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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 141-150, 2002
© 2002 American Thoracic Society

Differences in the Fibrogenic Response after Transfer of Active Transforming Growth Factor-ß1 Gene to Lungs of "Fibrosis-prone" and "Fibrosis-resistant" Mouse Strains

Martin Kolb, Philippe Bonniaud, Tom Galt, Patricia J. Sime, Margaret M. Kelly, Peter J. Margetts and Jack Gauldie

Department of Pathology and Molecular Medicine and Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada; Medizinische Klinik, Julius-Maximilians-Universität, Würzburg, Germany; Service de Pneumologie et Réanimation Respiratoire, CHU Dijon et Université de Bourgogne, France; and Department of Medicine, University of Rochester Medical School, Rochester, New York

Address correspondence to: Jack Gauldie, Ph.D., Department of Pathology and Molecular Medicine and Centre for Gene Therapeutics, McMaster University, 1200 Main Street West, Hamilton, ON, L8N 3Z5 Canada. E-mail: gauldie{at}mcmaster.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium, resulting in impaired lung function and respiratory failure. Investigation of the differences in individual susceptibility to the development of fibrosis may help to detect patients that are at risk to fibrosis when exposed to fibrogenic stimuli. In this study we used adenoviral gene transfer to transiently expose a fibrosis-prone (C57BL/6) and a fibrosis-resistant (Balb/c) mouse strain to high levels of active transforming growth factor (TGF)-ß1, a key profibrotic cytokine. Balb/c mice developed significantly less fibrosis compared with C57BL/6 mice in response to active TGF-ß1 despite higher levels of the transgene protein in the lung. This was not due to a general unresponsiveness of cells to TGF-ß1, because primary fibroblasts of both strains increased collagen synthesis upon stimulation with TGF-ß1 in vitro to the same degree. However, TGF-ß1 induced a strong upregulation of tissue inhibitor of metalloprotease-1 gene in pulmonary fibroblasts as well as in lungs of C57BL/6 mice, in contrast to a weak induction in Balb/c mice. These findings suggest that the differences in susceptibility to pulmonary fibrosis are downstream from TGF-ß1 and that fibrosis-prone individuals may have an altered collagen metabolism in the lungs that is balanced toward a "nondegrading" environment.

Abbreviations: acute respiratory distress syndrome, ARDS • bronchoalveolar lavage, BAL • bronchiolitis obliterans organizing pneumonia, BOOP • extracellular matrix, ECM • interleukin-1 receptor antagonist, IL-1RA • matrix metalloprotease, MMP • nonspecific interstitial pneumonia, NSIP • phosphate-buffered saline, PBS • transforming growth factor ß1, TGF-ß1 • tissue inhibitor of metalloprotease, TIMP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fibrosis is characterized by the abnormal accumulation of extracellular matrix (ECM) in the interstitium resulting in impaired and excessive tissue repair. This process may be initiated by acute or chronic injury as seen in acute respiratory distress syndrome (ARDS) or in hypersensitivity pneumonitis (1). It can also develop without apparent prominent inflammation such as in idiopathic pulmonary fibrosis–usual interstitial pneumonia (IPF-UIP) (2). In cases of known etiology, the nature and the duration of exposure to the underlying cause have an effect on the severity of pulmonary fibrosis. In addition to exogenous stimuli there is evidence that genetic factors can influence the development of fibrosis. Numerous reports of familial cases of IPF and evidence of association of certain genes with IPF suggest an important inherited component. A recent study showed a higher risk for fibrosing alveolitis in patients with tumor necrosis factor (TNF)-{alpha} and interleukin-1 receptor antagonist (IL-1RA) polymorphism, and suggested a role of these cytokine genes in pulmonary fibrosis (3). Another study reported that progression of IPF might be linked to functional polymorphisms of TNF-receptor II and IL-6 genes (4).

The most striking evidence for an important genetic component to fibrosis derives from studying disease models in various animal species and strains. C57BL/6 and CBA mice respond to stimuli such as bleomycin, asbestos, silica, or irradiation with marked fibrosis and are considered to be "fibrosis-prone." Balb/c and C3H/He mice develop very little fibrosis when exposed to these agents and are "fibrosis-resistant." 129 mice and mice crossbred between C57BL/6 and Balb/c are intermediate responders (5, 6). Different factors might contribute to this observation. It has been shown that fibrosis-resistant mice have lower levels of certain chemokines (lymphotactin, RANTES, monocyte chemotactic protein-1, IP-10) (7), and transforming growth factor ß (TGF-ß) (8–11) in lung tissue following irradiation or bleomycin compared with fibrosis-prone animals.

TGF-ß is a key profibrotic cytokine that promotes myofibroblast differentiation, enhances synthesis of collagen and other matrix components, and reduces collagen degradation by downregulation of matrix metalloproteases (MMPs) and upregulation of tissue inhibitors of metalloproteases (TIMPs) (12, 13). We have previously shown that transient overexpression of active TGF-ß1 in the rat lung using adenoviral gene transfer induces severe and progressive pulmonary fibrosis (14). In C57BL/6 mice, overexpression of active TGF-ß1 induced similar fibrotic responses in a recently published study (15).

The purpose of the current study was to compare the response of fibrosis-resistant mice (Balb/c) and fibrosis-prone mice (C57BL/6) to the overexpression of active TGF-ß1 using adenoviral gene transfer. We report here that even in the presence of high local concentrations of active TGF-ß1, Balb/c mice develop a substantially weaker pulmonary fibrotic response as compared with C57BL/6. This is not due to an inability of Balb/c fibroblasts to respond to TGF-ß1, because lung fibroblasts from both strains showed a similar increase of collagen synthesis when stimulated with TGF-ß1 in vitro. TGF-ß1 induced a strong upregulation of TIMP-1 gene in C57BL/6 derived fibroblasts in vitro and in C57BL/6 lungs in vivo in contrast to weak induction in Balb/c fibroblasts and lungs, suggesting that the differences in degree of tissue fibrosis might be in part due to altered extracellular collagen metabolism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant Adenovirus
For the current study, we constructed a replication-deficient adenovector by cloning a mutant TGF-ß1 gene into a shuttle vector with a human cytomegalovirus promoter and cotransfection with a virus rescuing vector (14). The gene had mutations of cysteine to serine at positions 223 and 225, rendering the expressed TGF-ß1 biologically active. The resulting replication-deficient virus AdTGF-ß1223/225 was amplified and purified by CsCl gradient centrifugation and PD-10 Sephadex chromatography, and finally plaque titered on 293 cells. The construction of adenoviral vectors is described in detail elsewhere (16). Control vectors (AdDL70) with no insert in the E1 region were produced in the same way.

Animal Treatment
Six-week-old female C57BL/6 and Balb/c mice were obtained from Charles River Laboratories (Montreal, PQ, Canada) and housed under specific pathogen-free conditions. Rodent laboratory food and water was provided ad libitum. The animals were treated in accordance to the guidelines of the Canadian Council of Animal Care. All animal procedures were performed with inhalation anesthesia with isoflurane (MTC Pharmaceuticals, Cambridge, ON, Canada). Three different doses of AdTGF-ß223/225 in phosphate-buffered saline (PBS) at a total volume of 25 µl were administered by intranasal injection: 5 x 107, 1 x 108, and 5 x 108 pfu, respectively. In one group of animals, 5 x 108 pfu of AdTGF-ß223/225 was delivered by intratracheal injection to adjust for strain differences in the ability to aspirate fluids into the lungs following intranasal administration. Control animals of both strains received intranasal AdDL70 (5 x 108 pfu in 25 µl PBS) or PBS only. Mice were killed by abdominal aortic bleeding at Days 3, 7, and 28 after injection of adenovectors (4–6 mice per group).

Bronchoalveolar Lavage
After opening the chest cavity, the lungs were removed and rinsed with PBS. Bronchoalveolar lavage (BAL) was performed as described previously (17). A total of 0.6 ml PBS was injected intratracheally and retrieved. The fluid was centrifuged at 1,500 rpm for 10 min and the supernatant removed for determination of TGF-ß1. Samples were stored at -70°C. BAL cells were counted using a hemocytometer, centrifuged in a cytospin and stained for differential cytology (Hema3-solution, Biochemical Sciences Inc., Swedesboro, NJ).

The right main bronchus was tied and the right lung removed, rinsed in PBS again, and frozen immediately in liquid nitrogen, and then stored at -70°C for hydroxyproline determination. The left lung was inflated with and fixed in 10% formalin for histologic examination.

Determination of TGF-ß Levels in BAL
Total TGF-ß1 was determined after acid activation using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), performed according to the recommendations of the manufacturer. The level of active TGF-ß1 was measured using the same assay without acid activation. The sensitivity of the assay is 7 pg/ml.

Cell Lines from Primary Lung Fibroblasts
The lungs of untreated mice (Balb/c and C57BL/6) were removed under sterile conditions after exsanguination of the animals. The pulmonary vasculature was perfused blood free with 10 ml PBS through the right ventricular cavity. Large bronchi and vessels were removed, the lung parenchyma was sliced into small portions (~ 2–3 mm3) and plated onto culture dishes. Eagle's minimum essential medium containing 10% fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin was added and the tissue was left for 3 d without further handling to ensure attachment to the bottom. After 5–7 d stromal cells were seen to be growing out of the tissue and cells were grown to confluence. Cells at passage 5–10 were used for the experiments.

Collagen Synthesis Assay
For analysis of collagen synthesis we used L-2,3-3H-proline (NEN Life Sciences, Boston, MA) and an incorporation assay as described previously (18). Equal numbers of lung fibroblasts of each strain (5 x 104 cells/well) were grown in 12-well plates and after reaching 90% confluence starved in serum-free medium supplemented with 50 µg/ml ascorbic acid. Cells were exposed to 0, 0.5, and 1.0 ng/ml recombinant human TGF-ß1 (R&D Systems) in serum-free medium (plus 50 µg/ml ascorbic acid) for 72 h. Collagen and total protein was determined in extracellular matrix in the cell layer. Briefly, supernatant was removed and cells were washed with TCP buffer (50 mM Tris-Cl, 1 mM CaCl2, 1 mM proline, pH 7.5). Fibroblasts were then lysed with 25 mM NH4OH and fixed with 75% ice-cold ethanol. After two additional washes with TCP buffer, cell lysates and associated ECM were incubated for 4 h with type-III collagenase (50 units/well; Sigma Chemicals, Oakville, ON, Canada) in collagenase buffer (50 mM Tris-Cl, 5 mM CaCl2, 62.5 mM N-ethylmaleimide in methanol, pH 7.5) at 37°C. Separate wells were exposed to collagenase buffer without collagenase. The amount of released 3H-proline after the incubation period was measured by a liquid scintillation counter (Rackbeta; LKB, San Francisco, CA). The difference in counts between collagenase-digested and collagenase-free samples reflects the amount of collagen in the specific cell layer. The exact procedure and equations for calculation of results are described elsewhere (18).

In Vitro Expression of TIMP-1 mRNA in Fibroblasts
Cells were grown to 80–90% confluence in 75 cm2 flasks. They were starved overnight in serum-free medium and then exposed to 0, 1.0, or 5.0 ng/ml recombinant human TGF-ß1 (R&D Systems) in serum-free medium. After 24 h cells were lysed using Trizol reagent (Gibco Life Technologies, Burlington, ON, Canada). Chloroform was added and the samples centrifuged at 3,000 rpm for 30 min. Total RNA was precipitated with isopropanol and dissolved in RNAse-free water. Northern blot technique was used to detect mRNA specific for murine TIMP-1 in treated cells. Twenty micrograms of total RNA extracts was separated on a 1% formaldehyde gel and transferred to a nylon membrane (ICN Pharmaceuticals, Montreal, PQ, Canada). Blots were hybridized with {gamma}-32P-adenosine triphosphate labeled antisense oligonucleotides corresponding to base pairs 164–191 of mouse TIMP-1 cDNA (5'-CTTATAAC GCTGGT ATAAGGTGGTCTCG-3') (Mobix; McMaster University, ON, Canada), stringently washed, and exposed to film for 5 d (Kodak XAR, Rochester, NY). Blots were analyzed by densitometry using NIH software (Scion Image; Scion Corporation, Frederick, MD).

In Vivo Expression of TIMP-1 mRNA in Lungs
For analysis of TIMP-1 in lungs, three mice of each strain were injected with 5 x 108 pfu AdTGF-ß223/225 and killed at Day 3. Blood-free frozen lung samples were homogenized in Trizol reagent and RNA was extracted. Reverse transcription was performed following DNase treatment by Super Script preamplification system, following the protocol of the supplier (Gibco Life Technologies). The following probes and primers were used: probe for murine TIMP-1 FAMCCG GTA CGC CTA CAC CCC AGT CAT GTAMRA (Applied Biosystems, Foster City, CA), forward primer 5' GTG GGA AAT GCC GCA GAT 3', reverse primer 5' GGG CAT ATC CAC AGA GGC TTT 3' (both Mobix; McMaster University) and rodent glyceraldehyde-3-phosphate dehydrogenase control reagents (VIC probe; Applied Biosystems). Quantitative PCR was initiated by adding 12.5 µg of TaqMan Universal PCR Master Mix (Applied Biosystems), 5.25 µg of nuclease-free water (Ambion RNA Diagnostics, Austin, TX), 1 µg of forward and reverse primers, 5 µg of probe, and 5 µg of the RT product. Water and RNA that had not been subjected to the RT step were used as negative control. Forty cycles were performed with the ABI PRISM 7,700 Sequence Detector.

Histology
After fixation in 10% buffered formalin for 24 h, a longitudinal section of the lung was paraffin-embedded, sectioned, and stained with hematoxylin and eosin and Masson-Trichrome.

Hydroxyproline Assay
Frozen lung samples were homogenized in 5 ml deionized water. One milliliter of the homogenate was hydrolyzed in 2 ml 6 N HCl for 16 h at 110°C. Hydroxyproline content was determined by a colorimetric assay described previously (19). The results were calculated as µg hydroxyproline per mg wet lung weight using hydroxyproline standards (Sigma Chemicals).

Statistical Analysis
Data are shown as mean ± standard error of the mean . For evaluation of group differences the Student's t test was used assuming unequal variances. A P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-ß1 Level in BAL Fluid after Injection of AdTGFß223/225
TGF-ß1 in BAL fluid was determined 3 and 7 d after intranasal injection of AdTGF-ß223/225. In Balb/c mice concentration of active TGF-ß1 peaked by Day 3 at 3.49 ± 0.52 ng/ml; total TGF-ß1 was 18.45 ± 1.38 ng/ml. By Day 7, levels of both active and total TGF-ß1 were similar to those of Day 3 (see Figure 1A) . In contrast, TGF-ß1 concentration in BAL fluid of C57BL/6 mice was significantly lower than in Balb/c mice and was maximal 7 d after intranasal injection at 0.76 ± 0.28 ng/ml active (P < 0.05) and 6.26 ± 1.73 ng/ml total (P < 0.0001). A low level of total TGF-ß1 was detected in animals of both strains treated with empty control vector AdDL70 (< 0.01 ng/ml, see Figure 1A). Active TGF-ß was at the lower detection limit of the assay in control vector–treated mice. By Day 28, TGF-ß1 levels were all at the lower level of detection in both strains of mice and in all treatment groups (< 0.1 ng/ml).




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  		Figure 1. (A) Level of total and active TGF-ß1 in BAL fluid after intranasal injection of AdTGF-ß1223/225 or control vector AdDL70 in Balb/c mice (blank bars) and C57BL/6 mice (solid bars). (B) TGF-ß1 in BAL fluid after intratracheal injection of AdTGF-ß1223/225. AdTGF-ß1223/225 versus AdDL70, P < 0.0001, in both strains for total and active TGF-ß1, **P < 0.001, *P < 0.05.

 
In a separate group of animals AdTGFß223/225 was administered by intratracheal injection (n = 5). Again, levels of active and total TGF-ß1 3 d after injection were significantly higher in Balb/c compared with C57BL/6 mice (active TGF-ß1: 4.49 ± 1.32 versus 0.45 ± 0.15 ng/ml, P < 0.05, see Figure 1B).

Differential Cytology in BAL Fluid after Injection of AdTGF-ß223/225
All animals treated with adenovectors showed a significant increase in total cell numbers in BAL fluid 3 and 7 d after injection compared with PBS control mice (see Table 1) . Animals injected with AdTGF-ß223/225 had higher total cell counts by Day 7 than AdDL70 controls. At Day 3 there were more cells present in BAL fluid of Balb/c than in C57BL/6 mice, but the differences were not significant by Day 7. The major cell type present was the alveolar macrophage, often with signs of activation (enlarged nuclei, cytoplasm, cytoplasmic vacuoles). No differences in cell differential counts were observed between AdTGF-ß223/225 and AdDL70 treatment and between the two mouse strains.


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  		TABLE 1 Total cells and differential counts in BAL fluid from Balb/c and C57BL/6 mice following administration of AdTGF-ß1223/225 or control vector AdDL70 (intermediate dose, 1 x 108 pfu, cells per ml fluid)

 
Inflammatory and Fibrotic Tissue Response in the Lung after Injection of AdTGF-ß223/225
The lungs of mice from both strains showed mild to moderate inflammatory reactions in peribronchial and perivascular areas 7 d after injection of AdTGF-ß223/225 at a time when the TGF-ß levels were falling to normal. As in BAL fluid, macrophages were the predominant inflammatory cell type, with some lymphocytes and only a few granulocytes present. The extent of acute inflammation was only moderately more than in control animals treated with empty adenovector AdDL70. No difference was observed between the lungs of Balb/c and C57BL/6 mice (Figures 2A–2C) . There were no differences in the inflammatory responses between animals injected by the intranasal or intratracheal route (data not shown).



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  		Figure 2. (A) Lungs of C57BL/6 mice treated with empty control vector AdDL70 (1 x 108 pfu) showed almost normal histology 7 d after treatment (identical picture in lungs of Balb/c mice, not shown). Mild peribronchial inflammation in mice treated with AdTGF-ß1223/225 (intermediate dose, 1 x 108 pfu) after 7 d, no difference between Balb/c (B) and C57BL/6 (C) mice. All sections H&E, magnification x25.

 
The fibrotic tissue response by Day 28 showed marked differences between Balb/c and C57BL/6 mice. The lungs of Balb/c mice exposed to the lower dose of AdTGF-ß223/225 were not distinguishable from animals injected with empty control vector AdDL70; the intermediate dose resulted in a slight peribronchial thickening (Figures 3A and 3B) . Only the highest dose induced a moderate fibrotic reaction in the lungs, mainly in peribronchial tissue (Figure 3C). In contrast, C57BL/6 mice showed profound pulmonary fibrosis after administration of intermediate doses of AdTGF-ß223/225 and extensive fibrotic changes at the highest dose (Figures 3D–3F). Morphometric analysis of tissue specimen was not performed in this study, because we recently demonstrated a significant correlation between hydroxyproline analysis and a semiquantitative histologic fibrosis score (17).



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  		Figure 3. Lung histology of lungs 28 d after administration of adenovectors in Balb/c (A–C) and C57BL/6 mice (D–F ). Normal lung architecture in mice treated with empty control vector AdDL70 (A and D). Balb/c mice showed only mild peribronchial collagen accumulation following the intermediate dose of AdTGF-ß1223/225, whereas C57BL/6 mice had substantial interstitial fibrosis after the same dose (B and E ). The highest dose of AdTGF-ß1223/225 induced a fibrotic response in both strains; however, the response was more severe in C57BL/6 compared with Balb/c mice (C and F ). All sections Masson's Trichrome, magnification x25.

 
Tissue fibrosis was quantified by analysis of lung hydroxyproline concentration (Figure 4) . Balb/c mice had increased amounts after injection of the highest dose of AdTGF-ß223/225, 1.83 ± 0.16, compared with 1.38 ± 0.11 µg/mg in AdDL70 control (P < 0.05). Hydroxyproline concentration in the lungs of C57BL/6 mice 28 d after injection of the same dose was significantly higher than in AdDL70 controls (2.40 ± 0.11 versus 1.32 ± 0.09 µg/mg, P < 0.0001) and in Balb/c mice (P < 0.01).



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  		Figure 4. Lung hydroxyproline concentration of mouse lungs 28 d after administration of AdTGF-ß1223/225 or control vector AdDL70. Blank bars, Balb/c; and solid bars, C57BL/6. **P < 0.001, *P < 0.05.

 
Collagen Synthesis of Pulmonary Fibroblasts In Vitro
Pulmonary fibroblasts of both mouse strains synthesized a baseline amount of collagen in vitro. Interestingly, Balb/c fibroblasts had a higher baseline production of collagen compared with C57BL/6 fibroblasts (incorporation of 474 ± 74 compared with 229 ± 51 counts per minute in 3H-proline assay over a period of 72 h), although an equivalent number of cells were plated. Collagen synthesis was significantly increased in fibroblast cell lines when recombinant human TGF-ß1 was added to serum-free medium and no difference was observed in the increase between the different strains. 3H-proline incorporation was 1,234 ± 163 cpm in Balb/c and 699 ± 71 cpm in C57BL/6 mice after exposure to 1.0 ng/ml rhTGF-ß1 for 72 h, representing a 2.3- and 3.1-fold increase, respectively, in collagen production over control (P < 0.01, see Figure 5) .



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  		Figure 5. Collagen synthesis of primary lung fibroblasts derived from Balb/c (blank bars) and C57BL/6 mice (solid bars) after stimulation with rhTGF-ß1. Shown is the increase of 3H-proline incorporation into collagen above baseline synthesis (baseline = 1). *P < 0.01 versus baseline.

 
TIMP-1 mRNA in Pulmonary Fibroblast Cell Lines
TIMP-1 is a protease inhibitor with an important role in collagen turnover and is partly regulated by TGF-ß1. Pulmonary fibroblasts derived from C57BL/6 mice showed a 1.4- and 1.8-fold upregulation of TIMP-1 mRNA following stimulation with 1.0 and 5.0 ng/ml rhTGF-ß1 for 24 h in serum-free medium (see Figure 6) . In contrast, fibroblasts from Balb/c mice did not show any significant increase in TIMP-1 mRNA in response to rhTGF-ß1 stimulation (P = 0.04 versus C57BL/6).



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  		Figure 6. Northern blot of TIMP-1 mRNA in cultured lung fibroblasts from Balb/c and C57BL/6 mice after 24 h stimulation with rhTGF-ß1. Increase of density based on unstimulated cells (density = 1) represent the average (± SEM) of five experiments with cell lines derived from three different mice per strain. *P < 0.05 versus same dose in Balb/c fibroblasts.

 
TIMP-1 mRNA in Lungs after Injection of AdTGF-ß223/225
To determine whether the in vitro findings with TGF-ß1 stimulation of fibroblasts is seen in vivo, we examined TIMP-1 gene expression in the lung by Taqman analysis after intranasal AdTGF-ß1 administration. To ensure the distribution of vector does not influence the outcome of total tissue examination, 3 Balb/c and 3 C57BL/6 mice were killed at 3 d after vector administration, and the left and right lung were removed and treated separately after PBS perfusion to remove blood components from the tissue. The samples were individually examined for TIMP-1 mRNA content. Figure 7 shows TIMP-1 gene expression is consistent between left and right lung and that there are significant differences between C57BL/6 and Balb/c mice (P < 0.001). A 2-fold increase in TIMP-1 mRNA is seen in the C57BL/6 mice compared with Balb/c.




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  		Figure 7. TIMP-1 gene mRNA analysis by Taqman. Balb/c and C57BL/6 mice (n = 3) were treated with AdTGF-ß i.n. (5 x 108 pfu) and left and right lung removed at 3 d. mRNA was isolated from the separate samples and examined for TIMP-1 gene expression. (A) Individual data points from separate animals and separate left (dotted) and right (hatched) lungs. (B) Collective data for Balb/c and C57BL/6 mice with separate left (dotted) and right (hatched) lung. (C) Collective data for total lung Balb/c (blank) and C57BL/6 mice (solid). *P < 0.01 C57BL/6 versus Balb/c. (Continued on next page.)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fibrosis may be considered as pathologic wound repair following acute or chronic injury with excessive accumulation of ECM. Not only exogenous but also genetic factors play a major role in the initiation and development of a fibrotic tissue response. Numerous reports suggest an important inherited component in IPF and other fibroproliferative disorders (2–4, 20). Investigation of disease patterns in different inbred mouse strains can provide valuable information about the reasons why certain individuals are susceptible to fibrogenic stimuli whereas others are not.

A variety of cytokines and growth factors are involved in the pathogenesis of tissue fibrosis. TGF-ß1 is a key profibrotic cytokine that stimulates collagen accumulation in several ways. It induces the differentiation of fibroblasts into myofibroblasts, increases collagen synthesis by these cells and reduces collagenase activity by downregulation of MMP mRNA and upregulation of TIMP mRNA (12, 13).

Here we report differences in the fibrogenic response between a fibrosis-prone and a fibrosis-resistant mouse strain following transient overexpression of active TGF-ß1 using adenoviral gene transfer. Pulmonary fibrosis is commonly induced in animal models based on C57BL/6 mice and these mice respond to agents such as bleomycin, asbestos, and silica, or to irradiation with acute alveolar or interstitial inflammation followed by rapidly developing fibrosis. In this study we generated fibrosis by overexpression of a single profibrotic cytokine that induced a transient and moderate inflammatory response in peribronchial areas but no major acute tissue destruction as seen with other initiating agents.

The data seen in BAL cell recoveries reflect the known early proinflammatory properties of adenovirus vectors and the later (7 d) immune responses associated with these vectors. However, differences seen between C57BL/6 and Balb/c mice do not immediately suggest this could account for the differences in susceptibility to fibrogenesis. Indeed, the administration of bleomycin to both strains induces a marked inflammatory response with abundant neutrophils, but still maintains the strain difference in susceptibility. We observed severe fibrotic tissue reactions when C57BL/6 mice were exposed to high local concentrations of active TGF-ß1 in the lung for a period up to 7 d, similar to what we described in a recently published study (15). In contrast, Balb/c mice, a strain that is thought to be resistant to fibrogenic stimuli, showed a significantly lesser degree of pulmonary fibrosis after injection of the same dose of AdTGF-ß223/225 despite 10-fold higher levels of transgene protein detected in BAL fluid. We cannot explain why the levels of transgene protein were so different between the animal strains, but assume that strain-specific factors in the ability to utilize the human cytomegalovirus promoter of the adenoviral construct may play an important role. This aspect is worthy of further investigation, but does not appear to contribute to the strain-specific susceptibility of fibrosis. Moreover, the trend toward a more pronounced inflammatory response in Balb/c compared with C57BL/6 mice could have some impact on fibrogenesis and has to be followed up.

Our results are discordant with the hypothesis that the inability of Balb/c mice to develop pulmonary fibrosis is due to reduced TGF-ß upregulation in the tissue following injury. This hypothesis has been suggested after several studies demonstrated strain differences in TGF-ß expression upon stimulation. Bleomycin treatment increased TGF-ß mRNA in fibrosis-prone C57BL/6 but not in fibrosis-resistant Balb/c mice (10, 11). Similar findings were reported when C57BL/6 were irradiated and showed an upregulation of TGF-ß mRNA after 8 wk but C3H/He mice, a resistant strain, did not (9). In a study using immunohistochemistry, C57BL/6 mice showed significant presence of active TGF-ß in early fibrotic lesions following irradiation and subsequently developed fibrosis, whereas C3HeB/Fe mice had only minimal active TGF-ß in the lung tissue and were resistant to fibrosis (8). In contrast, our data show that Balb/c mice do not develop severe pulmonary fibrosis even in the presence of abundant active TGF-ß1 in the lungs. One possible reason would be that the two mouse strains respond differently to TGF-ß. Lasky and coworkers have reported that C57BL/6 but not Balb/c mice expressed CTGF mRNA in the lung following bleomycin instillation (21). They suggested that the difference in the ability of the two strains to respond to TGF-ß is because bleomycin-induced lung fibrosis is TGF-ß-mediated and CTGF is a profibrotic cytokine downstream of TGF-ß. They also demonstrated that the differences are not due to the inability of pulmonary cells to respond to TGF-ß, because primary lung fibroblasts from both strains showed a comparable upregulation of CTGF in vitro when exposed to TGF-ß. We isolated lung fibroblasts from both strains and analyzed them for collagen synthesis upon stimulation in vitro with recombinant TGF-ß1. Interestingly, both strains of murine cells responded to addition of TGF-ß in the same way, with an approximate 3-fold increase of collagen production. These data and the observations of Lasky and coworkers (21) confirm that the fibrogenic effector cells of a fibrosis-prone and -resistant strain are indeed responsive to TGF-ß, and suggest that the differences in fibrogenesis are more likely caused by factors downstream of TGF-ß.

Extracellular matrix is not an inert substance, but is turned over at a considerable rate every day, and there is a subtle equilibrium between synthesis and degradation. It is estimated that 10% of the total pulmonary collagen is degraded and newly synthesized every day (22). Degradation of collagen is a complex process involving a group of zinc-containing proteinases, the MMPs (23). At least 18 members of the MMP family are known and divided into four major subgroups depending on substrate specificity. Interstitial collagenases degrade mainly fibrillar collagen, stromelysins digest proteoglycans and glycoproteins, gelatinases degrade basement membrane type IV collagen and denatured collagen, and membrane-type metalloproteases have a broader substrate affinity (24). MMP activity is closely regulated through endogenous inhibitors, mainly through TIMPs (23). Both MMPs and TIMP can be induced or inhibited by different cytokines. TGF-ß1 is known to downregulate MMP-1 and upregulate TIMP-1 expression (12, 25), thus supporting a "nondegrading" environment. In a model of experimental silicosis in rats, early granulomas showed both substantial TIMP and MMP expression, whereas late and fibrotic granulomas were positive for TIMP only and not for MMP (24). In humans, it has been demonstrated that TIMP gene expression is at higher levels than MMPs in the interstitium of lungs of patients with UIP (26), and that myofibroblasts in UIP but not in nonspecific interstitial pneumonia (NSIP) or bronchiolitis obliterans organizing pneumonia (BOOP) tissue express TIMP-2 (27). Patients with asthma and chronic bronchitis with signs of airway remodeling have higher TIMP-1 and a reduced MMP-9/TIMP-1 ratio in sputum (28), supporting a role of collagenase/collagenase-inhibitor imbalance in peribronchial ECM accumulation. Further, in a model of pulmonary hypertension in rats, transient overexpression of TIMP-1 using adenoviral gene transfer aggravated pulmonary arterial pressure compared with control animals and increased periadventitial collagen deposition (29).

In this study, we found differences in collagen accumulation in the lungs of Balb/c and C57BL/6 mice following overexpression of active TGF-ß1. In vitro, pulmonary fibroblasts of either strain increased collagen production when they were stimulated with recombinant TGF-ß1, showing that fibroblasts of both are responsive to TGF-ß in terms of collagen synthesis. To investigate whether the differences in matrix accumulation may be caused by altered degradation of collagen, we analyzed mRNA for TIMP-1, the most important regulator of MMP and collagenolytic activities. TIMP-1 expression was increased significantly in primary fibroblasts of C57BL/6 following stimulation with TGF-ß. This finding was expected and has been previously described (25). Surprisingly, primary fibroblasts derived from Balb/c mice did not show significantly increased TIMP-1 mRNA upon stimulation with TGF-ß. We found consistent data regarding TIMP-1 gene expression in vivo with C57BL/6 mice expressing TIMP-1 mRNA at levels greater than 2-fold above those seen in Balb/c mice after AdTGF-ß1 administration. Moreover, the data show that the intranasal route of administration results in equal distribution of effects throughout the lung and that selection of right or left lung is no different than taking whole lung for mRNA isolation. In addition, differences were seen at the individual lung as well as individual mouse level. A difference of 2-fold in mRNA expression of TIMP-1 in vivo agrees with the in vitro findings with isolated fibroblast lines and provides evidence for the hypothesis that strain differences in susceptibility to fibrosis may be due to differences in response to TGF-ß stimulation of TIMP genes and can result in a preferentially noncollagenolytic microenvironment in lungs of C57BL/6 compared with Balb/c mice, resulting in excess and prolonged collagen deposition. In this context it has to be considered that TIMPs do not only have anticollagenolytic activities, but also promote growth and proliferation of various cells, including fibroblasts, thus further promoting profibrotic mechanisms (30).

Other data shown in rodent models of liver fibrosis agree with the supposition that excess inhibition of MMPs through TIMP gene expression leads to fibrogenesis, and that during the reversible stage of experimental liver fibrosis in the rat, there is a decrease in TIMP gene expression, increase in MMP activity, and a microenvironment that favors collagen lysis and mobilization (31, 32). This would suggest that strain susceptibility to fibrosis could occur in a similar manner and that our findings both in vitro and in vivo support such a supposition.

The experiments performed here focus on the first and fourth week following initiation of the fibrotic response. Although results from our previous work showed a continuous development and progression of fibrosis in both rats and C57BL/6 mice (14, 15), it cannot be excluded that the time course of remodeling after overexpression of TGF-ß1 differs between Balb/c and C57BL/6 mice and may contribute in part to our observations. However, we find a consistent lack of experimental evidence of an underlying low-grade fibrogenesis in the fibrosis-resistant Balb/c mouse.

In summary, we have demonstrated that a fibrosis-prone and a fibrosis-resistant mouse strain respond differently to transient overexpression of active TGF-ß1 in the lung. Whereas C57BL/6 mice develop sustained pulmonary fibrosis, Balb/c mice appear to be relatively resistant to fibrosis even in the presence of abundant active TGF-ß1. We showed that lung fibroblasts from both strains have similar ability to synthesize collagen upon stimulation with TGF-ß1, but show marked differences in the regulation of expression of TIMP-1, both in vitro and in vivo. Our findings suggest that fibrosis in susceptible mouse strains might be the result of prevalence of a noncollagenolytic, nondegrading microenvironment in the lungs. Further in vivo and in vitro studies have to elicit the differences in collagen metabolism between fibrosis-prone and -resistant mice and could increase the knowledge about individual susceptibility to excessive wound repair and fibrosis.


   Acknowledgments
 
The authors thank Jane Ann Schroeder, Xueya Feng, Mary Jo Smith, and Duncan Chong for outstanding technical help. M.K. is supported by Deutsche Krebshilfe, P.B. by the Bourses Lavoisier, P.J.S. by the Wilmot Foundation, and P.J.M. by Kidney Foundation Canada. This study was also supported by CIHR Canada, Hamilton Health Sciences, and St. Joseph's Healthcare.

Received in original form July 12, 2001

Received in final form March 4, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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