Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase– and cathepsin-dependent emphysema

Cigarette smoke exposure is the major cause of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop significant COPD, and patients with asthma or asthma-like airway hyperresponsiveness or eosinophilia experience accelerated loss of lung function after cigarette smoke exposure. Pulmonary inflammation is a characteristic feature of lungs from patients with COPD. Surprisingly, the mediators of this inflammation and their contributions to the pathogenesis and varied natural history of COPD are not well defined. Here we show that IL-13, a critical cytokine in asthma, causes emphysema with enhanced lung volumes and compliance, mucus metaplasia, and inflammation, when inducibly overexpressed in the adult murine lung. MMP-2, -9, -12, -13, and -14 and cathepsins B, S, L, H, and K were induced by IL-13 in this setting. In addition, treatment with MMP or cysteine proteinase antagonists significantly decreased the emphysema and inflammation, but not the mucus in these animals. These studies demonstrate that IL-13 is a potent stimulator of MMP and cathepsin-based proteolytic pathways in the lung. They also demonstrate that IL-13 causes emphysema via a MMP- and cathepsin-dependent mechanism(s) and highlight common mechanisms that may underlie COPD and asthma.

Chronic obstructive pulmonary disease (COPD) is a generic term that encompasses chronic bronchitis (CB), small airway disease, and emphysema. Patients with CB have airway mucus metaplasia and mucus gland hypertrophy, and emphysema is characterized by tissue destruction with resulting alveolar enlargement. COPD affects 16 million people in the United States alone and is the fourth leading cause of death worldwide (1).

COPD occurs predominately in cigarette smokers (1). However, the relationship between cigarette smoking and COPD is complex, with only 10–15% of active smokers developing clinical COPD (2, 3) and estimates of cumulative smoking exposure explaining only 10–15% of the variation in pulmonary function seen in population-based investigations (2, 4, 5). Studies of this heterogeneity have also demonstrated that the presence of concurrent asthma or asthma-like airways hyperresponsiveness (AHR) or eosinophilia correlates with the development of or acquisition of a COPD phenotype characterized by accelerated loss of pulmonary function and chronic symptomatology (2, 6–9). They also highlighted the large number of patients that manifest features of COPD and asthma (1). These observations led, in 1961, to the formulation of the “Dutch Hypothesis.” This hypothesis proposes that the distinctions between COPD and asthma are not absolute and that similar mechanisms can contribute to the pathogenesis of both disorders (10, 11). They also led Burrows et al. to suggest, over a decade ago, that chronic airflow obstruction in adults should be differentiated on the basis of whether or not there are accompanying asthmatic features (12, 13). To date, however, the mechanisms responsible for the AHR and eosinophilia seen in COPD, and the existence of and nature of the biologic responses that might be common to COPD and asthma have not been elucidated.

The protease/antiprotease hypothesis has dominated pathogenetic thinking in COPD for more than 35 years. It proposes that an antiprotease “shield” protects the normal lung from locally elaborated proteases and that emphysema is the result of an abnormal increase in proteases and/or reduction in pulmonary antiproteases (1). Inflammation, characterized by increased numbers of macrophages, lymphocytes, neutrophils, and/or eosinophils is a characteristic feature of lungs from patients with COPD (1, 14–19). However, the nature of the mediators involved in this inflammation and the ability of these mediators to generate the emphysema and mucus changes, protease/antiprotease alterations, and varied natural history of COPD have not been investigated.

Because Th2-dominated inflammation underlies the pathogenesis of asthma and generates AHR and eosinophilia (20–22), we hypothesized that Th2 cytokines can also activate proteolytic pathways that could contribute to the pathogenesis of COPD. To test this hypothesis, we used an inducible overexpression transgenic modeling system to target IL-13, a Th2 cytokine that is strongly implicated in the pathogenesis of asthma and causes AHR and eosinophilia (20, 23), to the adult murine lung. These studies demonstrate that IL-13 causes a phenotype that mirrors human COPD including emphysema with enhanced lung volumes and pulmonary compliance; mucus metaplasia; and macrophage-, lymphocyte-, and eosinophil-rich inflammation. They also define the MMP and cathepsin abnormalities that generate the emphysema and demonstrate the efficacy of proteolytic blockade in ameliorating this response.

Rationale for and generation of inducible transgenic mice. Mice and humans are born with immature lungs that contain large sack-like structures. Normal alveolar size and number are acquired only after a growth and septation process that occurs during the first month and years of life, respectively (36). Thus, enlarged alveoli in adult mice or humans can be caused by processes that alter alveolar development or processes that destroy otherwise normal adult alveolar septae (1, 24, 37). The emphysema in COPD is caused by the latter (1). The need to differentiate development-dependent and adult-onset phenotypes confounds the use of transgenic modeling to study the pathogenesis of COPD. Specifically, any overexpression system in which a protein is expressed during lung development cannot be appropriately interpreted to represent destructive emphysema unless it is shown that appropriate lung development occurs before the emphysema is seen. To our knowledge, this has not been accomplished to date using conventional overexpression modeling systems.

To successfully use overexpression modeling to characterize the effects of IL-13 in the adult lung, we used a novel, externally regulable, dual-construct overexpression transgenic system developed in our laboratory (24). The constructs required for these transgenics were prepared, purified, and microinjected (Figure 1). Four dual transgene–positive CC10-rtTA-IL-13 founder mice were identified, and lines were obtained after breeding with C57BL/6 animals. These mice were kept on normal water until they were 1 month of age. They were then randomized to normal water or dox water. In the absence of dox administration, BAL IL-13 levels were less than or equal to 75 pg/ml. Increased levels of BAL IL-13 were noted within 48 hours, and steady-state levels between 0.5 and 1.5 ng/ml were seen within 96 hours of dox administration (data not shown). In all cases, organ blot analysis demonstrated that IL-13 was produced in a lung-specific fashion (Figure 2). Qualitatively, similar phenotypes were seen in all transgenic lines. They are summarized as follows:

Figure 2

Organ specificity of IL-13 expression. CC10-rtTA-IL-13 mice were given dox water for 2 weeks. RNA was then isolated from the lung and other organs and IL-13 mRNA quantitated by Northern analysis.

Histological, structural, and physiological evaluation. Lungs from nontransgenic mice on normal or dox water did not show any histological abnormalities. In addition, they could not be distinguished on H&E evaluation from lungs from double transgenic mice on normal water (Figure 3a). In contrast, dox-induced IL-13 caused inflammatory cell infiltration and pronounced emphysema in transgenic animals. The emphysema manifested as histological (Figure 3, a and b) and morphometrically obvious (Figure 3c) alveolar enlargement with loss of the orderly appearance of the acinus (38). It was prominent after 7 days and continued to progress throughout the first month of dox administration (Figure 3b). The inflammation contained increased numbers of macrophages, lymphocytes, and eosinophils in peribronchial and parenchymal tissues and BAL. It could be seen within 7 days and was more prominent after 1 month of dox administration (Figure 3d, and data not shown).

Figure 3

Characteristics of CC10-rtTA-IL-13 mice. (a) Compares H&E-stained lungs from 6-week-old nontransgenic [Tg (–)] and double transgenic [Tg (+)] mice that received normal water [Dox (–)] or dox water [Dox (+)] for the last 2 weeks of their lives. Emphysema is seen only in double transgenic animals receiving dox water. Unless otherwise stated, all photos are at ×25 original magnification. (b) Illustrates the kinetics of this response. Alveolar enlargement was seen after 1 week and was more pronounced at 1 month. (c) Illustrates the chord lengths of nontransgenic and double transgenic mice on normal or dox water for 4 weeks. ^AP < 0.01 versus all other conditions. (d) Higher-power (×100) view comparing the histological features (H&E) of nontransgenic and double transgenic mice that had received normal or dox water for 1 month.

Enhanced lung volumes and increased pulmonary compliance are characteristic features of human emphysema (1). To determine whether this murine model recapitulated these important clinical features of COPD, we compared the lung volumes and compliances of nontransgenic and transgenic mice before and after dox-induced IL-13 production. As seen in Figure 4, similar pressure-volume relationships were seen in lungs from nontransgenic mice that received normal water and dox water. A slight increase in compliance was seen in transgenic mice on normal water. In contrast, marked increases in pulmonary compliance and lung volumes were seen in transgenic mice in which IL-13 was induced with dox water (Figure 4, and data not shown). When viewed in combination, these studies demonstrate that IL-13 causes emphysema with lung volume enlargement and enhanced pulmonary compliance analogous to that seen in human COPD.

Figure 4

Effect of IL-13 on lung compliance. In these experiments, 1-month-old transgene negative (Neg) and dual transgene positive (Pos) mice were randomized to water with (+) or without (–) dox for 4 weeks before evaluation. Pressure-volume relationships were evaluated as described in Methods.

Mucus evaluation. To determine whether IL-13 caused mucus abnormalities comparable to those in COPD, we compared the goblet cells in nontransgenic and transgenic mice receiving normal and dox water using D-PAS staining and calculated HMIs. Lungs from nontransgenic animals receiving normal water or dox water did not contain D-PAS⁺ cells. In accord with their low-level IL-13 production, small numbers of D-PAS–positive cells were noted in transgenic animals receiving normal water (HMI = 10–20). In contrast, impressive increases in the levels of D-PAS staining were appreciated with dox-induced IL-13 production (HMI = 60–80) (Figure 5). Given that mice do not have intrapulmonary submucosal mucus glands (39), IL-13–induced alterations in these structures could not be assessed. These studies demonstrate that IL-13 is a potent inducer of airway mucus metaplasia.

Figure 5

Mucus responses in nontransgenic and transgenic mice. The mucus metaplasia in nontransgenic (left) and transgenic mice (right) that received dox for 2 weeks are compared using PAS stains.

Effect on MMPs. To define the mechanism(s) of IL-13–induced alveolar septal destruction, we compared the levels of mRNA encoding COPD-relevant MMPs in 2-month-old inducible CC10-rtTA-IL-13 mice and their nontransgenic littermate controls that had been randomized to normal water or dox water for 1 month. These studies demonstrated that IL-13 is a potent stimulator of MMP-9 and -12 mRNA accumulation (Figure 6). MMP-2, MMP-13, and MMP-14 were also induced, whereas comparable alterations in MMP-7 were not appreciated (Figure 6). Kinetic evaluations demonstrated that the increases in the levels of mRNA encoding MMP-12; MMPs -2, -13, and -14; and MMP-9 were seen after 1 day, 10 days, and 10–30 days of dox administration, respectively (data not shown). Western blot analysis confirmed the induction of MMP-2, MMP-12, MMP-13, and MMP-14 (Figure 7a, and data not shown) and immunohistochemistry demonstrated that MMP-12 was seen predominantly in macrophages, and MMP-9 was seen in parenchymal locations in epithelial cells and interstitial fibroblast-like cells (Figure 7, b–f, and data not shown). Zymography also demonstrated IL-13–induced BAL moieties that comigrated with the unprocessed and/or fully processed forms of MMP-9, -12, and -2 (Figure 8, a and b). The activities in these bands were inhibited by EDTA, which establishes them as metalloproteinases. Thus, IL-13 is a potent inducer of a variety of MMP moieties that collectively have potent elastolytic and collagenolytic activities.

Figure 6

Effect of IL-13 on respiratory MMP mRNA. RT-PCR was used to compare the levels of mRNA encoding a variety of MMPs in lungs from 2-month-old nontransgenic and transgenic CC10-rtTA-IL-13 mice on normal water (–) or dox water (+) from 1 to 2 months of age.

Figure 7

Western analysis and immunohistochemistry of BAL fluids and tissues from CC10-rtTA-IL-13 mice. BAL fluids and tissues were obtained from nontransgenic and transgenic CC10-rtTA-IL-13 animals on normal or dox water from 1–2 months of age. (a) Immunoblots were performed on BAL fluids using antibodies against MMP-12. (b–f) Immunohistochemistry was used to localize MMP-12 in the lung. In the immunohistochemical evaluation, photomicrograph b is from double transgenic mice treated with dox and stained with preimmune antiserum; c and d are from nontransgenic mice treated with normal and dox water, respectively, and stained with antiserum against MMP-12, and e and f are from double transgenic mice treated with normal and dox water, respectively, and stained with anti–MMP-12.

Figure 8

Zymography of BAL fluid from IL-13–overexpressing mice. BAL fluids were obtained from nontransgenic and transgenic CC10-rtTA-IL-13 animals on normal or dox water at 1–2 months of age. Casein zymography (a) and gelatin zymography (b) were undertaken. The presence of MMP-9, -12, and -2 can be appreciated in the zymograms.

Effects of MMP antagonist. Studies were next undertaken to define the importance of the induced MMPs in the pathogenesis of the lesions in our animals. This was done by determining whether the broad-spectrum MMP antagonist GM-6001 (32, 33) altered the IL-13–induced phenotype in our transgenic mice. GM-6001 did not alter the lung or alveolar size of nontransgenic animals receiving normal or dox water. It did, however, abrogate the IL-13–induced increase in lung volume by greater or equal to 70% (Figure 9a) and caused comparable decreases in lung and alveolar size assessed visually, histologically, and via chord length measurements (P < 0.001 for all comparisons) (Figure 9, b and c, and data not shown). GM-6001 also caused impressive alterations in pulmonary inflammation, decreasing BAL cell recovery and eosinophil and lymphocyte numbers by more than 90% (P < 0.001 for all comparisons). C1006, an inactive peptide control, and DMSO, the vehicle control, did not cause similar alterations (Figure 9, a–c, and data not shown). GM-6001 also did not inhibit IL-13–induced mucus metaplasia (data not shown). In these experiments, GM-6001 also did not alter the levels of IL-13 in the BAL fluids from double transgenic animals on dox water. This demonstrates that the effects of GM-6001 were not due to an inhibitory interaction with the CC10 promoter or drug-induced anorexia with a decrease in dox water intake by our animals (data not shown). Lastly, GM-6001 did not inhibit the levels of MMP-2, -12, -13, and -14 gene expression in lungs from nontransgenic or double transgenic mice on normal or dox water (Figure 10, and data not shown). This demonstrates that although GM-6001 decreases inflammatory cell influx by more than 90%, these inflammatory cells do not contribute, in a major way, to the induction of these MMP moieties in IL-13–treated lungs. When viewed in combination, these studies demonstrate that IL-13 induces emphysema and tissue inflammation via an MMP-dependent pathway. They also demonstrate that IL-13 utilizes a different MMP-independent pathway to induce airway mucus metaplasia.

Figure 9

Effect of MMP antagonist on IL-13–induced emphysema. One-month-old nontransgenic and transgenic CC10-rtTA-IL-13 mice on dox water were randomized to receive daily intraperitoneal doses of GM-6001 (200 mg/kg), C1006, and or DMSO. The effects of these agents on lung volume and size (a and b) and histology (c) are illustrated. The ability of GM-6001, but not its controls, to inhibit lung volume, decrease lung size, and decrease alveolar size can be readily appreciated. ^AP < 0.05 versus transgenic mice on dox water that received and did not receive C1006 or DMSO.

Figure 10

Effect of GM-6001 on IL-13–induced MMP mRNA. One-month-old nontransgenic and transgenic CC10 rtTA-IL-13 mice were randomized to receive daily doses of GM-6001 or DMSO (–) starting 1 day before and continuing for a 10-day cycle of dox administration. The effects of these interventions on the levels of mRNA encoding MMP-2, -12, -13, and -14 are illustrated.

IL-13 regulation of cysteine proteinases. Studies were also undertaken to determine whether cysteine proteases were induced by IL-13 in our modeling system. This was done by comparing the levels of mRNA encoding a number of airway-relevant cysteine proteases in lungs from CC10-rtTA-IL-13 animals and littermate controls. As shown in Figure 11a, IL-13 caused impressive increases in the levels of mRNA encoding cathepsins B, H, K, S, and L. This increase occurred predominantly in resident lung cells because GM-6001, which caused a greater than 90% decrease in inflammatory cell influx, did not alter the ability of IL-13 to induce these cathepsin moieties (data not shown). Active site probe analysis, which identifies bioactive cathepsin moieties (30), also demonstrated increased levels of these cathepsins in lung lysates from IL-13–expressing mice and free cathepsin S in BAL fluids from these animals (Figure 11, b and c). Thus, IL-13 is a potent inducer of cysteine proteinase production in the lung.

Figure 11

Effect of IL-13 on respiratory cathepsins. (a) Northern analysis was used to compare the levels of cathepsin mRNA in lungs from 8-week-old nontransgenic and transgenic CC10-rtTA-IL-13 mice that received normal water or dox water for the 4-week interval before evaluation. (b and c) Illustration of the levels of active cathepsin proteins (assessed with active site probe analysis) in lung lysates and BAL, respectively, from these animals.

Effects of cysteine proteinase antagonists. To define further the role(s) of the induced cysteine proteinases, we determined whether the cysteine protease inhibitors E-64 and leupeptin altered the phenotype of double transgenic CC10-rtTA-IL-13 mice. In these studies, neither E-64 nor leupeptin altered the size of the lungs or alveoli of nontransgenic mice on normal water or dox water. In contrast, they were potent inhibitors of IL-13-induced emphysema, causing a 65% or greater decrease in the IL-13–induced increase in lung volume (Figure 12a) and comparable decreases in lung and alveolar size assessed visually, histologically, or morphometrically (P < 0.01 for all comparisons) (Figure 12, b and c, and data not shown). E-64 and leupeptin also caused an approximately 60% decrease in BAL cell recovery and an 80% or greater decrease in BAL eosinophils and lymphocytes (P < 0.001 for all comparisons). Importantly, they did not alter IL-13–induced mucus metaplasia, IL-13–induced MMP gene expression, or the levels of BAL IL-13 (data not shown). These studies demonstrate that cysteine proteases play a major role in the pathogenesis of IL-13–induced emphysema and tissue inflammation, but not in IL-13–induced mucus metaplasia.

Figure 12

Effects of cysteine protease inhibitors on IL-13–induced emphysema. One-month-old nontransgenic and transgenic CC10-rtTA-IL-13 mice on dox water were given daily intraperitoneal injections of saline, E-64 (7.5 mg/kg), or leupeptin (15 mg/kg). The effects of these interventions on lung volume and size (a and b) and histology (H&E) (c) were evaluated. The ability of E-64 and leupeptin to decrease lung volume, lung size, and alveolar size can be readily appreciated. ^AP < 0.05 versus transgenic mice on dox that received or did not receive saline vehicle control.

Effects of cysteine proteinase antagonists and MMP antagonists in combination. As noted above, IL-13–induced alveolar enlargement was partially abrogated by treatment with cysteine proteinase antagonists or MMP antagonists individually. Studies were next undertaken to determine whether treatment with these protease inhibitors in combination would alter the IL-13 phenotype in a fashion that was quantitatively and/or qualitatively different than would be seen with the antagonists individually. This was done by comparing the size of the lungs and alveoli from double transgenic mice that received daily doses of GM-6001, leupeptin, or the antagonists in combination starting the day before and throughout a 10-day course of dox water administration. GM-6001 and leupeptin individually decreased lung volume and alveolar size (Table 2, and data not shown). Mice receiving both antagonists manifest a further decrease in alveolar and lung size (Table 2, and data not shown). The lungs from mice that received the antagonists in combination were significantly smaller than the lungs from mice that were treated with the antagonists individually or with the appropriate vehicle controls (P < 0.02 for all comparisons). Similar results were obtained with experiments in which GM-6001 and E-64 were used (data not shown). In no case, however, did the proteases in combination restore alveolar volume or lung size to normal (Table 2, and data not shown).

Table 2

Effects of protease inhibitors in combination on lung volume

IL-13 regulation of antiproteases in the lung. To understand fully the balance of proteases and antiproteases in the lungs of these mice, the levels of expression of lung-relevant antiproteases in IL-13 overexpressing mice and appropriate controls were also evaluated. IL-13 did not alter the expression of TIMP-2, TIMP-3, TIMP-4, SLPI, or cystatin C. Interestingly, IL-13 did cause an approximately 80% decrease in pulmonary α₁-AT expression (Figure 13). It also caused a significant increase in TIMP-1 expression that was seen after as little as 1 day and was readily apparent with longer periods of dox administration (Figure 13, and data not shown) (P < 0.05 for all comparisons).

Figure 13

Effect of IL-13 on respiratory antiproteases. RT-PCR was used to compare the levels of mRNA encoding respiratory relevant antiproteases in 2-month-old nontransgenic and transgenic CC10-rtTA-IL-13 mice that received either normal water (–) or dox water (+) from 1 month of age. The lack of effect of IL-13 on TIMPs-2, -3, and-4, SLPI, and cystatin C and the ability of IL-13 to inhibit α₁-AT and stimulate TIMP-1 can be appreciated.

Increases in total cell recovery, macrophages, neutrophils, lymphocytes (often CD8+), and eosinophils are characteristic findings in BAL fluids and/or lung biopsies from patients with COPD (15–17, 19, 40, 41). We hypothesized that mediators involved in these inflammatory responses play central roles in the pathogenesis of this disorder(s). We also hypothesized that mediators that generate Th2-type tissue inflammatory responses also activate proteolytic pathways that contribute to these diseases based on evidence that concurrent asthma, AHR, and eosinophilia correlate with accelerated respiratory deterioration in COPD. To test this hypothesis, we used a regulatable overexpression transgenic modeling system to target IL-13 to the adult murine lung. These studies demonstrate, for the first time, that the overexpression of IL-13 causes a phenotype that mirrors human COPD with prominent emphysema, enlarged lungs, enhanced pulmonary compliance, mucus metaplasia, and a mixed tissue inflammatory response. They also demonstrate that MMPs and cathepsins are induced by IL-13 and play important roles in the pathogenesis of the emphysema in these animals. These are the first studies to use inducible overexpression modeling to investigate the cellular and molecular events involved in the pathogenesis of emphysema. They are also the first to link, in a cause and effect fashion, any mediator of tissue inflammation and emphysema and define the protease/antiprotease alterations that generate this response. When viewed in combination, these studies suggest that IL-13 is an important upstream regulator of pulmonary protease/antiprotease balance that can contribute to the pathogenesis of COPD. Because IL-13 is strongly implicated in the pathogenesis of asthma and can also induce AHR and tissue eosinophilia (20, 23), these studies provide molecular support for the Dutch contention that common mechanisms underlie asthma and COPD in some individuals.

IL-13 is a pleiotropic cytokine with remarkable effects in the lung. This was illustrated in previous work from our laboratory that demonstrated that the constitutive overexpression of IL-13 causes a macrophage-, lymphocyte-, and eosinophil-rich pulmonary inflammatory response, mucus metaplasia, crystal deposition, subepithelial fibrosis, alveolar enlargement and AHR (20). In these CC10-IL-13 animals, the transgene was activated by the CC10 promoter in utero, and the phenotype that was induced by IL-13 was evaluated when the mice were 1–3 months of age. Thus, these studies did not enable us to define the early events or the kinetics of the IL-13–induced phenotype. In addition, as the majority of the alveoli in the murine (and human) lung are formed by developmental processes that occur after birth, these studies did not enable us to determine whether the alveolar enlargement was due to an alteration in alveolar development or to the destruction of otherwise normally formed alveoli. To address these issues, we used an externally regulatable, lung-targeted overexpression transgenic modeling system. This system allowed us to define the kinetics of the IL-13–induced phenotype in the adult, versus the developing, lung. The validity of this approach was established in early studies that demonstrated that indistinguishable phenotypes were noted in lungs from CC10-rtTA-IL-13 double transgenic animals that received dox for extended intervals (>2 months) and 1- to 2-month-old CC10-IL-13 mice (Z. Zhu et al., unpublished observation). The studies with the CC10-rtTA-IL-13 mice also demonstrated that IL-13–induced alveolar enlargement is not development dependent because it occurs when IL-13 is produced in adult lungs with normally formed alveoli. These studies also demonstrated that alveolar enlargement and mucus metaplasia are early responses, whereas pulmonary fibrosis and crystal deposition require more extensive periods of IL-13 exposure. When viewed in combination, these studies highlight the remarkable and complementary nature of the mechanistic insights that can be obtained from the combined use of constitutive and externally regulatable lung-targeted overexpression transgenic approaches.

A number of investigators have raised the possibility that MMPs and cathepsins are involved in alveolar destruction in emphysema. These speculations are based on studies from our laboratories and others that demonstrated that MMP-12 is essential for cigarette smoke–induced inflammation and emphysema in the murine lung (28), exaggerated MMP-12 expression in alveolar macrophages from smokers and patients with emphysema (39), and increased levels of MMP-9 and the induction and activation of the MMP-2–MMP-14–TIMP-2 complex in patients with COPD (42, 43). Similarly, macrophages also utilize cathepsins S, L, and K to degrade elastin, and there are increased levels of cathepsin L in BAL cells and fluids from smokers compared with nonsmokers (44–47). With the exception of MMP-12, however, the pathogenetic importance of each of these moieties in animal models of COPD has not been established and the roles of all of these moieties in human COPD have not been defined. Our studies demonstrate that MMP-2, -9, -12, -13, and -14 and cathepsins B, H, S, K, and L are induced by IL-13 in the murine lung. They also demonstrate that interventions that block MMPs or cysteine proteases have remarkable ameliorative effects on IL-13–induced emphysema and tissue inflammation without significantly altering IL-13–induced mucus metaplasia. The MMP findings are particularly impressive because tetracycline-like compounds, like the doxycycline we used to activate our transgenes, have been reported to inhibit MMP activity in a variety of systems (48, 49). These are the first studies to demonstrate that IL-13 is a potent activator of tissue proteolysis and define the components of this proteolytic pathway. They are also the first studies to demonstrate the efficacy of MMP- or cathepsin-based interventions in an inflammation-based model of emphysema. Thus, these studies demonstrate with certainty that MMPs and cathepsins play major roles in the generation of emphysema in our modeling system. They also raise the possibility that IL-13 or the proteolytic pathways it activates play important roles in other pulmonary proteolytic responses. This could be particularly important in diseases characterized by eosinophilia and tissue destruction or remodeling including pulmonary infections and pulmonary eosinophilic and hypersensitivity syndromes such as invasive and semiinvasive aspergillosis and allergic bronchopulmonary aspergillosis. It is important to point out, however, that these studies do not define the contributions that individual MMPs and/or cathepsins make to the IL-13 phenotype. In addition, they do not address the possibility that proteases, other than those that were studied, contribute in important ways to the generation of the IL-13 phenotype. The latter concern may be particularly relevant to ADAM (a disintegrin and metalloproteinase) family members like TNF-α–converting enzyme (TACE) which are also inhibited by hydroxamic-acid–based MMP antagonists (50, 51).

To define the contributions of MMPs and cysteine proteases to the IL-13 phenotype, the effects of MMP and cysteine proteinase inhibitors on IL-13–induced emphysema were evaluated. These studies demonstrated that the MMP antagonists and cysteine proteinase antagonists, individually, partially abrogated the IL-13–induced emphysematous response. They also demonstrated that the protease inhibitors in combination were more effective at reducing lung volume and alveolar size than either inhibitor individually. To our surprise, however, the inhibitors in combination did not totally abrogate IL-13–induced emphysematous alterations. These studies suggest that the MMP and cysteine proteinase pathways contribute in complementary and at least partially separable ways to the pathogenesis of the IL-13–induced emphysematous phenotype. They also suggest that IL-13–regulated proteolytic pathways that are not inhibited by the MMP and cysteine proteinase inhibitors that were used in our studies also contribute to the IL-13 phenotype.

As already noted here, the MMP inhibitor GM-6001 inhibited IL-13–induced emphysema while simultaneously inhibiting IL-13–induced tissue and BAL inflammation. These observations suggest that GM-6001 could mediate its effects by directly inhibiting MMP-induced matrix degradation. Alternatively, inflammatory cells could be the major sources of the MMPs, cathepsins, and other degradative enzymes involved in IL-13–induced pulmonary proteolysis and GM-6001 could mediate its effects solely by decreasing inflammatory cell influx into the lung. In an attempt to address this issue, studies were undertaken to determine whether GM-6001 altered the levels of expression of mRNA encoding IL-13–induced MMPs and cysteine proteinases. These studies demonstrated that GM-6001, although inhibiting emphysema and inflammation, did not significantly decrease the ability of IL-13 to stimulate MMPs -2, -12, -13, and -14 and cathepsin S, H, L, B, and K mRNA accumulation. These observations demonstrate that GM-6001 does not mediate its effects by altering the ability of IL-13 to induce the expression of these MMPs or cathepsins. They also demonstrate that these MMP and cysteine proteinase-stimulatory effects of IL-13 occur predominantly in resident as versus elicited pulmonary cells and that the effects of GM-6001 on the IL-13 phenotype can not be attributed solely to the ability of GM-6001 to alter inflammatory cell recruitment.

To understand fully the protease/antiprotease alterations that lead to emphysema, we also characterized the levels of expression of a variety of important antiproteases in the lungs of IL-13–overexpressing animals. Significant alterations in TIMPs-2-4, SLPI, and cystatin C were not noted. Interestingly, significant stimulation of TIMP-1 was noted. Although this alteration would be expected to inhibit MMP-induced proteolysis, it was not of sufficient magnitude (or in the proper location) to abrogate the MMP-induced emphysema in our animals. In contrast, the expression of pulmonary α₁-AT was decreased in our animals. At first blush, this would be expected to contribute to the pathogenesis of IL-13–induced emphysema. The biologic significance of an isolated decrease in pulmonary α₁-AT production, however, must be evaluated carefully because it is well known that the levels of α₁-AT secreted into the systemic circulation by the liver greatly exceed those produced in pulmonary tissues. In addition, humans with only a single functional α₁-AT allele and levels of circulating α₁-AT approximately 50% of normal do not manifest an increased prevalence of emphysema (1).

The Dutch Hypothesis emphasizes the importance of endogenous host factors in the development of COPD and asthma. In this view, AHR is considered a marker of an endogenous predisposition that contributes to the development of both disorders. This contrasts with the “British Hypothesis,” which emphasizes the importance of exogenous factors such as cigarette smoke exposure and recurrent infections in the generation of COPD (10). Our studies demonstrate that IL-13, a cytokine that is produced in large quantities by Th2 cells and is strongly implicated in the pathogenesis of asthma, AHR, and tissue eosinophilia (20, 23), causes emphysema when expressed in the adult murine lung. These findings support key features of the Dutch Hypothesis and suggest that at least part of the “basic disturbance” described in the hypothesis is the predisposition to produce exaggerated amounts of IL-13 or activate IL-13–like pulmonary proteolytic pathways. It is important to point out, however, that not all patients with COPD have concurrent asthma or manifest eosinophilia or AHR. This would suggest that the Dutch Hypothesis can account for only a subpopulation of patients with COPD and that the IL-13–based transgenic model described in this report is relevant only to these individuals. Interestingly, it may also be applicable to the emphysema that has recently been described in HIV-infected individuals (52), as evolution from a Th1 to Th2 cytokine profile has been associated with progression to AIDS in HIV-infected individuals (53) and HIV induces the expression of the IL-13 receptor on Kaposi’s sarcoma cells (54).

The data in this manuscript demonstrate that IL-13 is a potent stimulator of pulmonary proteolysis. Because IL-13 is produced in large quantities by Th2 and T cell 2 (TC2) cells, these findings support the hypothesis that type II–dominated tissue inflammation can contribute to the pathogenesis of pulmonary emphysema. This interpretation, however, must be viewed with caution because IL-13 can also be produced by type I CD4+ and CD8+ T cells (55), and CD8+ cells are found in large quantities in lungs from patients with COPD (40, 41). Thus, IL-13 may also be produced and activate proteolytic pathways in type I inflammatory responses in the lung. Additional experimentation will be required to determine whether IL-13 is produced in an exaggerated fashion in COPD and if this production is seen in the setting of type I or type II tissue inflammation. If exaggerated levels of IL-13 are noted in tissues from patients with COPD, investigations will also be needed to determine why IL-13 is associated with and potentially causally related to emphysematous responses in some individuals (patients with COPD with asthmatic features) but not in others (patients with “classic” bronchial asthma). These studies will need to determine whether the different outcomes can be attributed, at least in part, to: (a) differences in the location or magnitude of IL-13 or antiproteinase production; (b) individual host susceptibility factors such as polymorphisms of IL-13 or the IL-13 receptor; (c) the possibility that antiproteinase paradigms are induced in asthmatics that negate the proteolytic effects of IL-13; and (d) the possibility that cigarette smoke induces a variety of proteolytic responses, with emphysema being the result of the combined effects of the IL-13 and other proteolytic pathways. Consideration will also need to be directed at the possibility that diagnostic transference is responsible, at least in part, for the belief that asthmatics do not develop emphysema. This issue is raised because physicians tend to diagnose young people with eosinophilia and airways obstruction as asthmatics (regardless of their smoking status) and then change their diagnosis to COPD later on in life when emphysema or irreversible airways obstruction are appreciated.

In summary, these studies demonstrate that the targeted expression of IL-13 in the adult lung causes emphysema, mucus metaplasia, and inflammation that mirror, in many ways, the lesions seen in human COPD. They also demonstrate that this emphysematous response is mediated, via matrix metalloproteinase and cathepsin-dependent pathways. IL-13 may play an important role in the pathogenesis of COPD, particularly in patients with asthma-like features including AHR and eosinophilia.

The authors thank Kathleen Bertier for her excellent secretarial and administrative assistance. This work was supported by the following grants from the NIH: HL-56389, HL-61904, and HL-64242 (J.A. Elias).

Tao Zheng and Zhou Zhu contributed equally to this work.

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