Relationships Between Early Inflammatory Response to Bleomycin and Sensitivity to Lung Fibrosis: A Role for Dipeptidyl-Peptidase I and Tissue Inhibitor of Metalloproteinase-3?

By Pottier, Nicolas Chupin, Cecile; Defamie, Virginie; Cardinaud, Bruno; Sutherland, Rachel; Rios, Geraldine; Gauthier, Francis; Wolters, Paul J; Berthiaume, Yves; Barbry, Pascal; Mari, Bernard

Rationale: Different sensitivities to profibrotic compounds such as bleomycin are observed among mouse strains. Objectives: To identify genetic factors contributing to the outcome of lung injury.

Methods: Physiological comparison of C57BL/6 (sensitive) and BALB/ c (resistant) mice challenged by intratracheal bleomycin instillation revealed several early differences: global gene expression profiles were thus established from lungs derived from the two strains, in the absence of any bleomycin administration.

Measurements and Main Results: Expression of 25 genes differed between the two strains. Among them, two molecules, not previously associated with pulmonary fibrosis, were identified. The first corresponded to dipeptidyl-peptidase I (DPPI), a cysteine peptidase (also known as cathepsin C) essential for the activation of serine proteinases produced by immune/inflammatory cells. The second corresponded to tissue inhibitor of matrix metalloproteinase-3, which also inhibits members of the ADAM (a disintegrin and metalloproteinase) family, such as the tumor necrosis factor- converting enzyme. In functional studies performed in the bleomycin- induced lung fibrosis model, the level of expression of these two genes was closely correlated with specific early events associated with lung fibrosis, namely activation of polymorphonuclear neutrophil- derived serine proteases and tumor necrosis factor-a- dependent inflammatory syndrome. Surprisingly, genetic deletion of DPPI in the context of a C57BL/6 genetic background did not protect against bleomycin-mediated fibrosis, suggesting additional function(s) for this key enzyme.

Conclusions: This study highlights the importance of the early in- flammatory events that follow bleomycin instillation in the development of lung fibrosis, and describes for the first time the roles that DPPI and tissue inhibitor of matrix metalloproteinase-3 may play in this process.

Keywords: microarray; tumor necrosis factor-a; neutral serine proteases; matrix metalloproteinase; apoptosis

Idiopathic pulmonary fibrosis (IPF) is a heterogeneous progressive group of disorders characterized by the loss of alveolar structure through the apoptosis of epithelial and endothelial cells, infiltration of inflammatory cells into interstitial and alveolar spaces, proliferation of fibroblasts, and excessive deposition of interstitial collagens (1). The pathogenesis of pulmonary fibrosis is poorly understood and has been the subject of many investigations. Pulmonary fibrosis can indeed be driven by a wide variety of causes, such as drug toxicities, environmental exposure, and connective tissue diseases (reviewed by Taskar and Coultas [2]). Bleomycin is a drug leading to pulmonary fi- brosis, and one of the best established animal models of pulmonary fibrosis involves bleomycin instillation (3). Bleomycin is a useful drug in chemotherapy, as it does not induce major myelosuppression, but induction of lung fibrosis is a major side effect that has strongly impeded its use (4). In rodents as well as in guinea pigs and rabbits, a single bleomycin administration recapitulates most of the events observed in human pulmonary fibrosis (5). In mouse, it induces lung inflammation followed by a progressive destruction of the normal lung architecture. Interestingly, previous studies have demonstrated a genetic susceptibility to bleomycin-induced pulmonary toxicity: C57BL/6 mice are considered to be fibrosis prone, whereas BALB/c mice are relatively fibrosis resistant. Differences in bleomycin pharmacokinetics, susceptibility to oxidative stress, ability to repair DNA damage, immune system responses to lung injury, or in the pattern of cytokines produced during the inflammatory and/ or repair process have been invoked to explain such differences (6-10).

Several studies have analyzed the coordinated gene expression response of the lung after bleomycin instillation, in order to identify major traits leading to fibrosis. They analyzed changes in gene expression in response to bleomycin, using microarrays, and provided new insights into the progression of fibrotic lung diseases (11-13). However, these studies focused mainly on the late part of the time course (1-15 d), that is, when fibrosis was already established. In addition, interpretation of the gene expression modifications was obscured by major cellular modifications such as leukocyte infiltration or massive epithelial cell death. In line with previous studies that suggested the existence of early differences between resistant and sensitive mice (9, 14, 15), the aims of the current study were to (1) compare the expression profiles of sensitive and resistant mouse strains in the absence of bleomycin in order to characterize early traits driving the fibrogenic stimulus and (2) provide a molecular description of the early inflammatory response induced by bleomycin administration.

METHODS

Animal Treatment

Nine- to 12-week-old male C57BL/6 and BALB/c mice were purchased from Charles River (Lyon, France). Dipeptidyl-peptidase I (DPPI) knockout mice were backcrossed 10 generations into C57BL/6 mice as previously described (16). Mice were anesthetized by intraperitoneal injection of 1% ketamine and 0.25% xylazine. A 1-cm midline cervical incision was made to expose the trachea followed by intratracheal instillation of bleomycin (0.75 unit/ml) or vehicle (phosphate- buffered saline [PBS]), in a volume of 80 [mu]l. The cervical incision was closed with 2-octyl cyanoacrylate (Dermabond; Ethicon, Inc., Somerville, NJ) and the animals were returned to their cages to recover. Mice were killed 7 hours, 1 day, 8 days, and 15 days after instillation and lungs were removed for further analysis.

Wet-to-Dry Ratio

The lungs were blotted dry and weighed. They were then desiccated by incubation at 70[degrees]C for 24 hours in a vacuum oven and reweighed to determine their dry weight. The wet-to-dry ratio was then calculated.

Hydroxyproline Quantification

Lung hydroxyproline content was assayed as previously described with modifications (17). Briefly, the right lung of each mouse was dissected free of extraneous tissue, homogenized in 2 ml of water, and then incubated in 0.5% trichloroacetic acid on ice for 20 minutes. After centrifugation, the pellet was resuspended once in 1.0 ml of 12 N HCl and baked at 110[degrees]C for 24 hours, and then in 2.0 ml of distilled H2O. Fifty microliters of each sample was incubated for 20 minutes at room temperature in 100 ml of a solution containing 1.4% chloramine-T, 0.5 M sodium acetate, and 10% isopropanol, before addition of 100 ml of Ehrlich’s solution (1.0 M p-dimethylaminobenzaldehyde dissolved into 70% isopropanol plus 30% perchloric acid). Optical density was measured after a 15-minute incubation at 65[degrees]C. Purified hydroxyproline standards were used to convert optical density values to hydroxyproline concentrations

Bronchoalveolar Lavage

After intratracheal instillation, mice were killed and the trachea was exposed as described above. A catheter was inserted through a small incision in the trachea. Bronchoalveolar lavage (BAL) was performed by filling the lungs in situ with 0.5 ml of PBS and then gently aspirating the fluid back. This operation was repeated three times. The samples were then pooled and total cell counts were determined with a hemacytometer. After centrifugation (900 rpm for 8 min at 4[degrees]C) the supernatants were analyzed for albumin content, using a Lab-on-a-Chip protein kit (2100 Bioanalyzer; Agilent Technologies, Massy, France) and stored at – 80[degrees]C until use. The pellet was resuspended in 1 [mu]l of PBS and cells were then cytocentrifuged (Shandon Cytospin cytocentrifuge; Thermo Fisher Scientific, Waltham, MA) at 250 rpm for 3 minutes and stained by the May-Grunwald Giemsa method. Differential cell counts of 200 cells, using standard morphological criteria, were assessed.

Histopathology

The lungs and trachea were exposed by thoracotomy. Lungs were inflation-fixed through the trachea with 4% paraformaldehyde-PBS (at a transpulmonary pressure of 25 cm H2O) for 3 hours and then were fixed with 4% paraformaldehyde-PBS overnight at 4[degrees]C. Lung tissue was then cryoprotected in PBS containing 30% sucrose. Frozen sections (4 mm) were cut on a Leica cryostat (Leica Microsystems, Wetzlar, Germany) and stored at -70[degrees]C. Sections of lung were stained with Masson trichrome stain to assess the degree of fibrosis. The major criteria examined included interstitial thickening of alveolar or bronchiolar walls, collagen deposition, and inflammatory cell infiltration.

RNA Extraction and Gene Expression Profiling

Lungs removed from nontreated mice (n = 8 for both strains) were used for assessment of gene expression.

Total RNA was extracted with TRIzol reagent (Roche Diagnostics, Meylan, France), in accordance with the manufacturer’s recommended protocol. RNA integrity was assessed by electrophoresis on the Agilent 2100 bioanalyzer (Agilent Technologies). Hybridization solutions were prepared with a low RNA input fluorescent linear amplification kit (Agilent Technologies). Briefly, first- and second- strand cDNAs were synthesized from 500 ng of total RNA, using the Moloney murine leukemia virus reverse transcriptase and T7 promoter primer by retrotranscription at 40[degrees]C for 2 hours. cRNA was synthesized and labeled with cyanine 3 (Cy3)- and Cy5-labeled CTP by in vitro transcription (40[degrees]C for 2 h), using the T7 promoter- coupled double-stranded cDNA as template and the T7 RNA polymerase. The labeled cRNA was separated from unincorporated ribonucleotides by passage through an RNeasy mini spin column (Qiagen, Courtaboeuf, France). The cRNA was then fragmented and hybridized for 17 hours at 60[degrees]C to Agilent whole mouse genome oligo microarray G4122A containing 41,174 unique 60-mer probes. After washing, microarrays were scanned with a GenePix Autoloader 4200AL (Molecular Devices, Sunnyvale, CA). The two red and green lasers operated at 633 and 543 nm to excite Cy5 and Cy3, respectively. Intensity was measured at 670 nm for Cy5 and at 570 nm for Cy3. Laser power was set to 100%, and photomultiplier tube power was set between 60 and 70% depending on the slides.

Bioinformatics

TIF images containing the data from each fluorescence channel were quantified with the GenePix Pro 6.0 program (Molecular Devices), using an ”irregular features” quantification method. All microarrays had low background, and hence background subtraction was not used. All statistical and graphical analyses were carried out in the R computing environment (version 2.0.1), using the graphical user interface for the Limma microarray package (LimmaGUI, version 1.3.6), which is part of the Bioconductor project (www.Bioconductor.org) (18). Global loess normalization was carried out for each microarray, followed by quantile normalization among microarrays. Differential expression of genes was determined by an empirical Bayes approach (19). Genes were ranked as being differentially expressed in decreasing order of the B-statistics (i.e., the log-odds of differential expression).

Total Protein Extract

Lung tissue was homogenized in lysis buffer (50 mM N-2- hydroxyethylpiperazine- N9-ethanesulfonic acid [HEPES, pH 7.4], 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10 mM Na^sub 3^VO^sub 4^, 100 [mu]M NaF, 1% Triton X-100) at 4[degrees]C, using a homogenizer ( Polytron PT 2100; Kinematica AG, Littau, Switzerland). The samples were gently rocked at 4[degrees]C for 2 hours and then centrifuged at 13,000 rpm for 20 minutes at 4[degrees]C. Aliquots were stored at 280[degrees]C.

DPPI Activity

DPPI activity was quantified as previously described (20). Briefly, 5 [mu]l of BAL fluid supernatant in 45 [mu]l of incubation buffer (50 mM Tris [pH 7.4], 20 mM NaCl) was added to 50 [mu]l of reaction buffer (50 mM Tris [pH 7.4], 20 mM NaCl, 400 mM AMC substrate). Assays were carried out in 96-well microtiter plates at 37[degrees]C for 1 hour. The production of 7- amino-4- methylcoumarin (AMC) from the enzyme-catalyzed cleavage of GF-AMC was measured with a microplate fluorometer (Fluoroskan Ascent; Thermo Fisher Scientific, Vantaa, Finland) in the absence or presence of 250 mg of antipain, a reversible inhibitor of cathepsin (excitation wavelength of 390 nm and emission wavelength of 460 nm). To determine specific DPPI activity, the value of the absorbance signal of the inhibitor sample was subtracted from that of the noninhibited sample.

Neutrophil Elastase and Cathepsin G Activities

Polymorphonuclear neutrophil-derived serine protease activities were quantified as previously described (21). Briefly, 5 [mu]l of BAL fluid supernatant in 45 [mu]l of incubation buffer (50 mM HEPES, pH 7.4) was added to 50 [mu]l of reaction buffer (50 mM HEPES [pH 7.4], 5 [mu]M Abz- APEEIMDRQ-EDDnp substrate specific for neutrophil elastase or 10 [mu]M Abz-TPFSGQ-EDDnp substrate specific for cathepsin G) (Abz, 2-aminobenzyl group; EDDnp, ethylenediamine-2,4- dinitrophenyl group). Assays were carried out as described above, using an excitation wavelength of 320 nm and an emission wavelength of 420 nm.

Caspase-3, Caspase-8, and Caspase-9 Activities

Twenty-five micrograms of protein (lung homogenate) in 50 ml of incubation buffer (50 mM Tris [pH 7.4], 20 mM NaCl) was added to 50 ml of reaction buffer (50mMTris [pH 7.4], 20mMNaCl, 400 mMAMC substrate, 10 mM dithiothreitol). Assays were carried out as described above, using an excitation wavelength of 390 nm and an emission wavelength of 460 nm (Ac-DEVD-AMC for caspase-3, Ac- IETDAMC for caspase-8, or Ac-LEHD-AMC for caspase-9). For caspase- 3, activity was measured in the absence or presence of caspase-3 inhibitor (Ac-DEVD-CHO). To determine specific caspase-3 activity, the value of absorbance of the inhibited sample was subtracted from that of the noninhibited sample.

Tumor Necrosis Factor-a-converting Enzyme and Matrix Metalloproteinase Activities

Twenty-five micrograms of protein (lung homogenate) in 50 [mu]l of incubation buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM CaCl^sub 2^) was added to 50 [mu]l of reaction buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM CaCl^sub 2^, 20 [mu]M AMC substrate). Assays were carried out as previously described, using an excitation wavelength of 390 nm and an emission wavelength of 460 nm (Mca-PLGL- Dpa-AR-NH^sub 2^ for matrix metalloproteinase [MMP] or Mca-PLAQAV- Dpa-RSSSR-NH2 for tumor necrosis factor [TNF]-alpha-converting enzyme [TACE]) [Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N-3-(2,4- dinitrophenyl)-L-2,3- diaminopropionyl] (22, 23). Activities were measured in the absence or presence of selective inhibitors (RXPO3 for MMP and TNF-alpha proteinase inhibitor [TAPI]-1 for TACE). To determine specific activities, the value of absorbance of the inhibited sample was subtracted from that of the noninhibited sample

Zymography

Twenty-five micrograms of nonreduced protein sample (lung homogenate) was loaded on 10% sodium dodecyl sulfate-polyacrylamide gels containing 0.3% type I collagen prepared from rat tail tendon as previously described (24). After electrophoresis, gels were then washed four times in 2.5% Triton X-100 and incubated in substrate buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM CaCl2) at 37[degrees]C for 24 hours with gentle shaking. Gels were stained with 0.25% Coomassie Blue R-250 (Sigma, St. Louis, MO) and destained in 70% acetic acid for 30 minutes. Enzymatic activities appear as cleared bands in a dark background.

Western Blot Analysis

Proteins were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes (GE Healthcare, Velizy, France). Filters were blocked in Tris-buffered saline containing 1% Tween-20 and 5% nonfat dry milk for 2 hours, and then incubated with primary antibody M42 against MT1-MMP (membrane type 1 matrix metalloproteinase) (a kind gift from M.-C. Rio, Institute of Genetics and Molecular and Cellular Biology, Illkirch, France) overnight at 4[degrees]C on a shaker. Filters were washed twice for 15 minutes in Tris-buffered saline containing 1% Tween-20 before incubation with the secondary antibody conjugated with horseradish peroxidase. After three final washes, immunological complexes were revealed with anti-mouse peroxidase antibodies, followed by electrochemiluminescence.

Cytokine Assay

Monocyte chemoattractant protein (MCP)-1, IL-6, IL-5, TNF-alpha, and IL-10 assays were performed with a BD FACSArray bioanalyzer (mouse inflammation kit; BD Biosciences, Le Pont de Claix, France) according to the manufacturer’s protocol. Cytokine assays were measured in 50 [mu]l of BAL supernatant.

Statistical Analysis

The statistical significance of the functional data and survival curves was estimated by two-tailed Student t test, as provided with the Excel program (Microsoft, Redmond, WA).

RESULTS

C57BL/6 and BALB/c Mice Exhibit Distinct Responses after Bleomycin Instillation

Bleomycin impact on two mouse strains was evaluated by measuring survival, lung edema, and collagen deposition. (1) Survival: 35 of 35 mice survived 15 days after bleomycin instillation in the BALB/c group, whereas 17 of 35 animals survived in the C57BL/6 group. (2) Wet-to-dry ratio: Figure 1A shows the development of lung edema over time after bleomycin instillation. The wet-to-dry ratio in the control groups was about 4.5. It rose to about 5.0 15 days after bleomycin instillation in the BALB/c group (P < 0.01, Student t test), indicative of mild edema, and to 6.5 in the C57BL/6 group, indicative of more severe edema (P < 0.01). (3) BAL fluid albumin content: Similar observations were made by measurements of the albumin concentration in the BAL fluid (Figure 1B): albumin concentration in the C57BL/6 group was twice the amount in the BALB/ c group. (4) Collagen deposition: Figure 1C shows the increase in collagen deposition, as measured by an increase in hydroxyproline content. This increase was greater in the C57BL/6 group than in the BALB/c group (P < 0.001). Collagen deposition was further evidenced by direct staining of collagen of lung sections: increased deposition of collagen and morphological alterations were patent in the C57BL/6 group 15 days after bleomycin instillation (C57BL/6, Figure 1I; BALB/c, Figure 1H). Significant variations were already observed one week after bleomycin instillation (C57BL/6, Figure 1G; BALB/c, Figure 1F).

Microarray Experiments Reveal a Discrete Gene Expression Signature That Distinguishes the Two Mouse Strains

To determine whether this differential susceptibility to bleomycin could be caused ab initio by differential expression of some specific factors in resistant versus sensitive strains, we performed microarray experiments on lung samples from nontreated adult BALB/c and C57BL/6 animals. Using an empirical Bayes approach, we characterized a list of the 25 most discriminating genes between both strains (Table 1). Only nine of these genes were functionally annotated in public databases. Interestingly, two of them appeared to be central coordinators in inflammatory processes: tissue inhibitor of matrix metalloproteinase- 3 (TIMP-3) and cathepsin C (CTSC, DPPI). Differences in expression levels between the two mouse strains were con- firmed for these two genes, using another microarray platform (25) (data not shown). DPPI was the most discriminating gene, with an approximately 16-fold higher expression in the C57BL/6 strain. DPPI is essential for the processing and activation of many serine proteinases in immune/inflammatory cells, in particular the polymorphonuclear neutrophil-derived serine proteases: neutrophil elastase (NE), cathepsin G (CTSG), and proteinase- 3 (PRTN3) (26). TIMP-3 showed approximately twofold decreased expression in the C57BL/6 strain. TIMP-3 is a member of the tissue inhibitor metalloproteinase family, which inhibits matrix remodeling by MMPs. Of this family of proteins, TIMP-3 is the only TIMP to inhibit members of the ADAM (a disintegrin and metalloproteinase domain) family, such as tumor necrosis factor- converting enzyme (TACE, ADAM- 17). Indeed, studies of TIMP-3 knockout animals have shown that TIMP-3 is a crucial innate negative regulator of TNF-alpha in both tissue homeostasis and tissue response to injury (27, 28). Overall, the relative expression of DPPI and TIMP-3 in the two mouse strains suggests that it could lead to a differential inflammation response to bleomycin. We thus carefully analyzed the potential functional implication of these two genes in the early inflammatory processes after bleomycin exposure. DPPI and Neutrophil Serine Protease Activities Are Detected at Higher Levels in BAL Fluid from C57BL/6 Mice after Bleomycin Treatment

DPPI is a lysosomal cysteine protease produced largely by pulmonary alveolar macrophages in lung parenchyma (29). To confirm microarray data, we first compared DPPI activity in BAL fluid supernatant from the two mouse strains 24 hours after bleomycin infusion, using the fluorogenic substrate GFAMC and the cathepsin inhibitor calpain. Figure 2A shows a significant fivefold increase (P < 0.05) in DPPI activity for the sensitive C57BL/6 strain, confirming that stress conditions lead to higher levels of active DPPI in the alveolar compartment of C57BL/6 mice.

If DPPI activity was increased in C57BL/6 mice after bleomycin treatment, we anticipated that the activities of downstream targets of DPPI, such as the serine proteases from neutrophils NE and CTSG, would differ. Both activities were measured in BAL fluid, using highly specific fluorogenic substrates (21). Results indeed showed a significant increase (P < 0.05) in both enzymatic activities for the sensitive C57BL/6 strain compared with the resistant BALB/c strain (Figures 2B and 2C). Increased neutrophil serine protease activity after bleomycin treatment is therefore consistent with the higher levels of DPPI expression observed in lungs from C57BL/6 animals.

Bleomycin Exposure Results in Induction of TACE Activity and TNF- alpha Release That Is Strain Dependent

Because TIMP-3 is associated with the extracellular matrix and thus difficult to quantify by Western blot, we indirectly tested the potential consequence of this difference in gene expression on TACE activity. Activity was measured 24 hours after bleomycin treatment in lung homogenates, as described in METHODS. Results show a significant increase (P < 0.01) in TACE activity by bleomycin for the sensitive C57BL/6 strain whereas no induction was detected in BALB/c animals (Figure 3A). We analyzed in parallel the level of TNF- alpha in BAL fluid obtained 24 hours after PBS or bleomycin treatment. In agreement with TACE activity data, a significant induction of the level of TNF-alpha by bleomycin was measured in BAL fluid only from C57BL/6 mice (Figure 3B). Thus, these data indicate that high levels of expression of TIMP-3 in BALB/c mice limit bleomycin-mediated TNF-alpha burst via inhibition of TACE activity.

Severe Inflammatory Syndrome and IL-5-dependent Eosinophilia in Sensitive Strain

According to these data, bleomycin treatment should induce a more pronounced inflammatory response in the C57BL/6 strain. To confirm this, we measured the levels of proinflammatory cytokines such as IL- 6 and MCP-1, and of antiinflammatory cytokines such as IL-10, in BAL fluid 24 hours after bleomycin exposure. The results clearly indicated that C57BL/6 mice produced significantly more IL-6 (Figure 4A; P < 0.01) and MCP-1 (Figure 4B; P < 0.05) than did BALB/c animals. Interestingly, a significant decrease in IL-10 expression was also detected in the sensitive strain whereas no significant variation was observed in the resistant strain (Figure 4C; P < 0.05).

Because several studies have also indicated the importance of TNF- alpha-mediated IL-5 secretion in eosinophil recruitment and pulmonary fibrosis (30, 31), we measured in parallel the levels of IL-5 in the same lavages. Figure 4D clearly shows that only the sensitive C57BL/6 strain produces a significant (P < 0.01) increase in IL-5 expression after bleomycin exposure. In agreement with these results, we could detect eosinophils in BAL fluid only from C57BL/6 animals after 24 hours of treatment (data not shown).

Bleomycin-induced Apoptosis Is More Pronounced in C57BL/6 Animals

Apoptosis of pulmonary epithelial cells has been shown to play a key role in the initiation of the lung fibrotic process (1). Because TNF-alpha could play a central role in this process as a strong inducer of apoptosis for many cell types (32, 33), we measured caspase-3 activity in lung homogenates 24 hours after bleomycin administration. As shown in Figure 5, bleomycininduced caspase-3 increase was more pronounced in C57BL/6 strain (Figure 5A: 6.5-fold increase in sensitive strain vs. threefold increase in resistant strain). As expected for receptormediated cell death, we also found higher levels of caspase-8 activity in the sensitive strain (Figure 5B), whereas caspase-9 induction was similar in the two strains (Figure 5C).

Early Differences in MMP Activities between Sensitive and Resistant Strains after Bleomycin Exposure

MMPs are the main proteases that initiate degradation of collagen. They are thought to be key enzymes in the lung fibrotic process (34, 35). TNF-alpha could play a central role by inducing the expression of MMPs such as MMP-9 (36). In this context, it was interesting to compare MMP activity between the two strains 24 hours after bleomycin treatment. Zymography (performed on lung homogenates) revealed a strong increase in the expression level of both pro-MMP-9 and pro- MMP-2 in the sensitive strain. Moreover, activation of these two MMPs appeared after bleomycin treatment (Figure 6A). On the other hand, only weak induction and activation of these two MMPs were detected in the resistant BALB/c strain.

Global MMP activity was quantified with a fluorogenic substrate with broad range specificity (see METHODS). This measurement confirmed that C57BL/6 mice produced significantly more active MMP after bleomycin exposure (Figure 6B).

Because TNF-alpha is a strong inducer of MT1-MMP, the main activator of MMP-2 (37), we also quantified the level of MT1- MMP by Western blot as previously described (38). Figure 6C shows a strong induction of MT1-MMP expression in the sensitive mice only, making TNF-alpha the likely initial factor involved in this proteolytic cascade.

Evaluation of Susceptibility of DPPI Knockout Mice to Bleomycin- induced Fibrosis

To directly address the function of DPPI during bleomycininduced fibrosis, further experiments were performed on knockout mice in a C57BL/6 genetic background (16). Edema (Figure 7A), collagen deposition (Figure 7B), and mortality (data not shown) were compared between DPPI knockout and wild-type mice. Surprisingly, no difference was observed between the two groups for any of these three parameters. This lack of significant difference was not likely due to compensating dipeptidyl-peptidase-like activities expressed in BAL fluid from DPPI null mice, as evidenced by direct measurements of DPPI activity in BAL 24 hours after bleomycin instillation (Figure 7C). Figure 7D also indicates that no alternative NE-activating dipeptidase compensates for DDPI in DPPI null mice, as previously described for granzyme activation (26, 39).

DISCUSSION

Several studies have used the experimental model of bleomycininduced pulmonary fibrosis to analyze the contribution of genetic factors to the development of fibrosis (5-7, 40). We reasoned that differential gene expression between sensitive C57BL/ 6 and resistant BALB/c strains could reveal altered expression of cellular factors involved in the early proinflammatory and profibrogenic responses to bleomycin. From a perspective of pharmacological intervention at the onset of fibrosis, we thought that it would be useful to track early events associated with the response to bleomycin in fibrosis-prone and fibrosis-resistant mouse strains, in order to propose new therapeutic interventions. Using a pan-genomic microarray, we found that monitoring a relatively limited number of differentially expressed genes was sufficient to discriminate control lungs from the two types of mice. A first observation was the absence of linkage between any of these genes and any previously described susceptibility locus (8, 10, 41). This was not totally surprising as most of these genetic studies were focused on late time points, that is, when fibrosis was already present, rather than on early time points. Only nine of these genes were functionally annotated in public databases. Interestingly, two of them appeared to be central coordinators in inflammatory processes: TIMP-3 and DPPI. We thus decided to explore further the role of those two genes during the response to bleomycin.

We found that the expression of TIMP-3 was higher in resistant BALB/c mice than in sensitive C57BL/6 mice. TIMP-3 is the major physiological inhibitor of TACE and a crucial innate negative regulator of TNF-alpha, in particular in the context of tissue injury (27, 28). Deletion of the gene in mice induces spontaneous pulmonary air space enlargement (42) and differential response to lung injuries (43, 44). Both TACE and TIMP-3 are expressed in normal and IPF lungs (45-47). Our data clearly indicate that the level of TIMP-3 expression in the two mouse strains was inversely correlated with both TACE activity and TNF-alpha release. Importantly, the level of TNF-alpha transcript was similar in both strains (data not shown), in agreement with previous studies (9), indicating that the elevated TNF-alpha concentration observed in C57BL/6 BAL fluid was not due to differential transcriptional gene regulation but rather was a consequence of increased release of active TNF-alpha mediated by TACE. The early increase in TNF-alpha release observed in C57BL/ 6 mice after bleomycin instillation is likely to induce a deleterious cascade leading to increased inflammatory cytokine production, epithelial cell apoptosis, and matrix degradation, all hallmarks of pulmonary fibrosis development. Consistent with this model, inflammatory cytokines such as IL-6 were up-regulated only in C57BL/6 mice, as described (9). IL-5, another downstream target of TNF-alpha (30), was induced only in the bleomycinsensitive strain, where it likely contributes to lung injury by driving eosinophils into the airspaces (31). Inhibition of the expression of IL-10, an immunosuppressive cytokine, was observed only in C57BL/6 mice exposed to bleomycin. Because of its marked antiinflammatory and antifibrotic properties, its decrease can lead to additional cellular damage (48, 49). The increase in receptor-dependent caspase- 8, observed only in C57BL/6 animals, suggests that bleomycin- induced cell death is indirect, caused by apoptosis after an increase in TNF-alpha, rather than by a nonspecific cytotoxic effect. Evidence showing that caspase inhibitors could protect against bleomycin-induced lung fibrosis (50, 51) supports the notion that apoptosis could contribute to the initiation of pulmonary fibrosis. These observations are consistent with our own study showing increased activity of caspase-3 in C57BL/6 mice (Figure 5).

These data support the central role played by TNF-alpha in the development of pulmonary fibrosis (41, 52). In contrast, data from animal studies using transgenic mice overexpressing TNF-alpha in the lungs are conflicting (53-57). These data indicate that TNF-alpha has pleiotropic effects: under some circumstances it promotes and under other conditions it inhibits the development of lung fibrosis. In fact, the impact of TNF-alpha on fibrosis probably depends on the amplitude and duration of its production, which leads to the generation of either prosurvival or proapoptotic signals (58). From that perspective, sustained and early TNF-alpha release is correlated with increased cell death, increased inflammation, and lung matrix remodeling. Accordingly, findings have demonstrated that antiinflammatory treatment is beneficial only when administered early after exposure to bleomycin (59).

Growing evidence indicates that members of the papain family of cysteine proteinases, including DPPI, play specific functions in matrix remodeling and regulation of the immune response, and are linked to several lung diseases (29). Consistent with these roles, DPPI, a lysosomal cysteine peptidase highly expressed by macrophages, cytotoxic T lymphocytes, and mast cells (29), was the most discriminating transcript between the bleomycin-sensitive and resistant strains, with a 16-fold increase in expression in C57BL/6 mice. This strong difference at the RNA level was confirmed by the increase in DPPI activity we found in BAL fluid from C57BL/6 mice 24 hours after exposure to bleomycin. Because of this dramatic difference, DPPI became a lead candidate as the mediator of the difference in bleomycin sensitivity between C57BL/6 and BALB/c mice. Mechanistically, DPPI could promote fibrosis by processing and activation of granule-associated proteases NE, CTSG, PRTN3, granzymes A and B, and mast cell chymase (60), which require DPPI for activation (61). These possibilities are further supported by studies demonstrating that (1) blockade of NE activity by natural (such as a1-proteinase inhibitor or secretory leukocyte proteinase inhibitor) (62, 63) or synthetic NE inhibitors (64-66) attenuates the severity of bleomycin-induced acute lung injury and reduces the severity of pulmonary fibrosis; and (2) mice lacking NE are resistant to bleomycin-induced pulmonary fibrosis through insufficient transforming growth factor-b activation (67).

Because our data and those cited above support a central role for DPPI in the development of lung fibrosis, we anticipated that deletion of DPPI would mimic the deletion of NE and reduce the severity of bleomycin-mediated fibrotic lesions. Surprisingly, our data on DPPI2/2 mice did not support this model as total elimination of DPPI activity had no impact on mortality, edema formation, or fibrosis induced by bleomycin, that is, it generated a phenotype distinct from the deletion of NE itself (67). As no residual NE activity was detected in DPPI knockout animals (Figure 7D), it is unlikely that alternative activation of NE explains the difference between the two models.

To reconcile our observations with those in NE-deficient mice (67), it is important to highlight differences between the two models:

1. The NE knockout lacks the protein, whereas the DPPI knockout mice express the proform of the protease. As other proteases such as PRTN3 display biological activities independent of their catalytic activity (68), a first, but unlikely, hypothesis would be that the proform of NE would be associated with biological activities participating in the development of fibrosis. The fact that several elastase inhibitors block the development of fibrosis induced by bleomycin (63, 65, 66) argues against this hypothesis.

2. Unlike NE knockout mice, which lack activity only in NE, DPPI knockout mice lack the activity of many proteases including NE, CTSG, PRTN3, granzymes A and B, and mast cell chymase (61). Therefore the absence of activity of one (or more) of the other proteases may be profibrotic and counterbalance the absence of NE activity. This possibility is similar to prior studies in which NE- deficient mice were found to have increased mortality (69, 70) and DPPI-deficient mice were found to have reduced mortality (despite the absence of NE activity) after bacterial infection (16).

3. Differences in DPPI activity measured between C57BL/6 and BALB/ c mice are relative, meaning that, unlike DPPI^sup -/-^ mice, BALB/ c mice still express a significant amount of DPPI activity. Thus, DPPI may play a pleiotropic role in the development of lung fibrosis. For example, excessive DPPI production could induce proteolytic processing of additional profibrotic molecules. On the other hand, reduction of DPPI activity to below a threshold level could protect against lung fibrosis whereas full DPPI inhibition may unveil additional compensatory pro- fibrotic effects. If true, this idea would favor considering DPPI as a pharmacologic target for lung fibrosis because it is unlikely an inhibitor would ever completely inhibit DPPI in vivo.

In conclusion, this study highlights the importance of early inflammatory events and describes for the first time the roles DPPI and TIMP-3 may play in bleomycin-mediated lung fibrosis. These results suggest that DPPI has both beneficial (via inactivation of NE) and harmful (via processing of yet to be identified proteases or proteins) effects in this model. In addition, our work also highlights the importance of TNF-alpha and of the regulation of its activity. Early or late TNF-alpha releases can indeed lead to opposite effects. The low level of expression of TIMP-3 observed in C57BL/6 mice could therefore be associated with a TNF-alpha- mediated inflammatory syndrome. Additional studies using DPPI and TIMP-3 null mice as well as selective inhibitors are required to improve our understanding of their roles in the bleomycin-mediated inflammatory syndrome and associated lung fibrosis.

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Acknowledgment: The authors thank Dr. Marie-Christine Rio for the MT1-MMP antibody. The authors also thank Julie Cazareth for excellent technical assistance concerning the flow cytometric array experiments and Franck Aguila for artwork. Microarray experiments were carried out using the facilities of the Marseille Nice- Sophia Antipolis Genopole platform.

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

Physiological alterations produced by bleomycin in lung are well characterized in human and animal models, but early mechanisms leading to lung injury have not been explored.

What This Study Adds to the Field

DPPI (cathepsin C), TIMP-3, and TACE appear to participate in early inflammatory events following bleomycin instillation into the lungs.

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Nicolas Pottier1, Cecile Chupin1,2, Virginie Defamie1, Bruno Cardinaud1, Rachel Sutherland3, Geraldine Rios1, Francis Gauthier4, Paul J. Wolters3, Yves Berthiaume1,2, Pascal Barbry1, and Bernard Mari1

1Centre National de la Recherche Scientifique and Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice Sophia Antipolis, UMR6097, Sophia Antipolis, France; 2Departement de Medecine, Centre de Recherche, Centre Hospitalier de l’Universite de Montreal Hotel-Dieu, Montreal, Quebec, Canada; 3Department of Medicine and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California; and 4Institut National de la Sante et de la Recherche Medicale U618, Proteases et Vectorisation Pulmonaires, Universite Francxois Rabelais, Tours, France

(Received in original form July 28, 2006; accepted in final form July 31, 2007)

Supported by the Association Vaincre la Mucoviscidose, the Association de Recherche contre le Cancer (subvention fixe 3355), and the Institut National du Cancer (PL079). Y.B. was a recipient of a visiting scientist award from the Canadian Institutes for Health Research and from the Canadian Cystic Fibrosis Foundation.

Correspondence and requests for reprints should be addressed to Pascal Barbry, Ph.D., IPMC, CNRS UMR6097, 660, route des Lucioles, F06560 Sophia Antipolis, France. E-mail: barbry@ipmc.cnrs.fr

Am J Respir Crit Care Med Vol 176. pp 1098-1107, 2007

Originally Published in Press as DOI: 10.1164/rccm.200607-1051OC on August 2, 2007

Internet address: www.atsjournals.org

Copyright American Thoracic Society Dec 1, 2007

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