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Am J Physiol Lung Cell Mol Physiol 294: L572-L581, 2008. First published January 4, 2008; doi:10.1152/ajplung.00291.2007
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Epithelial expression of TIMP-1 does not alter sensitivity to bleomycin-induced lung injury in C57BL/6 mice

Cheryl L. Fattman,1,* Federica Gambelli,1,* Gary Hoyle,2 Bruce R. Pitt,1 and Luis A. Ortiz1

1Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania; and 2Department of Environmental and Occupational Health Sciences, School of Public Health and Information Sciences, University of Louisville, Louisville, Kentucky

Submitted 25 July 2007 ; accepted in final form 2 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Matrix metalloproteinases (MMPs) are mediators of lung injury, and their activity has been associated with the development of pulmonary fibrosis. To understand how MMPs regulate the development of pulmonary fibrosis, we examined MMP expression in two strains of mice with differing sensitivities to the fibrosis-inducing drug bleomycin. After a single intratracheal injection of the drug, bleomycin-sensitive C57BL/6 mice showed increased expression for MMPs (-2, -7, -9, -13) at both 7 and 14 days posttreatment compared with the bleomycin-resistant BALB/c strain. In addition, TIMP-1, an endogenous inhibitor of MMPs, was upregulated in the lungs of C57BL/6 mice but not BALB/c mice. We designed two strategies to decrease MMP expression to potentially decrease sensitivity of C57BL/6 mice: 1) we engineered C57BL/6 mice that overexpressed TIMP-1 in their lungs via surfactant protein C (SP-C) promoter; and 2) we inhibited expression of MMPs independent of TIMP-1 by knocking out metallothionein (MT), a critical zinc binding protein. SP-C-TIMP-1 mice reduced MMP expression in response to bleomycin. However, they were equally sensitive to bleomycin as their wild-type counterparts, displaying similar levels of hydroxyproline in the lung tissue. MT null mice displayed decreased lung activity of MMPs with no change in TIMP-1. Nonetheless, there was no difference between the MT null and wild-type control littermates with regards to any of the lung injury parameters measured. We conclude that although TIMP-1 expression is differentially regulated in fibrosis-sensitive and fibrosis-resistant strains, epithelial overexpression of TIMP-1 does not appear to substantially alter fibrotic lung disease in mice.

matrix metalloproteinase; pulmonary fibrosis; metallothionein

INTERSTITIAL LUNG DISEASES (ILDs) are a heterogeneous group of over 150 entities characterized anatomically by epithelial injury, fibroblast proliferation, and the expansion of the ECM of the lung. Among these diseases, idiopathic pulmonary fibrosis (IPF) is the most frequent and lethal. Median survival of patients with IPF is 3–5 yr despite treatment with anti-inflammatory, antifibrotic, or immunosuppressive therapy (39). These phenomena are indicative of an aberrant wound healing process that eventually leads to fibrosis formation and compromised lung function. Although the precise mechanisms involved in the formation and progression of pulmonary fibrosis are poorly understood, imbalances in key regulatory elements such as oxidants/antioxidants or proteases/antiproteases are believed to play an important role in the pathology of this disease (22, 37).

The ECM of the lung is comprised of a variety of molecules including collagens, elastin, fibronectin, and proteoglycans. The ECM structure is tightly controlled by regulating the turnover of these molecules, with the goal of providing stable scaffolding for the proper growth and function of the numerous cell types within the lung. This scaffolding is particularly important in the context of wound healing as the proper assembly and disassembly of a provisional ECM is key to controlling the inflammation and reepithelialization processes at the wound site. In pathological conditions characterized by excessive ECM remodeling, such as ILD, the provisional matrices formed can provide signals that enhance the inflammatory response and epithelial cell proliferation and migration. This, in turn, leads to the expansion of the ECM and permanent matrix remodeling (4). For example, in IPF and animal models of pulmonary fibrosis such as intratracheal administration of bleomycin, aberrant ECM remodeling is widely evident and thought to be a key pathological feature of these diseases (1, 13, 18). Accumulation of ECM in the interstitial and alveolar spaces and disruption of basement membranes occur during progression of IPF and is closely associated with the deregulation of a number of enzymes that function in the turnover of ECM components (2, 33).

The matrix metalloproteinases (MMPs) are a large family of enzymes that function in the turnover of ECM proteins and, to a lesser extent, the generation of bioactive peptides. MMPs have been classified in six subgroups based on their substrate specificity: collagenases (MMP-1, MMP-8, and MMP-13), which degrade mainly fibrillar collagen; gelatinases (MMP-2 and MMP-9) that have affinity for basement membrane type IV collagen; matrilysin (MMP-7), which degrades proteoglycans, fibronectin, laminin, gelatins, and elastin; stromelysins; membrane-type MMPs; and other MMPs. MMPs regulate many of the pathological processes active in lung fibrosis including ECM remodeling, basement membrane disruption, apoptosis, and/or cell migration (40). MMPs can be induced by a variety of cellular signals that also play a role in the development or progression of pulmonary fibrosis including TGF-β, TNF-{alpha}, FGFs, and several interleukins (26). In particular, MMP-2 and MMP-9 are strongly expressed in lungs (12, 15, 51) and bronchoalveolar lavage fluid (BALF) of patients with IPF (41).

MMPs are synthesized and secreted as latent zymogens and are usually activated in the pericellular space through cleavage of the propeptide of the enzyme by serine proteases or other activated MMPs. The catalytic site of the MMPs is maintained in an inactive state by the presence of a cysteine-zinc bond in the prodomain. Cleavage of the prodomain or other disruptions of the cysteine-zinc bond activates the enzyme. MMP activity can also be regulated through the binding of tissue-specific inhibitors of the metalloproteinases (TIMPs; Ref. 27). The TIMP family consists of four members (TIMP-1, -2, -3, and -4) that bind to the active site of MMPs in a 1:1 ratio. TIMPs have been shown to stimulate cell proliferation, to induce or inhibit apoptosis, and to stimulate MMPs expression. Therefore, the concept has been advanced that the balance between the activated MMPs and the presence of the inhibitors would determine the degree of tissue remodeling.

In an effort to better elucidate the role of MMPs in pulmonary fibrosis, we took advantage of two mouse strains that have differing sensitivities to bleomycin-induced lung fibrosis: the sensitive C57BL/6 mouse and the resistant BALB/c mouse. Based on the available evidence, we hypothesized that differing levels of MMP activity may, in part, explain the observed difference in strain sensitivity to bleomycin-induced pulmonary fibrosis. Both C57BL/6 and BALB/c mice were exposed to bleomycin, and samples were collected at 7 and 14 days posttreatment. Using gelatin zymography and Western blotting, we found predominant increased activity for MMP-2, -9, and -13 in the lungs and alveolar lavage of bleomycin-treated C57BL/6 mice compared with BALB/c mice. Both mouse strains reacted to bleomycin with increased MMP-7 activity in their lungs. In contrast, increased expression of TIMP-1 following bleomycin exposure was only observed in the lungs of C57BL/6 mice.

Subsequently, we used two genetic strategies, overexpression of TIMP-1 in the lung epithelium or deletion of the zinc regulator protein metallothionein (MT), to determine the effects of inhibiting MMP activity in the lungs of bleomycin-exposed mice. Although the overexpression of the mouse TIMP-1 gene under the control of surfactant protein C (SP-C) promoter was successful in inhibiting activation of MMPs in the lung, it did not alter the inflammatory or collagen deposition in the mouse lung in response to bleomycin. Similarly, MT knockout mice showed decreased activity of MMPs (-2, -7, -9), but not TIMP activity, in their lungs in response to bleomycin. However, compared with their MT-competent littermates, MT-deficient mice did not differ in their lung inflammatory response or the deposition of collagen. We conclude that specific patterns of MMP and TIMP-1 activation characterize the murine strain sensitivity to bleomycin and that measures that promote MMP inhibition in the lung are not sufficient to overcome the sensitivity to fibrogenic agents such as bleomycin.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal exposure and sample collection. All animal experiments were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use committee. Eight- to ten-week-old female mice were given a single 60-µl intratracheal injection of 4 U/kg bleomycin, 0.2 g/kg freshly fractured crystalline silica, or 0.9% saline as previously described (10). Mice were killed at 7 or 14 days postbleomycin treatment as indicated. BALF was obtained, and total protein, total white blood cell counts, and differential counts were obtained as previously described (9). Lung tissue was either flash-frozen and stored at –80°C, dried for hydroxyproline analysis, or inflation-fixed with 10% buffered formalin and paraffin-embedded for histological analysis as previously described (44).

For MMP activity analysis, lung samples were thawed on ice, homogenized, and sonicated in 10 volumes of 50 mM potassium phosphate, pH 7.4, 0.3 M potassium bromide, 3 mM diethylenetriaminepentaacetic acid, and 0.5 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 20,000 g for 20 min, and supernatants were collected. The insoluble fraction remaining after supernatant recovery was extracted in 50 mM Tris·HCl, 150 mM NaCl, and 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer containing 100 µl of E-64 protease inhibitors. CHAPS detergent aids in the extraction of MMPs (50). All homogenates were stored at –80°C until use.

Gelatin zymography. Gelatin zymography was performed as described previously (41, 43). Briefly, equivalent amounts of protein in lung homogenates or BALF were resolved by nonreducing SDS-PAGE through gels containing 10% acrylamide and 0.1% gelatin gels (Invitrogen). Gels were then washed twice, for 20 min each, in 2.5% Triton X-100. Gels were then incubated in 50 mM Tris·HCl, pH 7.8, 5 mM CaCl2, 1 µM ZnCl2, and 1.25% Triton X-100 for 20–24 h and then stained with Coomassie blue R-250 (Fisher Scientific). Clear bands against a blue background indicated MMP activity. Identification of the bands as MMP-9 and MMP-2 were validated by comparison with the purified enzymes. For MMP-7 analysis, samples were resolved in 12.5% SDS gels containing bovine-conditioned medium transferrin (Invitrogen) and heparin as substrate (45). Recombinant human MMP-7 (Chemicon) was used as positive control. For analysis of MMP-13, 10% zymogram gels (Invitrogen) were used. For all experiments, identical gels were incubated in the presence of 20 mM EDTA to determine specificity of the MMP activation.

Western blots. Equivalent protein amounts of lung homogenate were resolved by SDS-PAGE as described previously (11) and blotted onto 0.2-µm polyvinylidene difluoride membranes. An antibody against TIMP-1 (1:500; Santa Cruz Biotechnology) or MMP-7 (1:1,000; Chemicon) and horseradish peroxidase-linked anti-rabbit secondary antibody (1:5,000; Cell Signaling Technology) were used to probe for TIMP-1 and MMP-7 where indicated. Bands of antibody reactivity were visualized by ECL (Amersham Biosciences).

Generation of TIMP-1 transgenic and MT null mice. Transgenic mice expressing TIMP-1 under the control of the human SP-C promoter were generated by microinjection of a SP-C-TIMP-1 DNA construct into fertilized B6SJLF2 mouse eggs as described previously (3). SP-C-TIMP-1 transgenic lines were backcrossed 7 generations to inbred C57BL/6 mice before exposure to bleomycin or silica. Transgenic mice were identified by dot hybridization of tail DNA with a radiolabeled human SP-C probe. Expression of the transgene message was assessed by Northern blot analysis of total lung RNA (16) with a radiolabeled simian virus 40 (SV40) probe that specifically detects the transgene message. TIMP-1 activity in lung lavage fluid was measured by reverse zymographic analysis (29) using gels containing 2.25% gelatin and 160 ng/ml MMP-2 (Oncogene Research).

MT-deficient (null) mice were obtained from The Jackson Laboratory. These mice are on a C57BL/6 background and MT I and MT II genes were deleted by target mutation as previously described (20, 26).

Hydroxyproline assay. Lung tissue was dried and acid-hydrolyzed in sealed, oxygen-purged glass ampoules containing 2 ml of 6 N HCl for 24 h at 110°C and subjected to hydroxyproline quantitation using chloramine T as previously described (10).

Histological scoring. Standard hematoxylin and eosin staining was performed as previously described (10). Slides were scored by a researcher (C. L. Fattman) blinded to treatment groups. Individual fields were examined by light microscopy using a high-powered objective (x400 magnification). Every other field was scored in the entire lung. Fields that contained terminal bronchioles/alveolar tissue in >50% of the fields were scored. Fields not meeting the criteria were not scored. Scoring in each field was based on the percentage of terminal bronchioles/alveolar tissue with interstitial fibrosis according to the following scale: 0 = no fibrosis, 1 = up to 25% of field, 2 = 25–50% of field, 3 = 50–75% of field, and 4 = 75–100% of field. Scores were calculated for each animal, and the group scores were averaged together for statistical comparison.

Statistical analysis. All values were expressed as means ± SD. Differences between treatment groups were measured by Student's t-test or ANOVA with Fisher's protected least significant differences test for pairwise comparison (StatView 4; Abacus Concepts). A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bleomycin induces activation of MMPs in the mouse lung. Previous studies have shown that MMPs are involved in the pathology of pulmonary fibrosis in both human and animal models. Therefore, we characterized the expression of these mediators in the lungs of bleomycin-exposed mice. We compared the expression of MMP-2, -7, -9, and -13 in the lungs (Fig. 1) and BALF (Fig. 1B) of fibrosis-sensitive C57BL/6 and fibrosis-resistant BALB/c mice. Bleomycin caused a time-dependent induction, as detected by gelatin zymography, of MMPs in the lungs of mice from both mouse strains. However, compared with BALB/c mice, lungs of C57BL/6 mice exhibited a greater increase in expression of MMP-2 (68 kDa) and MMP-9 (92 kDa) at 7 days (Fig. 1A, lanes 2 and 4) and 14 days (Fig. 1A, lanes 6 and 8) posttreatment. Similar results were observed in the BALF of these mice (Fig. 1B). Both mouse strains demonstrated an increased expression of MMP-7 (19 kDa) in their lungs in response to bleomycin (Fig. 1C). In contrast, activated MMP-13 (30 kDa) was almost exclusively increased in the lungs of C57BL/6 mice after bleomycin challenge (Fig. 1D).


Figure 1
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  		Fig. 1. Expression of matrix metalloproteinases MMP-9, MMP-2, MMP-7, and MMP-13 in fibrosis-sensitive C57BL/6 (C57) and fibrosis-resistant BALB/c mice. C57BL/6 and BALB/c mice were given a single intratracheal dose of 4 U/kg bleomycin and analyzed at 7 and 14 days posttreatment. Gelatin zymography for MMP-9 and MMP-2 in lung homogenates (A) and bronchoalveolar lavage fluid (BALF; B) was performed as described in MATERIALS AND METHODS. Identification of bands representing MMP-9 and MMP-2 was made by comparison with purified MMP-9 and MMP-2 (+). MMP-9 and latent and active MMP-2 were increased at both time points (B) compared with control (C). C: gelatin zymography for MMP-7 in BALF recovered from these mice. A single band representing MMP-7 is indicated. D: gelatin zymography of BALF for MMP-13 (30-kDa band representing MMP-13 is indicated). Representative gels are shown. STD, standard.

 
TIMP-1 expression is exclusively increased in C57BL/6 mice in response to bleomycin. We also examined the levels of an endogenous MMP inhibitor, TIMP-1, in the lungs (Fig. 2A) and BALF (Fig. 2B) of both C57BL/6 and BALB/c mice. Evidence of TIMP-1 activity was identified, by reverse zymography, in the lungs from bleomycin-treated C57BL/6 but not in BALB/c mice at 7 and 14 days posttreatment (Fig. 2A, lanes 3 and 7). This increased TIMP-1 expression was also observed by Western blotting in the BALF of bleomycin-treated C57BL/6 mice at either time point (Fig. 2B, lanes 2 and 3).


Figure 2
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  		Fig. 2. Levels of tissue-specific inhibitor of metalloproteinase-1 (TIMP-1) in fibrosis-sensitive C57BL/6 and fibrosis-resistant BALB/c mice. A: reverse zymography for TIMP-1 in lung homogenates from C57BL/6 and BALB/c mice was performed as described in MATERIALS AND METHODS. A 28-kDa band representing TIMP-1 is present in the bleomycin-treated (B) C57BL/6 mice but not in control or BALB/c mice (C). B: Western blot analysis of BALF from C57BL/6 and BALB/c mice. TIMP-1 is only present in BALF from bleomycin-treated C57BL/6 mice (lanes 2 and 3). Representative gels are shown. B-7, 7 days postbleomycin; B-14, 14 days postbleomycin.

 
Overexpression of TIMP-1 in mouse lung does not alter the fibrotic response. To further elucidate the role of TIMP-1 in pulmonary fibrosis, we generated a strain of mice that expresses TIMP-1 under the lung-specific SP-C promoter (Fig. 3A). Overexpression of the TIMP-1 transgene was confirmed by Northern blot analysis of total lung RNA using a radiolabeled human SP-C probe (Fig. 3B), and TIMP-1 inhibitory activity was confirmed by reverse zymography (Fig. 3C). Bleomycin challenge induced activation of the SP-C-TIMP-1 transgene, and therefore these SP-C-TIMP-1 mice demonstrated enhanced TIMP-1 expression and activation in their lungs (Fig. 4A) and BALF (Fig. 4B) compared with their nontransgenic littermates. This enhanced TIMP-1 activity observed in the lungs of SP-C-TIMP-1 transgenics was associated with a decreased expression of MMP-2 and -9 (Fig. 4C) in response to bleomycin.


Figure 3
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  		Fig. 3. Construction and verification of TIMP-1-overexpressing mice. A: the mouse TIMP-1 cDNA (8) was ligated downstream from the human surfactant protein C (SP-C) promoter (16). A DNA fragment from simian virus 40 (SV40) that includes an intron and polyadenylation signal was placed downstream from the TIMP-1 cDNA (TIMP-1SV). B: Northern blot analysis of total lung RNA from 4 independent SP-C-TIMP-1 transgenic lines using an SV40 probe specific for the TIMP-1 transgene. One line expressed the transgene at a high level (lane 1), and 1 line expressed the transgene at a lower level (lane 2). In 2 lines, no transgene expression was detected (lanes 3 and 4). No signal was detected in RNA isolated from wild-type mice (lane 5). Levels of 18S RNA are shown as loading control. C: reverse zymographic analysis of lavage fluid from 2 SP-C-TIMP-1 transgenic and 2 wild-type mice to assess TIMP-1 inhibitory activity towards MMP-2 was performed. Lane 1: recombinant mouse TIMP-1. Lane 2: loading buffer only. Lanes 3 and 4: SP-C-TIMP-1 transgenic mice. Lanes 5 and 6: wild-type mice. The recombinant TIMP-1 migrates more slowly (top arrow) than TIMP-1 from transgenic mice (bottom arrow), possibly as a result of differences in glycosylation. 45, 35, and 18, Molecular weight markers (kDa).

 

Figure 4
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  		Fig. 4. A: reverse zymography of lung homogenates from bleomycin (BLM)-treated SP-C-TIMP-1 mice and nontransgenic (NT) littermates for TIMP-1 and TIMP-2. Mice were given a single intratracheal dose of 4 U/kg of bleomycin and killed at 7 or 14 days posttreatment. Bands representing TIMP-1 and TIMP-2 are indicated. B: Western blot analysis of BALF from bleomycin-treated SP-C-TIMP-1 mice at 7 or 14 days posttreatment. A single band representing TIMP-1 is indicated. C: inhibition of MMP-9 activity by overexpression of TIMP-1. Bleomycin-treated SP-C-TIMP-1 mice had reduced MMP-9 activity as assessed by zymographic analysis compared with their NT littermates. Representative gels are shown for A, B, and C. D: levels of fibrosis in silica-treated and bleomycin-treated TIMP-1-overexpressing mice. C57BL/6 and TIMP-1-overexpressing mice were given a single intratracheal dose of either 4 U/kg bleomycin or 0.2 g/kg freshly fractured crystalline silica and analyzed at 14 days posttreatment. Hydroxyproline content of acid hydrolyzed lung tissue was assessed as a measure of fibrosis (see MATERIALS AND METHODS). Bleomycin or silica treatment of wild-type (WT) and TIMP-1-overexpressing mice resulted in a significant increase in lung hydroxyproline content compared with saline-treated control mice (*P < 0.05, Student's t-test). There was no difference in hydroxyproline content of lungs from bleomycin-treated wild-type compared with TIMP-1-overexpressing mice. However, a small but significant decrease in hydroxyproline content was noted in the TIMP-1-overexpressing mice compared with wild-type mice in response to silica (P < 0.05, Student's t-test). Tg, transgenic mouse (Sp-C-TIMP-1).

 
Subsequently, we examined the levels of fibrosis (as assessed by hydroxyproline content) present in the lungs of SP-C-TIMP-1-overexpressing mice after bleomycin treatment. Lungs from bleomycin-treated wild-type and TIMP-1-overexpressing mice showed increased hydroxyproline content compared with the saline-treated control mice (Fig. 4D). However, there was no difference in lung hydroxyproline content in TIMP-1-expressing mice compared with their wild-type counterparts. To further characterize the fibrotic response of SP-C-TIMP-1-overexpressing mice, we challenged these mice with silica, an environmental agent capable of inducing lung fibrosis. Exposure of SP-C-TIMP-1 mice to silica was followed by increased deposition of collagen in the lung (Fig. 4D). However, as was the case of bleomycin exposure, the magnitude of the hydroxyproline accumulation observed in the lungs of these mice was similar to that observed in the nontransgenic littermates exposed to this agent.

MT regulates expression of MMPs in response to bleomycin. Since both MMPs are dependent on zinc for their activity, we examined the effect of knocking out the zinc-regulatory proteins MT-1 and MT-2 on the activation of MMPs, lung inflammation, and fibrosis in response to bleomycin. MMP-9 and MMP-2 expression was increased in both wild-type and MT knockout mice as detected by gelatin zymography at 7 days (Fig. 5A, lanes 2 and 4) and 14 days (Fig. 5A, lanes 6 and 8) posttreatment. However, the magnitude of the MMP expression in the lungs of the MT knockout mice was much lower, at both time points, than their wild-type counterparts (Fig. 5A, lane 2 vs. 4 and lane 6 vs. 8) following bleomycin exposure.


Figure 5
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  		Fig. 5. MMP and TIMP expression in bleomycin-treated metallothionein (MT) null mice. Wild-type and MT null mice were given a single intratracheal dose of 4 U/kg bleomycin and analyzed at 7 and 14 days posttreatment. A: gelatin zymography of lung homogenates was performed as described in MATERIALS AND METHODS. Identification of bands representing MMP-9 and MMP-2 was made by comparison with purified MMP-9 and MMP-2 (+). MMP-9 and latent and active MMP-2 were increased at both time points (B) compared with control (C). In addition, both enzymes were significantly decreased in the MT knockout mice compared with wild-type mice (lanes 2 and 4 vs. lanes 6 and 8). B: Western blot analysis of BALF from control and bleomycin-treated C57BL/6 and MT null mice at 7 and 14 days posttreatment. A single band representing MMP-7 is indicated. C: TIMP expression in bleomycin-treated C57BL/6 and MT null mice at 7 and 14 days posttreatment. Bands representing TIMP-1 and TIMP-2 are indicated. Representative gels are shown. rmTIMP1, recombinant mouse TIMP-1.

 
Western blot analysis of MMP-7 in the BALF of bleomycin-treated wild-type and MT knockout mice showed a baseline level of expression in saline-treated control animals (Fig. 5B). Bleomycin treatment of either strain showed an increase in MMP-7 expression at 14 days posttreatment (Fig. 5B, lanes 6 and 8). However, similar to our observations of MMP-9 and -2 expression, MMP-7 levels were decreased in the MT knockout mice at both 7 and 14 days postbleomycin treatment compared with wild-type littermate mice (Fig. 5B, lane 6 vs. 8).

MT deficiency decreases TIMP-1 expression but does not alter bleomycin-induced lung injury. As discussed above, MT null mice show decreased lung MMP expression in their lungs following bleomycin treatment. In addition, and similar to the response observed in the lungs of BALB/c mice (Fig. 2), MT-deficient mice fail to show TIMP-1 activity, although they exhibited enhanced TIMP-2 activity, in response to bleomycin (Fig. 5C). These data would suggest that the MT null mice might be resistant to the effects of bleomycin even though they were developed in a C57BL/6 genetic background. Therefore, we determined the level of inflammation and fibrosis associated with bleomycin-induced lung injury in the MT null mice and their normal-expressing littermates. At 7 days postbleomycin treatment, both strains displayed increases in common indicators of pulmonary inflammation such as total BALF protein content and total numbers of polymorphonucleocytes (Fig. 6, A and B). However, there was no difference in any of these indicators between the MT null mice and their wild-type littermates. At 14 days postbleomycin treatment, BALF neutrophil number and total protein were still elevated (Fig. 6, A and B). In addition, there was a significant increase in the BALF total protein content in the MT null mice compared with the control mice (Fig. 6A). We also assessed total lung fibrosis in the two strains. The lung hydroxyproline content of both the MT competent wild-type and MT null mice was elevated 14 days after bleomycin treatment (Fig. 6D) compared with saline-treated control mice. However, there was no significant difference in hydroxyproline levels in the lungs of MT null mice compared with wild-type littermates.


Figure 6
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  		Fig. 6. Bleomycin-induced lung injury is similar in wild-type and MT null mice. Wild-type (black bars) and MT null (white bars) mice were given a single intratracheal dose of 4 U/kg bleomycin and analyzed at 7 and 14 days posttreatment. A: BALF total protein (used as a measure of lung injury) was increased in MT knockout mice at both 7 and 14 days posttreatment. B: numbers of BAL neutrophils were not significantly different in the MT knockout mice compared with the wild-type mice. C: histological analysis of the 2 strains showed no difference between MT knockout and wild-type mice at either time point. D: hydroxyproline analysis showed similar levels of fibrosis between the 2 strains at either time point.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
IPF is a debilitating fibrotic lung disease without well-defined cause or an effective therapy. Although the etiology of the disease has long been the subject of intensive investigation, the precise biochemical mechanisms underlying this disease have yet to be elucidated.

MMPs have been implicated in both the human disease (41) and in animal models of pulmonary fibrosis, including the utilization of bleomycin (46), silica (35), and asbestos (43) as the eliciting stimulus. In this context, MMP-7, MMP-2, and MMP-9 have been studied in some detail (12, 51). In this study, we wished to determine if the differences in sensitivity to bleomycin observed between the C57BL/6 strain and the BALB/c strain of mice (38) could be accounted for by differences in the activity or regulation of MMPs.

In their original report, Swiderski et al. (42) identified that a differential expression of MMPs and TIMP-1 was induced by bleomycin during the development of lung injury in C57BL/6 mice. These authors reported activation of gelatinase A (MMP-2) and macrophage metalloelastase, but not gelatinase B (MMP-9), stromelysin-1, or interstitial collagenase, in the lungs of these bleomycin-sensitive mice. In addition, TIMP-1 expression, but not expression for TIMP-2 or -3, was significantly induced in the C57BL/6 mouse lung after bleomycin exposure. In the present work, we extend these observations and find increased activity for gelatinases (MMP-2 as well as MMP-9), matrilysin (MMP-7), and interstitial collagenase (MMP-13) in the lungs of mice from both C57BL/6 as well as BALB/c mouse strains, although the level of expression appear to be substantially higher in bleomycin-sensitive C57BL/6 mice (Fig. 1). In contrast to the expression of MMPs, we only find increased activity for TIMP-1 in the lungs of C57BL/6 mice during bleomycin injury (Fig. 2). Similar results were reported by Manoury et al. (24), who localized the enhanced TIMP-1 expression to macrophages and epithelial cells in the lungs of C57BL/6 mice. Thus these data suggest that the enhanced expression of MMPs is effectively inhibited by increased TIMP-1 activity in bleomycin-sensitive C57BL/6 but not in bleomycin-resistant BALB/c mice.

To further investigate the hypothesis that a balance between MMP activity and the presence of the protease inhibitor TIMP-1 influences the fibrogenic response of the lung to bleomycin, we created mice, on a C57BL/6 genetic background, that overexpress the mouse TIMP-1 gene under the lung-specific SP-C promoter (Fig. 3). These mice developed normally and did not show spontaneous evidence of lung fibrosis 1 yr after birth. SP-C-TIMP-1 transgenic mice showed increased TIMP-1 mRNA and protein expression and demonstrated enhanced TIMP-1 activity both in lung as well as BALF compared with their wild-type littermates (Fig. 4). This enhanced activity of TIMP-1 in the lung of SP-C-TIMP-1 mice was further increased by exposure to bleomycin (Fig. 4, A and B) and resulted in reduced expression of MMPs (Fig. 4C). Examination of these mice at 14 days postbleomycin treatment revealed that the lungs of both the TIMP-1-overexpressing mice and their wild-type counterparts had increased hydroxyproline levels (a common measure of collagen deposition/fibrosis) compared with saline-treated mice (Fig. 4D). However, no statistically significant differences were found in hydroxyproline content between the SP-C-TIMP-1 and their littermate controls. These results were rather perplexing to us as previous reports indicated that overexpression of human TIMP-1 in liver, under constitutive promoters such as albumin, enhanced the liver fibrosis and decreased the spontaneous resolution of this injury in mice exposed to carbon tetrachloride (48, 49).

Several reasons could be invoked to explain this apparent difference in fibrosis response. First, the systemic rather than the tissue-specific expression of TIMP-1 determines the fibrotic response during injury. Thus our transgenic mice were engineered to express the mouse TIMP-1 gene under a lung epithelial-specific promoter, limiting its expression to lung tissue, whereas the mice described by Yoshiji et al. (48, 49) express the human TIMP-1 protein under the regulation of the mouse albumin enhancer, and significantly elevated levels of circulating as well as liver TIMP-1 were documented in these mice. Consistent with this hypothesis are data indicating that the inhibition of mammary tumor growth and angiogenesis correlate with the long-term exposure to elevated circulating levels of TIMP-1 rather than the tissue-specific, mammary-derived TIMP-1 levels (17, 47).

Second, it is conceivable that the expression of the SP-C-driven transgene was not efficient during the bleomycin-induced lung injury. It has been reported that expression of epithelial genes is altered in mouse lung in response to bleomycin-induced lung injury (6). However, in contrast to the expression of the Clara cell secretory protein (CCSP) that is decreased in response to bleomycin, SP-C expression does not appear to decrease but rather increase and locate at ectopic sites such as the airways (7). These data appear to correlate with our observation in which enhanced TIMP-1 expression was observed in both the lung as well as BALF in response to injury.

Third, we did not evaluate the expression of TIMP-1 in the interstitium of the lung, and therefore we cannot exclude the possibility that the cell-specific effects of TIMP-1 (i.e., TIMP-1 interaction with interstitial fibroblast) rather than global lung expression determine its fibrotic response. This concept is exemplified by the observation that TIMP-1 expression during liver fibrosis is decreased in hepatic stellate cells, which are fundamental for the resolution of fibrosis in response to carbon tetrachloride, rendering them susceptible to apoptosis (49). Thus further studies to clarify this concept are necessary.

Fourth, we could exclude the possibility that the agent used to induce lung injury determines the contribution of TIMP-1 to fibrosis. In the case of the liver system of TIMP-1 overexpression, carbon tetrachloride has been the only agent used to test the sensitivity of the animals. Here, in addition to bleomycin, we used silica, an environmental agent capable of inducing lung injury, to test the fibrosis phenotype of the SP-C-TIMP-1 mice. We find that, similar to bleomycin, SP-C-TIMP-1 mice reacted to silica with increased lung fibrosis. However, no significant differences were detected in response to silica between these SP-C-TIMP-1 transgenic mice and their littermate controls. Finally, it may be important to consider that although TIMP-1 expression is an important regulator of inflammation, it does not directly influence the formation of fibrosis in response to injury. This concept is supported by data provided by Kim et al. (19) showing that although mice with a TIMP-1 deficiency evidence a stronger degree of lung inflammation, they did not display an altered fibrotic response to bleomycin.

Two experimental limitations of the current studies are worth mentioning as they could impact our current results. The first one is that, in the current manuscript, we use a single dose of bleomycin to evaluate the effects of this drug on the expression of TIMP-1 and MMPs. However, although a more complete dose-response curve with bleomycin would indeed be the best approach to identifying the role of MMP, our focus was on modulating TIMP-1. As such, the selected dose of bleomycin (4 U/kg or 0.1 unit per animal) and route of administration (endotracheal instillation) facilitate a comparison with our previous work (30) or those of others examining the basis of murine strain differences in response to this drug (21, 31). This dose of bleomycin is also consistently used to test the efficiency of therapeutic interventions in modulating bleomycin-induced lung injury in mice (as an example, Ref. 36). More specifically, in designing the current experiments, we chose a dose that would facilitate comparison with existing reports on the effect of bleomycin in inducing TIMP-1 expression and enhancing MMP activity (23). Similarly, our dose of bleomycin is intermediate between those used (0.0035 or 0.007 U/g, equivalent to 0.175 unit per animal) to demonstrate that TIMP-1-deficient mice exhibit enhanced lung inflammation in response to bleomycin exposure (19).

In addition, although a more dynamic analysis with respect to time will indeed provide additional insight into the role of TIMP-1 and MMPs in bleomycin-induced fibrosis, we felt it was critical to focus on early time points to facilitate comparison with existing information. In this regard, it is noteworthy that Madtes and colleagues (23) showed that the maximal induction of TIMP-1 in the mouse lung was observed 2 days after bleomycin exposure. Similarly, the most significant inflammatory events in the lungs of TIMP-1-deficient mice were observed between 24 h and 7 days after bleomycin exposure (19). Furthermore, no significant differences in lung fibrosis were observed in TIMP-1-deficient mice over the 4-wk period that followed the exposure of these mice to bleomycin.

As mentioned above, most MMPs require the presence of zinc in their active site for efficient proteolytic activity. Since we observed no significant difference in the pathogenic response to bleomycin in the TIMP-1-overexpressing mice, we wished to examine other possible pathways of MMP regulation in response to bleomycin treatment. One possible mechanism involved MMP regulation by the MTs. MTs are low molecular weight metal binding proteins with redox capabilities that have been shown to impact a myriad of biological processes (reviewed in Ref. 5). These proteins have been shown to be vital in the sequestration of environmentally toxic or physiologically important metals, including copper and zinc. In addition, MTs, particularly MT-1 and MT-2, been shown to control the activity of enzymes through redox reactions and the maintenance of intracellular zinc pools. Since MMP-9 and MMP-2 are activated by oxidative stress and regulated by zinc (40), we employed MT null mice in our bleomycin model to examine the role of this protein in the activation of MMP-9 and MMP-2. Compared with wild-type littermates, MT null mice exhibited reduced lung and BALF MMP-2 and MMP-9 activity 7 and 14 days after challenge with bleomycin (Fig. 5). This result is consistent with previously published data indicating that the presence of MT can enhance MMP-2 activity (14).

We also examined the expression of MMP-7 as several studies have suggested a role for this MMP in the regulation of fibrosis. For example, microarray analysis of human IPF samples demonstrated that MMP-7 expression was significantly upregulated in affected patients (32). In addition, it has been shown that MMP-7 is upregulated in mouse models of pulmonary fibrosis induced by bleomycin (25). Furthermore, MMP-7 null mice are significantly protected against bleomycin-induced pulmonary fibrosis (34). In our current study, we also observed an increase in MMP-7 expression in wild-type and, to a lesser extent, the MT null mice (Fig. 5B). As the MMP expression patterns in the wild-type vs. MT null mice were similar to what we observed in the C57BL/6 vs. BALB/c mice, we hypothesized that MT null mice might be protected against bleomycin injury. However, when we examined a number of inflammatory and pathological criteria, there was no difference in the severity of bleomycin-induced lung disease between the wild-type and the MT null mice (Fig. 6).

Taken together, these results serve to reinforce the idea that a number of factors have been shown to either positively or negatively regulate the formation of lung fibrosis. Increased levels of MMPs have been associated with pulmonary fibrosis in both humans and animal models. However, other factors such as the level of inflammation present, which also contributes to the overall levels of MMP and TIMP activity, and the rate of apoptosis in the lung may have a greater influence on overall disease severity. For example, MT-1 and -2 have been implicated as negative regulators of bleomycin toxicity by their ability to bind copper (28), which would reduce the amount of fibrosis seen in our model. However, these proteins also negatively regulate apoptosis mediated by bleomycin (20), which has been shown to both promote and suppress pulmonary fibrosis. Our data would suggest that rather than any individual gene it is this combination of opposing profibrotic and antifibrotic stimuli that ultimately controls disease development and determines strain sensitivity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported, in part, by grants from National Institutes of Health to L. A. Ortiz (R01-HL-071953 and ES-010859).


   ACKNOWLEDGMENTS
 
We thank Prof. Dylan Edwards (University of East Anglia) for the kind gift of mouse TIMP-1 cDNA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Ortiz, Univ. of Pittsburgh, Graduate School of Public Health, Dept. of Environmental and Occupational Health, Bridgeside Point, 100 Technology Dr., Suite #328, Pittsburgh, PA 15219-3130 (e-mail: lao1{at}pitt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* C. L. Fattman and F. Gambelli contributed equally to this publication. Back


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 ABSTRACT
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 RESULTS
 DISCUSSION
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 REFERENCES
 

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