Requirement for Macrophage Elastase for Cigarette Smoke-Induced Emphysema in Mice

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Science  26 Sep 1997:
Vol. 277, Issue 5334, pp. 2002-2004
DOI: 10.1126/science.277.5334.2002


To determine which proteinases are responsible for the lung destruction characteristic of pulmonary emphysema, macrophage elastase–deficient (MME−/−) mice were subjected to cigarette smoke. In contrast to wild-type mice, MME / mice did not have increased numbers of macrophages in their lungs and did not develop emphysema in response to long-term exposure to cigarette smoke. Smoke-exposed MME / mice that received monthly intratracheal instillations of monocyte chemoattractant protein–1 showed accumulation of alveolar macrophages but did not develop air space enlargement. Thus, macrophage elastase is probably sufficient for the development of emphysema that results from chronic inhalation of cigarette smoke.

Pulmonary emphysema is a major component of the morbidity and mortality of chronic obstructive pulmonary disease (COPD), a condition that afflicts more than 14 million persons in the United States and has become the country's fourth leading cause of death (1). Cigarette smoking is the main risk factor for the development of COPD. With the rapid rise in cigarette smoking occurring now in many countries, COPD may become epidemic worldwide in years to come.

Emphysema is defined as the enlargement of peripheral air spaces of the lung, including respiratory bronchioles, alveolar ducts, and alveoli, accompanied by destruction of the walls of these structures (2, 3). Before the development of emphysema, smokers' lungs show an accumulation of macrophages, lymphocytes, and neutrophils in the walls and adjacent air spaces of the respiratory bronchioles, alveolar ducts, and alveoli (4). Inherited deficiency of α1-antitrypsin, which is the primary inhibitor of neutrophil elastase, predisposes individuals to early onset emphysema (5), and intrapulmonary instillation of elastolytic enzymes in experimental animals causes emphysema (6). Together, these findings have led to the hypothesis that emphysema results from proteolytic injury directed especially against elastin, the main component of elastic fibers.

Macrophages, which account for over 90% of the inflammatory cells in smokers' lungs, produce many proteolytic enzymes that are capable of degrading elastic fibers and other extracellular matrix components (7). One of these enzymes, which is designated macrophage elastase (MME) (8, 9), is a metalloproteinase that solubilizes many extracellular matrix proteins, including elastin.

To determine the contribution of macrophage elastase to the pathogenesis of emphysema due to cigarette smoke, we compared mice made genetically deficient in MME by gene targeting (MME / mice) (10) with wild-type littermates (MME+/+) for the development of emphysema in response to chronic exposure to cigarette smoke. We exposed adult mice to smoke from two nonfiltered cigarettes for 6 days per week for up to 6 months, using a smoking apparatus (11) adapted for mice (12). Carbon monoxide (peak carboxyhemoglobin blood concentrations were 10 to 14%) did not reach toxic levels, and the mice appeared grossly normal during the entire experimental period. On completion of the smoking protocol, the mice were killed and the lungs were inflated and fixed by intratracheal instillation of 10% formalin at 25-cm H2O pressure and sectioned for microscopic analysis.

To investigate the spectrum of elastolytic enzymes present in lung tissue in response to cigarette smoke, we made extracts of whole lung and analyzed them for elastase activity by zymography using κ-elastin as the substrate (Fig. 1A) (13). No elastase activity was found in extracts prepared from MME+/+ mice that had not been exposed to cigarette smoke or from any MME / mice. Extracts from smoke-exposed MME+/+ animals, however, exhibited two zones of elastolytic activity that comigrated at the same molecular size as the active 45-kD macrophage elastase and the 22-kD fully processed active form of macrophage elastase (9). The activity in both bands was inhibited by EDTA, which establishes them as due to metalloproteinases, and the identity of the enzymes responsible for the activity in these bands was shown to be macrophage elastase by protein immunoblot analysis. Immunohistochemistry of lung sections with affinity-purified antiserum directed against mouse macrophage elastase confirmed the presence of this enzyme specifically in macrophages within the lungs of MME+/+ mice exposed to cigarette smoke, whereas the enzyme was only weakly detected in a few macrophages of nonsmoking MME+/+ mice (Fig. 1B). As expected, no cells were immunoreactive in lungs of MME / mice regardless of whether the animals had been exposed to cigarette smoke. Thus, the expression of macrophage elastase was induced by cigarette smoke and appeared to be the only detectable elastase present in lung tissue of mice exposed to cigarette smoke.

Figure 1

Cigarette smoke exposure induces MME expression in alveolar and interstitial macrophages. (A) Lung tissue homogenates were subjected to κ-elastin zymography as described (13). Areas of elastin degradation were visualized as clear zones of lysis after Coomassie staining. Lytic bands were observed only in lungs of MME+/+ mice exposed to smoke. These bands were inhibited by EDTA and comigrated in this nondenatured gel with the 45-kD MME (NH2-terminal activated) and fully processed 22-kD MME (arrows). (B) Immunohistochemical staining for MME in lung tissue of MME+/+ mice exposed to cigarette smoke for 3 months (bottom) and age-matched controls (top). An affinity-purified polyclonal antibody generated against MME (9,10) (0.5 mg/ml) was used at 1:2000 dilution [as in (17)]. MME staining is limited to macrophages (arrows). In MME+/+control mice, few macrophages had detectable MME. Exposure of MME+/+ mice to cigarette smoke was associated with the induction of MME in most macrophages. No MME was detected in MME / mice.

MME+/+ mice exposed to cigarette smoke for 6 months had enlarged air spaces compared with age-matched littermate controls that were not exposed to smoke. Calculation of mean linear intercepts (Lm), an estimate of the average distance between the opposing walls of a single alveolus (14), showed a 32% increase (Table 1), a degree of enlargement comparable with that seen after intrapulmonary instillation of neutrophil elastase (5). In contrast, MME / mice did not show changes in alveolar dimensions after 6 months of exposure to cigarette smoke (Table 1). Cigarette smoke exposure also led to enlargement of alveolar ducts (15) in MME+/+mice (Table 1), whereas in smoke-exposed MME / mice, there was only a modest, insignificant increase in the size of these structures.

Table 1

Alveolar duct area and mean linear intercepts (Lm) of MME+/+ and MME / mice after long-term exposure to cigarette smoke. Values shown are the mean and standard deviation (values in parentheses) for groups of 10 mice each.P values were derived from a two-tailed Student'st test.

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Like human smokers, MME+/+ mice exposed to cigarette smoke showed increased macrophages in their lung tissue (Fig.2). Smoke-exposed MME / mice had no increase in the number of lung macrophages compared with MME / control mice. Also, the small accumulation of neutrophils in alveolar walls seen in MME+/+ animals exposed to smoke was not significantly altered in MME / mice throughout the 6-month study.

Figure 2

Macrophage accumulation in the lung in response to cigarette smoke is dependent on MME. Groups of 10 MME+/+and MME / mice were exposed to cigarette smoke for 6 months (Sm). Equal numbers of age-matched MME+/+ and MME / littermates were used as controls (C). Lung tissue was prepared as described (13). The number of macrophages and neutrophils were counted (17) and normalized per millimeter of alveolar wall. Error bars represent the standard deviation.

Because the lack of macrophages in the lung, and not the lack of elastase per se, could be the reason for the absence of emphysema development in MME / mice, we intratracheally instilled monocyte chemoattractant protein–1 (MCP-1), a monocyte chemokine, which resulted in substantial macrophage accumulation in lung interstitium and alveolar spaces in both MME+/+ and MME / mice (half-life for both ∼18 days). Thus, MME / monocytes could egress from the pulmonary vasculature if there was a stimulus. Monthly MCP-1 instillation in MME / smoke-exposed mice caused an increase in macrophage recruitment compared with wild-type MME+/+ mice exposed to cigarette smoke alone (Fig. 3, inset). However, despite the presence of MME / macrophages in lungs of mice exposed to cigarette smoke, the mean linear intercept (Fig.3) and alveolar duct areas remained unchanged (7.0 mm2 or 4% greater than mice that were not exposed to smoke). In contrast, MCP-1 treatment in MME+/+smoke-exposed mice enhanced emphysema (Lm = 76.2 μm,n = 4 or 15% greater than MME+/+ smoke-exposed mice without MCP-1).

Figure 3

Instillation of MCP-1 in the lungs of MME / mice exposed to cigarette smoke results in macrophage accumulation but not emphysema. MCP-1 (50 μg in 50 μl of saline) was given to MME / mice ( / MCP-1) by monthly intratracheal administration, starting at 30 days after the initiation of cigarette smoke exposure. (Inset) Macrophage recruitment to the interstitium and alveolar surface (macrophages per millimeter of alveolar wall, Mac./mm alv. wall). Despite significant macrophage recruitment, the Lm of MME / mice exposed to cigarette smoke in the presence of MCP-1 (MME / + MCP-1) was not different from that of MME / animals. n = 4 to 6 mice per intermediate time point (for MCP-1, the numbers are averages of counts before and after instillation). n = 10 for 6-month data. Error bars represent the standard deviation. •, −/−; ▪, +/+; and ○, −/− and MCP-1.

Because only macrophages, and not blood monocytes, express MME, we hypothesize that MME expressed by constitutive alveolar macrophages after exposure to cigarette smoke generates monocyte chemotactic activity and that this activity is responsible for the monocyte recruitment in response to smoke. Thus, macrophage-mediated lung destruction after exposure to cigarette smoke is directly related to the presence of MME, which most likely is required for direct degradation of lung tissue. Additionally, macrophage elastase can inactivate α1-antitrypsin (16), indirectly enhancing neutrophil elastase activity and potentially contributing to lung destruction.

In this study, wild-type mice exposed to cigarette smoke developed inflammatory cell recruitment and dilatation and destruction of alveolar walls and alveolar ducts. These changes mirror the pathology of smokers' lungs, although human lungs also have destruction and dilitation of respiratory bronchioles, a structure not present in mouse lungs. Unlike normal mice, mice without MME failed to recruit macrophages and did not develop lung destruction in response to cigarette smoke. These findings point to a primary role for macrophages and macrophage elastase in the development of emphysema induced by cigarette smoke. This study also sheds light on both the beneficial and harmful effects of macrophage elastase. On exposure to foreign material such as cigarette smoke, constitutive lung macrophages are activated to produce macrophage elastase, which modulates the host inflammatory response by recruiting more macrophages into the lung. However, production of macrophage elastase may overwhelm local inhibition and destroy the lung tissue it was designed to protect.

  • * To whom correspondence should be addressed. E-mail: sshapiro{at}


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