Role of Cathepsin S-Dependent Epithelial Cell Apoptosis in IFN-γ-Induced Alveolar Remodeling and Pulmonary Emphysema

Transgenic and genetically modified mice

CC10-rtTA-IFN-γ mice that had been generated in our laboratory were used in these studies (19). These are dual transgene-positive animals in which the reverse tetracycline transactivator (rtTA) drives the expression of the murine IFN-γ gene in a lung-specific and externally regulatable fashion. The transgene in these mice is activated by adding doxycycline (dox) to the animal’s drinking water. Construct 1 in these mice contains the Clara cell 10-kDa protein (CC10) promoter, rtTA, and human growth hormone intronic and polyadenylation sequences. The rtTA is a fusion protein made up of a mutated tetracycline repressor (rtet-R) and the herpesvirus VP-16 transactivator. The second construct contains a polymeric tetracycline operator, minimal CMV promoter, murine IFN-γ cDNA, and human growth hormone intronic and adenylation signals. In this system, the CC10 promoter directs the expression of rtTA to the lung. In the presence of dox, rtTA is able to bind in trans to the tetracycline operator and the VP-16 transactivator activates IFN-γ gene transcription. In the absence of dox, rtTA binding does not occur or occurs at very low levels, and no or very low-level gene transcription is noted. These mice were maintained as dual transgene (+) heterozygotes (referred to as transgene; Tg (+) hereafter). The details of both genetic constructs, the methods of microinjection and genotype evaluation, the inducibility, and the emphysematous and inflammatory phenotype of CC10-rtTA-IFN-γ mice have been previously described (19).

Cathepsin S (Cat S) null mutant (−/−) and caspase-3^−/− mice were generated as described by Shi et al. (29) and Kuida K et al. (30), respectively, and bred onto the C57BL/6 background. On this background, caspase-3^−/− mice are deaf but are otherwise phenotypically normal (31). CC10-rtTA-IFN-γ mice with wild-type^+/+ or null^−/− Cat S or caspase-3 loci were generated by breeding C57BL/6 IFN-γ-overexpressing mice with the Cat S^−/− or caspase-3^−/− animals. PCR was used to define the transgenic status of all offspring using primers that detected rtTA and/or the junction region of the murine IFN-γ-human growth hormone construct. The Cat S loci were evaluated by PCR using primers 5′ (5′-CTT GAA GGG CAG CTG AAG CTG-3′) and 3′ (5′-GTA GGA AGC GTC TGC CTC TAT-3′). The caspase-3 loci were evaluated using the following primers: 5′ (5′-TGC-TAA AGC GCA TGC TCC AGA CTG-3′) and 3′ (5′-GCG AGT GAG AAT GTG CAT AAA TTC-3′). The evaluation of QACRG/NEO was undertaken with the following primers 5′ (5′-GGG AAA CCA ACA GTA GTC AGT CCT-3′) and 3′ (5′-GCG AGT GAG AAT GTG CAT AAA TTC-3′).

dox water administration

CC10-rtTA-IFN-γ mice and littermate controls were maintained on normal water until they were 4–6 wk old. They were then randomized to normal water or water with dox as previously described (19).

Pharmacologic interventions

Four- to 6-wk-old transgene-negative and transgene-positive mice were randomized to receive the desired agent or the appropriate vehicle control. Two days later, they were randomized to normal or dox water and maintained on this regimen for 2–4 wk. At the end of this interval, the animals were sacrificed and pulmonary phenotype was assessed as described below. In experiments in which caspase-mediated apoptosis was being evaluated, N-benzylcarboxy-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) (3 μg/kg/day; Calbiochem) was given via an i.p. route. When cathepsins were being evaluated, the broad-spectrum cysteine protease inhibitors leupeptin (15 mg/kg/day; Sigma-Aldrich) or E-64 (7.5 mg/kg/day; Sigma-Aldrich) were given via an i.p. route. To evaluate Cat S, we used the selective Cat S inhibitor 05141 (10 mg/kg/day twice a day by gavage; Sanofi-Aventis). In all cases, comparisons to appropriate vehicle controls were undertaken.

Characterization of the Cat S inhibitor 05141

Solutions of Cat S inhibitor in varying concentrations were prepared in 10 μl of DMSO and then diluted into 40 μl of the appropriate assay buffer. The following assay buffers were used: Cat S assay: MES, 50 mM (pH 6.5); EDTA, 2.5 mM; NaCl, 100 mM; cathepsin K or L assay: MES, 50 mM (pH 5.5); EDTA, 2.5 mM; DTT, 2.5 mM; cathepsin B assay: N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 50 mM (pH 6.0); polyoxyethylenesorbitan monolaurate, 0.05%; and DTT, 2.5 mM. Human Cat S (0.158 pmol), cathepsin K (0.091 pmol), cathepsin L (0.05 pmol), or cathepsin B (0.025 pmol) in 25 μl of assay buffer were added to the compound dilutions. The assay solutions were incubated for 30 min at ambient temperature. Substrates for Cat S (Z-Val-Val-Arg-AMC, 9 nmol), cathepsin K (Z-Phe-Arg-AMC, 4 nmol), cathepsin L (Z-Phe-Arg-AMC, 1 nmol), or cathepsin B (Z-FR-AMC, 20 nmol) in 25 μl of assay buffer were added to the assay solutions. Hydrolysis was followed spectrophotometrically (l460 nm) for 5 min. Apparent inhibition constants (K_i) were calculated using a standard mathematical model. The characteristics of this inhibitor can be seen in Table I⇓.

TUNEL evaluations

End-labeling of exposed 3′-OH ends of DNA fragments in paraffin-embedded tissue was undertaken with the TUNEL in situ cell death detection kit AP (Roche Diagnostics) using the instructions provided by the manufacturer as previously described by our laboratory (32).

Type II alveolar epithelial cell isolation and apoptosis evaluation

Type II cells were isolated from wild-type (WT) and IFN-γ transgenic mice using the methods developed by Corti et al. (33). Annexin V and propidium iodine (PI) staining were undertaken with the Annexin V^FITC apoptosis detection kit (BD Biosciences) as described by the manufacturer. Analysis was undertaken by flow cytometry (BD Biosciences).

Bronchoalveolar lavage (BAL) and quantification of IFN-γ

Mice were euthanized, the trachea was isolated by blunt dissection, tubing was secured in the airway, and BAL with PBS was undertaken as described previously (32). The levels of IFN-γ were determined using a commercial ELISA (R&D Systems) as per the manufacturer’s instructions.

Histologic analysis

Animals were anesthetized, a median sternotomy was performed, and right heart perfusion was accomplished with calcium- and magnesium-free PBS to clear the pulmonary intravascular space. The lungs were then fixed to pressure (25 cm) with neutral-buffered 10% Formalin, fixed overnight in 10% Formalin, embedded in paraffin, sectioned at 5 μm, and stained. H&E stains were performed in the Research Histology Laboratory of the Department of Pathology at Yale University School of Medicine.

Lung volume, morphometric assessment, and compliance assessment

Lung volume, alveolar size, and lung compliance were assessed via volume displacement and morphometric chord length assessments as previously described (19, 32, 34).

mRNA analysis

mRNA levels were assessed using RT-PCR as previously described by our laboratory (19, 32). In the RT-PCR assays, gene-specific primers were used to amplify selected regions of each target moiety. The primers for the targeted genes are detailed in Table II⇓.

In selected experiments, RNase protection was also used. These assays were undertaken with mAPO-1, mAPO-2, and mAPO-3 multiprobe kits (BD Pharmingen) as per the manufacturer’s instructions.

Mouse lung immunohistochemistry

In selected experiments, TUNEL evaluations and immunohistochemistry for surfactant aproprotein C (SP-C) were simultaneously undertaken. In these experiments, slides were deparaffinized with xylene and graded ethanol and taken to water. Microwave Ag retrieval was done with DAKO pH 6 Ag retrieval solution, and slides were treated with DAKO protein block and rinsed. TUNEL (Roche) staining was then undertaken as described above and developed with alkaline phosphatase/5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (blue). Afterward, tissue was counterstained with goat anti-SP-C (Santa Cruz Biotechnology) overnight and developed with biotylinated anti-goat/streptavidin peroxidase/3-amino-9-ethyl carbazole (red).

Protein extraction and Western blot analysis

Whole lung lysates were prepared using lysis buffer as previously described (34). The lysates were then clarified by centrifugation at 10,000 × g for 15 min, and supernatant protein concentrations were determined with a Bio-Rad assay kit (Bio-Rad). The samples were then mixed with an equal volume of 2× SDS-PAGE sample buffer (100 mM Tris-Cl (pH 6.8), 200 mM DTT, 4% SDS, 0.2% bromphenol blue, and 20% glycerol), heated in a boiling water bath, and equal amounts were loaded onto 12% SDS-polyacrylamide gels (Bio-Rad) and transferred to immune-blot polyvinylidene difluoride membrane (Bio-Rad). After transfer the membranes were blocked for 1 h in nonfat dried milk, rinsed, incubated with the appropriate primary Abs for 1.5 h at room temperature or overnight at 4°C, washed, incubated with secondary Ab (diluted 1/1000–1/2000) for 1.5 h at room temperature, and washed in 0.05% Tween 20. Immunoreactive proteins were visualized using the 20× LumiGLO reagent and 20× peroxide according to the manufacturer’s instructions (Cell Signaling Technology). The membranes were exposed to BioMax MR films (Eastman Kodak). Rabbit polyclonal Abs reactive to caspase-3, caspase-9, Cat S, and β-tubulin were purchased from Santa Cruz Biotechnology and polyclonal anti-caspase-8 was obtained from BD Biosciences.

Assessments of caspase activity

The bioactivities of caspases-3 and -8 were measured with colorimetric assays using the CaspACE assay system (Promega) and the Caspase 8 Colorimetric Activity assay kit (Chemicon), respectively. In brief, lung tissues were homogenized and centrifuged, and the supernatants were incubated with the colorimetric substrate Ac-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) or N-acetyl-Ile-Glu-Thr-Asp-p-nitroanilide (Ac-IETD-pNA) for caspases 3 and 8, respectively. The release of pNA was measured at 405 nm using a spectrophotometer.

Statistics

Normally distributed data are expressed as mean ± SEM and were assessed for significance by Student’s t test or ANOVA as appropriate. Data that were not normally distributed were assessed for significance using the Wilcoxon rank sum test for groups of two or the Kruskal-Wallis statistic for groups of three. Statistical analysis was performed using Stata (version 7.0) and Deltagraph. Statistical significance was defined at a level of p < 0.05.

Effect of IFN-γ on lung cell apoptosis

We previously demonstrated that the transgenic overexpression of IFN-γ causes emphysema in the murine lung (19). To investigate the mechanism(s) of this response, TUNEL staining was used to determine whether transgenic IFN-γ caused DNA injury and/or apoptosis in this setting. In WT mice on normal or dox water and transgenic mice on normal water, ≤2.5% of all nuclei were TUNEL stain positive (Fig. 1⇓A and data not shown). In contrast, IFN-γ induction in transgenic mice caused a remarkable increase in the number of TUNEL⁺ nuclei (Fig. 1⇓A). These effects were seen after as little as 1 day and peaked after 14–28 days of dox administration (Fig. 1⇓A). At these latter time points, ∼33% of the nuclei in lung tissue sections were TUNEL stain positive (Fig. 1⇓A). The majority of these cells were airway epithelial cells and type I and type II alveolar epithelial cells on light microscopic evaluations (Fig. 1⇓, B and C). In accord with these observations, double staining with TUNEL and anti-SP-C Abs revealed a large number of alveolar type II epithelial cells that were TUNEL positive and SP-C positive (Fig. 1⇓, D). TUNEL-positive and SP-C-negative cells were also appreciated (data not shown).

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FIGURE 1.

Transgenic IFN-γ-induced DNA injury and apoptosis. CC10-rtTA-IFN-γ transgene (+) mice and their transgene (−) littermate controls were placed on normal or dox water for the noted intervals. Their lungs were then removed and TUNEL staining was evaluated. A illustrates the percentage of total nuclei that were TUNEL (+) in transgene (-) mice on normal water (▦), transgene (−) mice on dox water (▨), transgene (+) mice on normal water (▤), and transgene (+) mice on dox water (▪). Each value represents the mean ± SEM of a minimum of six animals (∗, p < 0.01 vs the other three groups). The TUNEL staining in lungs from transgene (−) and transgene (+) mice treated for 4 wk with normal water (dox (−)) or dox water (dox (+)) at 10× is seen in B. The TUNEL staining in lungs from transgene (−) (WT) mice and transgene (+) mice (γ (+)) treated with Z-VAD-fmk or PBS vehicle control (100×) after 4 wk of dox administration is seen in C. D, Double staining is used to localize TUNEL staining (blue, left), SP-C (red) (middle), and TUNEL (+)/SP-C (+) cells (right). Representative double staining cells are highlighted by the arrows. The experiments in A–C are representative of a minimum of 10 similar evaluations and the results in D are representative of the results obtained in four similar evaluations.

To define the cellular response(s) that was ongoing, dual annexin V and PI staining and FACS analysis were undertaken. Using whole lung cells from WT and transgenic mice, these studies demonstrated that many of the cells were undergoing apoptosis (increased annexin V staining only). Smaller numbers of cells manifest increased PI staining only and were undergoing necrosis or late-stage apoptosis with compromised plasma membranes. Small numbers of cells also had increases in annexin V and PI staining. These cells were undergoing apoptosis and necrosis. At the peak of this response, ∼15% of whole lung cells were undergoing cell death (Fig. 2⇓A). A similar increase in the number of cells undergoing apoptosis was seen with dual staining and FACS analysis of purified alveolar type II cells (Fig. 2⇓B) from WT and transgenic mice. These studies demonstrate that IFN-γ is a potent inducer of epithelial cell DNA injury and apoptosis in the murine lung.

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FIGURE 2.

Dual annexin V and PI staining. Apoptosis and necrosis were evaluated with annexin V and PI staining of total lung cells (A) and alveolar type II cells (B) isolated from WT and transgene (+) mice on dox water for 4 wk. The experiments in these panels are representative of the results obtained in four similar evaluations.

Role of apoptosis in IFN-γ-induced emphysema

To determine whether apoptosis contributed to the pathogenesis of IFN-γ-induced alveolar remodeling and emphysema, we compared the effects of IFN-γ in mice treated with the caspase inhibitor Z-VAD-fmk or vehicle control. In addition, we bred the CC10-rtTA-IFN-γ transgenic mice with caspase-3^−/− animals and compared the effects of IFN-γ in mice with (+/+) and (−/−) caspase-3 loci. As previously reported (19), IFN-γ caused emphysema with alveolar and lung enlargement and increases in pulmonary static compliance (Fig. 3⇓ and data not shown). The chemical (Z-VAD-fmk) and the genetic (caspase-3^−/−) interventions decreased the levels of IFN-γ-induced apoptosis by ≥85%. They simultaneously decreased the emphysema-generating effects of IFN-γ. These alterations were readily apparent in measurements of lung volume, alveolar morphometry, alveolar histology, and lung compliance in mice on dox for as little as 2 wk and as long as 4 mo (Fig. 3⇓ and data not shown). In all cases, these alterations, although significant, did not return the parameters to normal. (Fig. 3⇓ and data not shown). Thus, IFN-γ induces pulmonary emphysema via a mechanism that is at least partially apoptosis dependent.

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FIGURE 3.

Effects of inhibition of apoptosis on IFN-γ-induced emphysema. A, C, and E, Transgene (−) and transgene (+) mice were randomized to receive Z-VAD-fmk or PBS vehicle control and then placed on dox for 2 wk. B, D, and F, We compare transgene (+) mice with WT (+/+) and null mutant (−/−) caspase-3 loci. Lung volume (A and B), chord length (C and D), and alveolar histology (E and F) were evaluated. The values in A–D are the mean ± SEM of evaluations in a minimum of six animals. E and F, Representative of six similar experiments (∗, p < 0.01).

IFN-γ regulation of Cat S

We previously demonstrated that IFN-γ causes prominent alterations in protease-antiprotease balance in the lung (19). To define the role(s) of Cat S in this response, studies were undertaken to define the effects of IFN-γ on this important protease. As seen in Fig. 4⇓, IFN-γ was a potent stimulator of Cat S mRNA and protein. This stimulation was seen in lungs from transgenic mice after as little as 24 h of dox administration and persisted throughout the study interval (Fig. 4⇓A and data not shown). Immunohistochemical evaluations demonstrated that Cat S protein could only be appreciated in rare, random cells in lungs from WT mice on normal water or dox water and transgenic mice on normal water. In contrast, Cat S protein was readily appreciated in macrophages and alveolar epithelial cells in lungs from transgenic mice on dox water (Fig. 4⇓B). This staining was Cat S specific because it was not seen in the absence of primary Ab and was abrogated by excess unlabeled Cat S protein (data not shown). These studies demonstrate that IFN-γ is a potent stimulator of macrophage and epithelial cell Cat S.

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FIGURE 4.

Regulation of Cat S by IFN-γ. WT (transgene (−)) and transgene (+) mice were randomized to normal water or dox water. At the noted intervals, the levels of Cat S mRNA were evaluated (A). After 4 wk of dox, Western blot analysis (B) and immunohistochemistry were used to identify and localize Cat S (C, top panels, ×10; bottom panels, ×40). The open and solid arrows in C highlight Cat S staining macrophages and alveolar epithelial cells, respectively.

Role of Cat S in IFN-γ-induced apoptosis

Studies were next undertaken to determine whether Cat S played an important role in the pathogenesis of IFN-γ-induced DNA injury, apoptosis, and emphysema. This was done by comparing the levels of apoptosis and emphysema in transgenic mice treated with the selective Cat S inhibitor 05141 (Table I⇑) or its vehicle control. We also bred the IFN-γ transgenic mice with Cat S^−/− mice and compared the effects of IFN-γ in mice with (+/+) and (−/−) Cat S loci. As noted above, IFN-γ was a potent inducer of DNA injury/apoptosis and emphysema in the murine lung. Importantly, treatment with 05141 or a null mutation of Cat S significantly inhibited these injury/apoptosis and emphysematous responses (Fig. 5⇓). Similar decreases in DNA injury/apoptosis and emphysema were seen in mice treated with the broad-spectrum, noncaspase, cysteine protease inhibitors leupeptin and E-64 (data not shown). Thus, IFN-γ induces pulmonary epithelial cell DNA injury and apoptosis via a mechanism that is, at least in part, Cat S dependent.

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FIGURE 5.

Effects of 05141 or a Cat S null mutation on IFN-γ-induced DNA injury/apoptosis and emphysema. A, C, and E, Transgene (−) and transgene (+) mice were randomized to treatment with 05141 or PBS vehicle control and then placed on dox. B, D, and F, We compare transgene (+) mice with (+/+) and null mutant (−/−) Cat S loci. The percentage of cells that were TUNEL (+) (A and B), the chord length of the alveoli (C and D), and the histologic appearance of the tissues (E and F) were evaluated. The values in A–D are the mean ± SEM of evaluations in a minimum of six animals. E and F, Representative of six similar experiments (∗, p < 0.01).

Mechanism of IFN-γ-induced apoptosis and regulation by Cat S

To define the mechanism of IFN-γ-induced apoptosis and the mechanism by which Cat S regulates this response, we compared the expression of key mediators of apoptosis in IFN-γ transgenic mice with (+/+) and (−/−) Cat S loci. IFN-γ-induced apoptosis was associated with significant increases in the levels of mRNA encoding key components of the extrinsic (death receptor) and intrinsic (mitochondrial) apoptosis pathways, including Fas, Fas ligand (FasL), TNF-α. TNF-related apoptosis-inducing ligand (TRAIL), Bak, Bid, Bim, caspase-3, -6, -8, and -9, and protein kinase Cδ (PKCδ) (Fig. 6⇓A and data not shown). Interestingly, the Bcl-2 family apoptosis inhibitor A1 was also stimulated while apoptosis-inducing factor and Bcl-2 were not similarly altered (Fig. 6⇓A). Bioassays and Western blot evaluations also demonstrated that IFN-γ activated caspase-3, -8, and -9 (Fig. 6⇓, B and C). Interestingly, an absence of Cat S diminished the ability of IFN-γ to augment the levels of mRNA encoding Fas, FasL, TRAIL, TNF-α, Bid, Bim, PKCδ, and caspase-3, -8, and -9 (Fig. 6⇓A and data not shown). It also diminished the ability of IFN-γ to activate caspase-3, -8, and -9 (Fig. 6⇓, B and C). The Cat S mutation did not, however, alter the expression of Bak, caspase-6, Bcl-2, or A₁ (Fig. 6⇓A). Similar alterations were induced by 05141, leupeptin, and E-64 (data not shown). Importantly, the chemical and genetic Cat S manipulations and the leupeptin and E-64 treatments did not alter the levels of BAL IFN-γ, demonstrating that the alterations they induced were not due to decreases in transgenic cytokine production (data not shown).

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FIGURE 6.

Mechanisms of apoptosis and contributions of Cat S in IFN-γ transgenic mice. Transgene (−) and (+) mice with (+/+) and (−/−) Cat S loci were randomized to dox water or normal water for 4 wk. A, Whole lung RNA was extracted and the levels of mRNA encoding key apoptosis-regulating genes were evaluated. B, A bioassay (top) and Western blot (bottom) were used to evaluate the activation of caspase-3. C, A bioassay was used to quantitate caspase-8 activity (top) and Western blots (bottom) were used to assess caspase-8 and -9 activation. A—C, Representative of three similar evaluations.

Role of apoptosis in IFN-γ-induced inflammation and protease induction

To further understand the importance of apoptosis, studies were undertaken to determine whether blocking apoptosis altered the ability of IFN-γ to induce tissue inflammation. When compared with WT mice on normal or dox water, transgenic IFN-γ caused a significant increase in BAL total cell, neutrophil, lymphocyte, and macrophage recovery and a patchy mononuclear tissue inflammatory response. These alterations were seen in transgenic mice on dox for as little as 2 wk and as long as 4 mo (Fig. 7⇓ and data not shown). These responses were at least partially apoptosis dependent because Z-VAD-fmk treatment or a null mutation of caspase-3 ameliorated the IFN-γ-induced increases in BAL total cell, neutrophil, lymphocyte, and macrophage accumulation and tissue inflammation at all time points (Fig. 7⇓, A, B. and D, and data not shown). In accord with our demonstration that IFN-γ induces apoptosis via a Cat S-dependent mechanism, similar decreases in BAL and tissue inflammation were noted in transgenic mice with null vs (+/+) Cat S loci (Fig. 7⇓, C and E).

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FIGURE 7.

Roles of apoptosis and Cat S in IFN-γ regulation of inflammation. A, Transgene (−) and (+) mice were treated with Z-VAD-fmk (+) or vehicle and then given dox water. B and C, Transgene (−) and (+) mice with (+/+) and (−/−) caspase-3 and Cat S loci, respectively, were treated with dox. Two weeks later, BAL was undertaken and total cell recovery (A–C) and histology (D and E) were assessed. The values in A–C are the mean ± SEM of evaluations in a minimum of six animals. D and E, Representative of a minimum of four similar evaluations (∗, p < 0.05). Casp-3, Caspase-3.

When compared with WT mice on normal or dox water, transgenic IFN-γ caused a significant increase in the levels of mRNA encoding cathepsins B, S, K, L, and H and matrix metalloproteinase (MMP)-2, -9, -12, and -14 (Fig. 8⇓). These responses were partially apoptosis dependent because null mutations of caspase 3 or Cat S ameliorated the IFN-γ-induced increases in the levels of mRNA encoding MMP-9 and cathepsin K in mice on dox for as little as 2 wk and as long as 4 mo (Fig. 8⇓). In contrast, the levels of mRNA encoding cathepsin H and MMP-2 and -12 were not altered in these systems (Fig. 8⇓, A and B). When viewed in combination, these studies demonstrate that cathepsin-dependent DNA injury/apoptosis is a critical event in the pathogenesis of the inflammatory and the proteolytic responses at sites of IFN-γ-induced tissue remodeling.

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FIGURE 8.

Roles of apoptosis and Cat S in IFN-γ regulation of lung proteases. Transgene (−) and (+) mice with (+/+) and (−/−) caspase-3(A) and Cat S (B) loci were treated with dox. Two weeks later, the levels of mRNA encoding the noted cathepsins and MMPs were assessed. The panels are representative of a minimum of four similar evaluations.