-induced pulmonary fibrosis. TNF-{alpha} has a complex role in the development of pulmonary fibrosis. Endogenous TNF-{alpha} may be important in the development of fibrosis as indicated in other reports, but overexpression of TNF-{alpha} or exogenous TNF-{alpha} limits pulmonary fibrosis in mice.

Abbreviations: LacZ adenovirus, AdLacZ • adenovirus vector of TGF-ß, AdTGF-ß • bronchoalveolar lavage, BAL • bleomycin, BLM • enzyme-linked immunosorbent assay, ELISA • idiopathic pulmonary fibrosis, IPF • matrix metalloproteinase, MMP • prostaglandin E2, PGE2 • recombinant tumor necrosis factor-{alpha}, rTNF-{alpha} • ribonuclease protection assay, RPA • surfactant protein C, SP-C • transforming growth factor-ß, TGF-ß • tumor necrosis factor-{alpha}, TNF-{alpha} • TNF receptor, TNFR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary fibrosis is characterized by accumulation of extracellular matrix, collagen deposition, fibroblast proliferation and migration, and loss of alveolar gas exchange units. The etiology of idiopathic pulmonary fibrosis (IPF) is not known, and IPF remains a devastating disease with a > 50% 5-yr mortality rate. Unfortunately, the pathogenesis of IPF is also incompletely understood. One critical issue is to define what components of the inflammatory process regulate or modify the fibrotic reaction. Inflammatory cytokines are thought to be important role in the initiation and perpetuation of the fibrotic process. Tumor necrosis factor (TNF)-{alpha} is a proinflammatory cytokine with many biologic properties (1) and is thought to be critical in the development of pulmonary fibrosis (2). Anti–TNF-{alpha} antibody attenuates both bleomycin (BLM)-induced pulmonary fibrosis and silica-induced pulmonary fibrosis in mice (3, 4). In addition, a soluble receptor for TNF-{alpha} has also been shown to lessen bleomycin induced pulmonary fibrosis (5). TNF-{alpha} receptor knockout mice are also protected against pulmonary fibrosis due to silica, BLM, and asbestos (6–8). However, transgenic mice that overexpress TNF-{alpha} do not develop pulmonary fibrosis but instead develop alveolar enlargement and loss of elastic recoil, which are characteristic of pulmonary emphysema (9). In addition, instillation of an adenovirus that expresses TNF-{alpha} produces inflammation but only limited pulmonary fibrosis (10). Hence, TNF-{alpha} by itself appears not to be able to induce pulmonary fibrosis, but in conjunction with BLM or silica may accentuate the fibrotic process.

Because TNF-{alpha} overexpression alone does not produce pulmonary fibrosis (9), we sought to determine if TNF-{alpha} overexpression would make the mice more sensitive to fibrotic agents. Pulmonary fibrosis can be produced in mice by several means, and these include BLM, an adenovirus that expresses active TGF-ß, silica, asbestos, butylated hydroxytoluene and oxygen, or irradiation. We have chosen to evaluate BLM and overexpression of TGF-ß. BLM is a chemotherapeutic drug used to treat cancer, and can cause pulmonary fibrosis as a complication. BLM has been used by many investigators to produce pulmonary fibrosis in mice and is thought to produce an oxidative injury that results in DNA damage and destruction of alveolar epithelial cells (3). In addition, we wanted to evaluate another type of fibrotic agent that did not directly produce an oxidant injury and DNA damage. TGF-ß is a well-known stimulant of extracellular matrix production by fibroblasts and has been suggested to play an important role in the development of pulmonary fibrosis (11). Instillation of a recombinant adenovirus expressing TGF-ß produces extensive fibrosis in rats (12).

The purpose of this study was to determine whether BLM or an adenovirus that expresses TGF-ß would serve as an additional insult to produce pulmonary fibrosis in mice that overexpress TNF-{alpha}. The surprising finding was that these mice were protected against both BLM and TGF-ß. The phenotype of these TNF-{alpha} transgenic mice has been published (9, 13). These mice have large lung volumes, decreased elastic recoil, and pulmonary hypertension as well as chronic inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TNF-{alpha} Transgenic Mice
Surfactant protein (SP)-C/TNF-{alpha} transgenic mice were a kind gift of Y. Miyazaki (Kyushu University, Beppu, Japan) (14). Transgenic mice were crossed with C57BL/6 and bred in specific pathogen–free conditions in our animal facility. The transgenic mice were identified by polymerase chain reaction analysis of genomic DNA with primers reported previously (14, 15). Transgene-negative littermates were used as controls. All the mice were ~ 10 wk old at the time of the instillations.

BLM Instillation
BLM was purchased from Pharmacia (Kalamazoo, MI). After anesthesia with bromoethanol, a 22–gauge feeding tube with a tip bent to 45 degrees was inserted into the trachea and advanced into the left bronchus. Either 0.2 U BLM in 50 µl saline or 50 µl saline alone was instilled into the left lung. Before and after administration, body weights were measured weekly. At 1, 3, 7, 14, and 28 d after instillation, the mice were killed by an overdose of phenobarbital.

Adenoviral Vector Preparation and Instillation
An adenovirus vector expressing active TGF-ß (AdTGF-ß) (12) was a kind gift from Dr. J. Gauldie (Hamilton, ON, Canada). The construction of the LacZ adenovirus (AdLacZ) was reported previously (16). An adenovirus type 5 lacking the E1 region was used to construct these recombinant viruses. The recombinant adenoviruses were grown in 293 cells and purified by centrifugation on CsCl2 gradients, as described previously (16, 17). Either AdTGF-ß, AdLacZ, or 50 µl vehicle (saline containing 10% glycerol) was injected into the left lung at a dose of 3 x 108 plaque-forming units/mouse.

Simultaneous Injection of Recombinant TNF-{alpha} and BLM
Recombinant human TNF-{alpha} (rTNF-{alpha}) was a kind gift from Dainippon pharmaceuticals, Osaka, Japan. rTNF-{alpha} was injected into left bronchus by the method as previously described. The rTNF-{alpha} was biologically active as measured by neutrophil accumulation of bronchoalveolar fluids after rTNF-{alpha} instillation. Either 50 ng of rTNF-{alpha} in saline or saline only was administered with 0.05 U BLM. The mice were killed 28 d after installation, and histology and hydroxyproline content were evaluated.

Histology
Mice were killed by intraperitoneal injection of sodium pentobarbital. The lungs were inflated by intratracheal injection of 4% paraformaldehyde in phosphate-buffered saline at 25 cm H2O static pressure. After 1 h the lungs were immersed in same buffer and kept overnight at 4°C. The lungs were then sliced and processed for histology. Tissue sections were stained by hematoxylin and eosin, Sirius Red, trichrome, and pentachrome methods.

Hydroxyproline Determination
Hydroxyproline assay was performed as previously described (9). Briefly, after killing, the lungs were removed, snap frozen, and stored at -70°C. They were then lyophilized for at least 48 h, and 10 mg of pulverized ground lung was added to 500 µl of 6 N HCl and incubated overnight at 120°C. The acid hydrolysates (5 µl) and standards were applied to an enzyme-linked immunosorbent assay (ELISA) plate along with 5 µl of citric/acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% NaOH, 1.2% glacial acetic acid, pH 6.0) and 100 µl of chloramine T solution (564 mg chloramine T, 4 ml H2O, 4 ml n-propanol, 32 ml of the citrate/acetate buffer). The plates were incubated for 20 min at room temperature and then 100 µl of Ehrlich's solution (4.5 g 4-dimethylaminobenzaldehyde, 18.6 ml n-propanol, 7.8 ml 70% perchloric acid) were added and incubated further at 65°C for 15 min. Reaction product was read at OD 550 nm. Solutions of 0–200 µg/ml hydroxyproline (Sigma, St. Louis, MO) were used to construct the standard curve.

RNase Protection Assay
RNA was prepared by a guanidine isothiocyanate-cesium chloride (GITC) gradient procedure as described previously (18). For RNase protection assay (RPA), mouse multiprobe template sets were purchased from Pharmingen (San Diego, CA). RPA was performed according to manufacturer's protocol. Briefly, an antisense transcript was synthesized by using T7 RNA polymerase and [32P]CTP (ICN, Costa Mesa, CA). Samples of 5 ug of total lung RNA were hybridized at 56°C overnight. Hybrids were digested with RNAses A and T1. Protected fragments were separated on 5% polyacrylamide/8M urea gels and analyzed by autoradiography. The increase of mRNA levels was quantified by ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Prostaglandin E2 Measurement
Homogenized lung was used for prostaglandin (PG)E2 determination. Lungs were removed after thoracotomy, homogenized in phosphate-buffered saline, and centrifuged at 1,000 x g for 10 min. Four hundred microliters of methanol was added to 100 µl of the supernatant and centrifuged to remove proteins. The supernatant was removed, frozen, and used for measurement of PGE2 by ELISA (Cayman-Chemical Co, Ann Arbor, MI). An aliquot of the supernatant was also used for protein determination by the bicinchoninic acid method (Pierce, Rockford, IL).

Statistics
Data are expressed as mean ± SE. The statistical analyses were performed using a computer program (SAS software, version 6). ANOVA was performed, then the Tukey-Kramer method was applied to adjust for multiple comparisons. P < 0.05 was considered to indicate a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histology
BLM produced extensive inflammation in the littermate control mice, as reported previously (19). There was marked inflammation of the left lung between 3 and 7 d, and later the lung architecture was markedly altered and severe fibrosis developed. At 28 d, the left lung was smaller and fibrotic (Figure 1). For the littermate controls, there were clear and obvious differences between the saline- and the BLM-instilled lungs at all time points. On the other hand, the lungs from BLM-treated TNF-{alpha} transgenic mice showed only a limited fibrotic reaction even at 28 d (Figure 1). The general lung architecture was preserved, and there was no gross reduction in lung volume observed on macroscopic examination or in histologic sections. However, at 7 d the pleural surface of the BLM-treated transgenic mice became reddish such that the lungs of the saline- and BLM-instilled mice could be easily differentiated. Nevertheless, histologic examination revealed no apparent differences between saline and BLM treatment, in part because the TNF-{alpha} transgenic mice already have parenchymal inflammation as described previously (9, 13, 14). To demonstrate that the observations were not limited by the dose administered, we also administered a higher dose of 0.4 U BLM in 100 µl saline into the left lung of transgenic mice. With the higher dose, there was marked fibrosis in the littermate control mice and only a limited reaction in the TNF-{alpha} transgenic mice 14 and 28 d after instillation (data not shown). Saline-treated transgenic mice and littermates did not show any evidence of histologic alterations compared with nontreated mice.



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  		Figure 1. Histology of the lungs from BLM-treated and saline-treated mice. The animals were killed 28 d after the instillation of BLM or saline. The lungs were stained with hematoxylin and eosin. (A and E) Saline-treated control mice. (B and F) BLM-treated control mice. (C and G) Saline-treated SP-C/TNF-{alpha} transgenic mice. (D and H) BLM-treated SP-C/TNF-{alpha} transgenic mice. Magnifications: A–D, x7.5; F–H, x40. The transgenic TNF-{alpha} lungs are larger and do not develop the contracture and dense cellular infiltration as seen in the controls in response to BLM.

 
Hydroxyproline Content
Collagen deposition was evaluated by measurement of hydroxyproline 28 d after the instillation of BLM, and the data are expressed as hydroxyproline content per left lung (Figure 2A). The TNF-{alpha} transgenic mice have significantly larger lungs than the littermate controls and hence more hydroxyproline per lung (9, 14). BLM increased the hydroxyproline content of the control mice but not that of the TNF-{alpha} transgenic mice (Figures 2A and 2B). BLM did not alter the hydroxyproline content or the dry weight of the transgenic mice, whereas it did in control mice. The reduction of dry weight in the control mice after BLM administration reflects the lung contracture and gross reduction in size (Figure 1B).





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  		Figure 2. Hydroxyproline contents of the lungs from BLM-treated and saline-treated mice. Tg(-) indicates control mice and Tg(+) indicates SP-C/TNF-{alpha} transgenic mice. The animals were killed 28 d after instillation. Bleomycin increased hydroxyproline in the control mice but not in the TNF-{alpha} transgenic mice. (A) Data are expressed per lung and as noted previously, the tg(+) lungs are larger and contain more hydroxyproline. However, there is no change in hydroxyproline content with bleomycin treatment. (B) The results are expressed per mg dry weight and demonstrate fibrosis in the tg(-) mice. C reports the dry weight and demonstrates the lung contracture in the control mice treated with BLM. For these analyses there were four untreated littermate controls, three untreated TNF-{alpha} transgenic mice, five saline-instilled littermate controls, six saline-instilled TNF-{alpha} transgenic mice, five BLM-instilled littermate controls, and six BLM-instilled TNF-{alpha} transgenic mice. Asterisks indicate statistically different (P < 0.05). NS indicates no statistical difference.

 
PGE2 Content
PGE2 has been shown to be a potent inhibitor of fibroblast proliferation (20, 21). Therefore, we measured PGE2 concentration in homogenized lung as well as bronchoalveolar lavage (BAL) fluid in untreated transgenic mice and untreated transgenic negative mice to determine if changes in PGE2 may in part account for milder fibrotic reactions in the TNF-{alpha} transgenic mice. After BLM instillation, homogenized lungs from transgenic mice showed significantly higher concentrations of PGE2 than those from control mice as summarized in Table 1. PGE2 was below the level of detection in BAL fluids and was not evaluated further.


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  		TABLE 1 PGE2 levels in homogenized lungs

 
TNF-{alpha} Receptor and Cytokine Expression after BLM Administration
Because the TNF-{alpha} transgenic mice were protected against the effect of bleomycin, TNF-{alpha} receptor and cytokine expression was evaluated to determine if there was a reduction in receptor expression or a decrease in the expected increase of cytokine expression after the administration of bleomycin. TNFRI mRNA expression was downregulated, whereas TNFRII was increased in transgenic mice compared with littermates (Figure 3).



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  		Figure 3. TNF-receptor mRNA expression measured by RPA. Total lung RNA was isolated from uninstilled mice as stated in the methods section. TNF-receptor mRNA was measured by an RPA using the mCK-4 kit according to manufacturer's directions (Pharmingen, San Diego, CA). Tg(-) indicates control mice and Tg(+) indicates SP-C/TNF-{alpha} transgenic mice. The mRNA levels were normalized with the constitutive probes L32 and GADPH. (A) Animals 1–4 were transgenic-negative and animals 5–8 were transgenic-positive. The calculated differences are shown in B (n = 4 for each group). Asterisks indicate a statistical difference (P < 0.05).

 
AdTGF-ß Experiments
The results above indicate that TNF-{alpha} transgenic mice are protected against pulmonary fibrosis induced by BLM. To extend this observation, we evaluated another fibrogenic agent, an adenovirus that expresses active TGF-ß (12). TGF-ß protein was measured in BAL fluid by ELISA. Compared with a comparable viral load of an adenovirus expressing LacZ (AdLacZ), the AdTGF-ß produced high levels of TGF-ß in both transgenic and transgene-negative littermates. There were no significant differences in TGF-ß in lavage fluid between transgenic mice and transgene-negative littermates (transgenic mice, 155.9 ± 77.9 pg/ml TGF-ß1; transgene-negative littermates, 127.8 ± 73.8 pg/ml TGF-ß1). TGF-ß1 was not detected in lavage fluid of mice injected with Ad LacZ. As shown in Figure 4, AdLacZ caused slight inflammation but no fibrosis, whereas AdTGF-ß induced severe pulmonary fibrosis in the littermates. In the case of TNF-{alpha} transgenic mice, AdTGF-ß induced inflammation but only slight fibrosis. Hydroxyproline measurements also demonstrated that TNF-{alpha} transgenic mice showed a reduced fibrotic reaction in response to AdTGF-ß (Figure 5). In the littermate controls AdTGF-ß but not AdLacZ increased hydroxyproline content per lung (Figure 5). However, neither AdLacZ nor AdTGF-ß increased the hydroxyproline content in the TNF-{alpha} transgenic mice.



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  		Figure 4. Lung histopathology of adenovirus vector-treated mice. The lungs were removed for histology 21 d after instillation and were stained with hematoxyin and eosin. (A) AdLacZ-treated control mice. (B) AdTGF-ß–treated control mice (Tg(-)). (C) AdLacZ-treated SP-C/TNF-{alpha} transgenic mice (Tg(+)). (D) AdTGF-ß–treated SP-C/TNF-{alpha} transgenic mice. All micrographs are submitted at x40 magnification. The mice were instilled with 3 x 108 plaque-forming units into the left lung. AdLacZ produced inflammation as commonly seen when adenoviruses are instilled in vivo. However, the AdTGF-ß produced much more fibrosis. This was more apparent in the control mice than in the TNF-{alpha} transgenic mice.

 


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  		Figure 5. Hydroxyproline content of the lungs from adenovirus vector–treated mice. Tg(-) indicates control mice and Tg(+) indicates SP-C/TNF-{alpha} transgenic mice. In this series of experiments, AdTGF-ß increased hydroxyproline per left lung in the control mice but not in the Tg(+) mice. The mice were killed 21 d after instillation. For these analyses there were seven vehicle-instilled littermate controls, six vehicle-instilled TNF-{alpha} transgenic mice, seven AdlacZ-instilled littermate controls, eight AdlacZ-instilled TNF-{alpha} transgenic mice, six AdTGF-ß–instilled littermate controls, and five AdTGF-ß–instilled TNF-{alpha} transgenic mice. Asterisks indicate statistical differences (P < 0.05). N.S. indicates no statistical difference.

 
rTNF-{alpha} Injection
To investigate the role of increased TNF-{alpha} further, we instilled wild-type C57B6 mice with bleomycin and rTNF-{alpha}. Recombinant human TNF-{alpha} increased neutrophils in lavage fluid in a dose-dependent manner. After instillation of 1 µg of rTNF-{alpha}, neutrophils accounted for ~ 50% of lavage cells. After saline instillation neutrophils account for < 5% of the cells. A single injection of 50 ng rTNF-{alpha} produced no pathologic finding in the lungs at 28 d, whereas injection of 0.05 U BLM caused severe pulmonary fibrosis. Simultaneous injection of 50 ng of rTNF-{alpha} with 0.05 U BLM produced limited pulmonary fibrosis (Figure 6). Changes in hydroxyproline content were consistent with the histologic appearance (data not shown). Hence, the results with exogenous TNF-{alpha} support the concept that high levels of TNF-{alpha} can protect the lungs from pulmonary fibrosis produced by bleomycin.



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  		Figure 6. Left lung hydroxyproline content from recombinant human TNF-{alpha} and BLM-treated mice. Mice were administered BLM with rTNF-{alpha} simultaneously. The mice were killed 28 d after the instillation. TNF-{alpha} reduced the hydroxyproline per left lung when compared with saline instillation. For these analyses there were three untreated mice, three mice instilled with BLM only (sham), nine mice instilled with saline and BLM, and eight mice instilled with rTNF-{alpha} and BLM. Asterisks indicate statistical differences (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is a considerable evidence to suggest that TNF-{alpha} is important in the development of pulmonary fibrosis. Most importantly, anti–TNF-{alpha} antibody attenuates the development of pulmonary fibrosis due to bleomycin or silica (3, 4). Therefore, there was a great deal of interest in the transgenic mice that overexpressed TNF-{alpha} under the control of the SP-C promoter and were reported to develop fibrosing alveolitis (14). We have studied the same transgenic mice and have very similar results but different conclusions (9, 13). We agree with the original description that there is chronic inflammation, especially in the younger mice, with lymphocytic infiltration, and focal areas of increased collagen deposition, and an increase in hydroxyproline per lung. In addition, we observed air space enlargement resulting from loss or rupture of alveolar septa. However, by staining with hematoxylin and eosin, pentachrome, sirius red, and Masson's trichrome stain we found very little fibrosis (9, 14, and unpublished observations). There was an increased hydroxyproline per lung but no increase when the hydroxyproline values were corrected for lung weight (9). The lungs of the transgenic mice are larger than controls. However, the most important difference is that we measured lung volumes and elastic recoil, which demonstrated large lung volumes and decreased elastic recoil similar to emphysema and not diffuse pulmonary fibrosis. Although chronic overexpression of TNF-{alpha} by itself did not produce pulmonary fibrosis, we hypothesized that chronic overexpression of TNF-{alpha} might make the lungs more susceptible to BLM or TGF-ß.

This is the first report on the susceptibility of chronic overexpression of TNF-{alpha} to fibrotic agents and demonstrates that these mice are less susceptible to BLM and AdTGF-ß. Phan and coworkers previously reported that simultaneous injection of LPS, a strong inducer of TNF-{alpha}, reduced BLM-induced fibrosis (22). Although TNF-{alpha} had been reported to be a major cytokine for the development of pulmonary fibrosis, recently several studies have shown that TNF-{alpha} can both induce and protect against disease processes (23, 24). For example, although TNF-{alpha} contributes to the pathogenesis for multiple sclerosis, TNF-{alpha} has a protective role in autoimmune-mediated demyelination, a mouse model of multiple sclerosis (25, 26). Moreover, TNF abrogates the autoimmune process in the late phase of virally induced diabetes, whereas TNF-{alpha} promotes the autoimmune process in the early phase (24). TNF-{alpha} has also been reported to be important for the development of hepatic fibrosis. However, anti–TNF-{alpha} antibody enhances hepatic fibrosis in mice injected with Shistosoma mansoni eggs and interleukin-12 (27). Furthermore, growth of hepatic stellate cells, which play an important role in the pathogenesis of liver inflammation and fibrosis, is inhibited by TNF-{alpha} (28). More recently, Kuroki and coworkers demonstrated that TNF-{alpha} can lessen pulmonary inflammation after bleomycin in TNF-deficient mice by inducing apoptosis of infiltrating inflammatory cells (29). With respect to pathogenesis of lung diseases, TNF-{alpha} has been reported to be responsible for several lung diseases such as pulmonary fibrosis, acute lung injury, and pulmonary emphysema (2, 10, 30). However, TNF-{alpha} also protects rats against oxygen toxicity even in the presence of lung disease (31). There are also many reports that TNF-{alpha} has antifibrotic effects in vitro (32–35). In the present study, chronic overexpression of TNF-{alpha} did not enhance the fibrogenic effects of BLM or AdTGF-ß. Moreover, rTNF-{alpha} attenuated the development of BLM-induced pulmonary fibrosis. Taken together, these data indicate that TNF-{alpha} may both induce and protect in the development of pulmonary fibrosis and that the ultimate response will be determined by a variety of host factors that are not clearly determined at this time.

We do not believe that the failure to develop pulmonary fibrosis due to BLM was simply that we gave too little bleomycin to the left lung. We administered 0.2 U BLM, which is higher than used in other reports (19). Moreover, 0.4 U BLM did not produce significantly more pulmonary fibrosis than 0.2 U in these transgenic mice. Intrabronchial administration was done after confirmation of respiratory movements to ensure that the BLM or AdTGF-ß entered the respiratory system and not the esophagus. We can also exclude the possibility that the BLM spilled over into right lung, because we visually inspected the right lung at the time of killing and examined the right lung macroscopically and histologically. Finally, we demonstrated that this dose produced severe fibrosis in the littermate controls.

There are several possibilities as to why TNF-{alpha} overexpression protects against pulmonary fibrosis by BLM or TGF-ß. (i) The neutrophils present in the airspace might protect against the development of pulmonary fibrosis. There is a paradigm shift in the possible pathogenesis of idiopathic pulmonary fibrosis (IPF) (36). A new hypothesis is that IPF is not a disease of chronic inflammation but a disease of faulty epithelial repair and epithelial dysfunction. Hence, chronic inflammation may not be as critical in pathogenesis of pulmonary fibrosis as previously thought. In addition there are data that collagen synthesis and deposition is greater after BLM treatment in neutrophil-depleted animals (37, 38). Neutrophils also help clear necrotic and apoptotic epithelial cells and repair the epithelium after ozone exposure (39, 40). In the TNF-{alpha}–overexpressing mice there is a marked increase in the number of neutrophils in lavage fluid (9). (ii) It is possible that part of the reason for the diminished response to BLM and AdTGF-ß in the transgenic mice is due to the altered histology and airspace enlargement. Burkhardt and colleagues suggested that an important step in the development of pulmonary fibrosis and loss of gas exchange units is due to appositional atelectasis of adjacent alveolar walls and subsequent fusion by the inflammatory exudate (41). This is a possible explanation of why pulmonary fibrosis develops more slowly or is less progressive in emphysematous lungs. This might be an explanation why another transgenic mouse (SP-C/TGF-{alpha}) that has airspace enlargement is also less susceptible to injury (42). (iii) TNF-{alpha} affects expression of other cytokines. Recently, several reports pointed out that a Th2 shift is responsible for pulmonary fibrosis (43–45). Hoffmann and coworkers hypothesized that TNF-{alpha} may be antifibrogenic in hepatic fibrosis by stimulating a Th1 cytokine response (27). A dominant Th1 response was observed in the TNF-{alpha} transgenic mice, as indicated by an increase of interferon-{gamma} and interleukin-12 (9). Interferon-{gamma} has been reported to be antifibrogenic by suppressing TGF-ß expression and inhibition matrix production and fibroblast proliferation (46, 47). This is another plausible explanation of why TNF-{alpha} transgenic mice are less sensitive to BLM- and TGF-ß–induced pulmonary fibrosis. (iv) BLM usually causes a transient increase of TNF-{alpha} from Day 3 to Day 14. Some species of mice, such as BALB/c mice, are resistant to BLM-induced fibrosis. BALB/c mice have an increase in TNF-{alpha} early after BLM administration, whereas CBA/2J mice, which are sensitive to BLM, show an increase of TNF-{alpha} late in the course of the response (23). The concentration of TNF-{alpha} induced by BLM was lower than that of TNF-{alpha} transgenic mice in this study. Thus it is possible that a change in TNF-{alpha} concentration or a low level of TNF-{alpha} may contribute to a fibrotic reaction, whereas a sustained high level may inhibit to fibrotic reaction. (v) TNF-{alpha} receptor knockout mice are insensitive to the development of pulmonary fibrosis induced by BLM, silica, or asbestos fibers (6–8). Chronic exposure of TNF-{alpha} also reduces TNF-{alpha} receptor expression. We demonstrated that TNF receptor I (TNFRI) was downregulated in the transgenic mice, whereas TNFRII was upregulated. (vi) The downregulation of the TNFRI should reduce apoptosis induced by TNF-{alpha}. This might account for the accumulation of inflammatory cells seen in histologic sections, but would also decrease apoptosis of epithelial cells, which are necessary for alveolar repair (19, 48). TNF-{alpha} also induces PGE2 production (49), which inhibits fibroblast proliferation (20, 21). Recent studies have suggested that reduced PGE2 production may be important in the development of fibrosis. Patients with IPF have reduced levels of PGE2 in their alveolar lavage fluid (50). In addition, fibroblasts cultured from patients with IPF have a diminished capacity to produce PGE2 (51). Furthermore, mice deficient in cyclooxygenase-2 have an enhanced response to bleomycin (52). In this study, we found that transgenic mice showed higher pulmonary PGE2 levels. (vii) Matrix metalloproteinases (MMPs) and their inhibitors have also been suggested to be important in the development of pulmonary fibrosis (53). These transgenic mice have increased MMPs, specifically MMP 2 and MMP 9 (9), and these MMPs may have prevented the development of pulmonary fibrosis. (8). As stated above, TNF-{alpha} may also facilitate clearance of inflammatory cells after the instillation of bleomycin (29).

Hence, there are many possible explanations of why TNF-{alpha} transgenic mice failed to develop pulmonary fibrosis. One interesting possibility is that the airspace neutrophils present in the TNF-{alpha}–overexpressing mice protected against the development of pulmonary fibrosis. In addition, the airspace enlargement and alveolar architecture may in itself diminish the fibrotic response by decreasing appositional atelectasis. Chronic overexpression of TNF-{alpha} may also inhibit pulmonary fibrosis by modifying the immunologic reaction, increasing PGE2 production, activating MMPs, or downregulating TNF-{alpha} receptors. Although it is difficult to state what factor(s) are responsible for the inhibition of fibrosis, this study strongly suggests that TNF-{alpha} can both stimulate and inhibit the development of pulmonary fibrosis. Further understanding the mechanism of inhibition of pulmonary fibrosis in this and similar murine models could lead to new insight into the development of pulmonary fibrosis.


   Acknowledgments
 
The authors thank Y. Miyazaki for providing the SP-C/TNF-{alpha} transgenic mice. They are appreciative of Karen Edeen for her excellent technical help, Lynn Cunningham for the histology, Carlyne Cool for reviewing the pathology, Karen Sheff for statistical analysis, and Jay Westcott for performing the PGE2 analyses. They also thank Dainippon pharmaceuticals for providing the rTNF-{alpha}. This study was supported by an NIH grant HL56556, as part of a Specialized Center of Research in Pulmonary Fibrosis.

Received in original form April 12, 2002

Received in final form June 13, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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