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

Abstract

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.

Figure 1

Constructs utilized in the generation of CC10-rtTA-IL-13 mice.

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.

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.

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.

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.

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.

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.

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.

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.

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.