This longitudinal characterization of the evolution of lung disease in βENaC-overexpressing mice in vivo revealed sequential pathogenetic events in airways disease caused by airway Na+ hyperabsorption and ASL dehydration. Our morphometric studies defined severe airway mucus obstruction in the first days of life as the earliest and death-causing lesion in βENaC-overexpressing mice. The initial mucus plugging was restricted to the trachea and occurred in the absence of goblet cell metaplasia and elevated mucin gene expression compared with wild-type mice (see ). These results indicate that Na+ hyperabsorption-induced mucus accumulation was caused by deficient clearance of constitutively secreted mucus and demonstrate that ASL depletion alone (i.e., in the absence of mucus hypersecretion) is sufficient to cause severe airway mucus obstruction with airflow limitation resulting in systemic hypoxia and pulmonary mortality.
The striking similarity between initial mucus plugging in βENaC-overexpressing neonatal mice and infants with CF (27
), both exhibiting severe mucus plugging in the absence of goblet cell metaplasia and mucus hypersecretion, indicates that dehydration of airway surfaces may be sufficient to cause severe airway obstruction in the human lung. The regional differences in initial mucus plug formation between βENaC-overexpressing mice and patients with CF, in whom mucus plugging is not restricted to the trachea but is observed initially in the small airways, may reflect species differences in the anatomic structure of the tracheobronchial tree. In mice, due to the paucity of airway branching, the narrowest cross-sectional surface area of the tracheobronchial tree is found at the level of the trachea, whereas the extensive branching of human airways produces significant restrictions in surface area at the level of the terminal bronchiole (28
). Accordingly, the locus of initial mucus plaque formation in infants with CF and neonatal βENaC-overexpressing mice is consistent with the concept that ASL volume depletion–induced mucus obstruction occurs in regions of the tracheobronchial tree with critical reductions of total airway caliber. Alternatively, the observed differences in localization of early mucus plugging between patients with CF and βENaC-overexpressing mice may be related to regional differences in relative expression of abnormal Na+
transport, goblet cell numbers, and/or mucin secretory rates.
The histopathologic search for early changes in the intrapulmonary airways revealed that epithelial cells of βENaC-overexpressing neonates were depleted of glycogen stores and that a subset of cells subsequently underwent hydropic degeneration and necrosis in the absence of detectable intrapulmonary mucus obstruction (). The following observations suggest that epithelial necrosis is a direct consequence of increased airway Na+
absorption. First, glycogen depletion and epithelial degeneration were confined to CCSP-positive Clara cells (i.e., the cell type in which CCSP-driven overexpression of βENaC was induced). Second, epithelial degeneration was not observed in airways from mice that overexpressed αENaC or γENaC and did not exhibit increased airway Na+
absorption (data not shown). Third, previous studies in human CF airways demonstrated that increased airway Na+
absorption caused increased epithelial energy consumption (29
). Collectively, these data suggest that reduced oxygen tension due to formation of tracheal mucus plugs in the first days of life (see
and ), coupled with increased O2
demands of βENaC-overexpressing Clara cells, produced cellular hypoxia () that resulted in necrotic degeneration of a susceptible cell population (likely Clara cells) in βENaC-overexpressing neonates.
We speculate that release of proinflammatory stimuli by necrotic epithelial cells played a key role in the recruitment of macrophages and in the initiation of airway inflammation in neonatal βENaC-overexpressing mice (, , and ). Macrophages are a major source of TNF-α, which acts as a potent proinflammatory cytokine in the lung. Therefore, Na+
hyperabsorption–induced epithelial necrosis generated a previously unrecognized trigger for an inflammatory response (30
). Such a mechanism could contribute to early inflammation observed in a number of infants with CF in the absence of detectable bacterial infection. Although necrosis is not a commonly mentioned feature of CF pathogenesis, ultrastructural studies have detected necrotic debris in the small airways of patients with CF in the absence of bacterial infection (31
In addition to macrophage recruitment and elevation of TNF-α, the initial inflammatory response caused by ASL depletion was dominated by an elevation of airway neutrophils and the neutrophil chemoattractant KC, as previously described for adult βENaC-overexpressing mice (11
) ( and ). Juvenile βENaC-overexpressing mice also exhibited a transient airway eosinophilia and increased expression of the key Th2 signaling molecule IL-13 that was markedly exaggerated as compared with wild-type animals (32
). In wild-type and βENaC-overexpressing mice, eosinophil numbers in BALF and IL-13 waned with age, followed by an increase in pulmonary mRNA expression of the Th1 cytokine IFN-γ. Consistent with the data of George and colleagues (23
), our data suggest that the immune system of juvenile wild-type mice during normal development is temporarily biased/polarized toward a Th2 response (24
). To our knowledge, these are the first data demonstrating that ASL depletion, in addition to producing chronic airway neutrophilia, exaggerates Th2-driven airway inflammation in a Th2-biased host. We propose that ASL depletion impaired clearance of inhaled airborne allergens and triggered Th2-driven inflammation in young βENaC-overexpressing mice. Release of IL-13, which induced expression of eotaxin-1, resulted in the recruitment of eosinophils into the lung (35
The chronic lung disease in adult βENaC-overexpressing mice, including airway mucus plugging, goblet cell metaplasia, epithelial hypertrophy, elevated mucin gene expression, and airway neutrophilia, persisted after necrotic cells were detectable and Th2-dependent mechanisms waned (, , , and ). In this context, the present study supports the idea that ASL depletion–induced goblet cell metaplasia and mucus hypersecretion are secondary changes that develop in parallel to, or consequent to, persistent airway inflammation (11
). The mechanisms of persistent neutrophilic inflammation and goblet cell metaplasia are not known. We previously hypothesized that chronic airway inflammation and mucus hypersecretion may be perpetuated by a failure to clear inhaled environmental particles and irritants triggering the release of proinflammatory chemokines like MIP-2 and KC from macrophages and/or airway epithelia in βENaC-overexpressing mice (11
). We predict that future studies in which βENaC-overexpressing mice are exposed to defined intrapulmonary doses of particulates will help to further elucidate the relative role of air pollution in the pathogenesis of chronic obstructive lung disease caused by ASL depletion and reduced mucus clearance.
Chronic airway inflammation in βENaC-overexpressing mice was also associated with the formation of eosinophilic crystals and increased expression of various members of a recently identified family of mammalian chitinases and chitinase-like proteins, including Ym1, Ym2, and AMCase (18
) (). Early studies of these proteins in the lung identified elevated expression of Ym1 in mouse models with chronic granulomatous disease, where Ym1 was shown to be a neutrophil granule protein, and it was suggested that crystal formation was due to excess neutrophil turnover at sites of inflammation (25
). Subsequently, Ym1 was detected in alveolar macrophages from wild-type mice (39
). Recent studies supported the involvement of these chitinases in airway inflammation by demonstrating that (1
) all three proteins are up-regulated in the context of Th2-driven airway inflammation, (2
) AMCase expression is elevated in lungs from patients with asthma, and (3
) AMCase polymorphisms are associated with human asthma (19
). Because chitin is expressed in the walls of fungi and parasites and chitinases are expressed in alveolar macrophages, neutrophils, and the airway epithelium, it is possible that chitinases take part in the innate immune defense against these pathogens (42
). Conversely, high expression of chitinases during inflammation may have deleterious effects, as indicated by studies showing that inhibition of AMCase activity ameliorates disease severity in murine asthma models (19
). Indeed, precipitation and formation of crystals, frequently greater that 100 μm in size, in the airways of βENaC-overexpressing mice may cause mechanical injury of epithelial cells and phagocytes and thus promote chronic inflammation. Although the focus of recent studies on the role of chitinases in airway disease has been on classic Th2-driven inflammatory models, our studies suggest that alternative stimuli (e.g., dehydration) can promote chitinase crystal formation.
Our longitudinal studies demonstrate for the first time that increased airway Na+ absorption and ASL depletion cause emphysema (). Although lung volumes, alveolar architecture, and alveolar size were normal in βENaC-overexpressing neonates, the subsequent increase in lung volume and relative distal airspace enlargement together with an increase in lung compliance show that βENaC-overexpressing mice develop emphysema in the first weeks of life.
We speculate that several mechanisms may contribute to the development of emphysema in vivo
in βENaC-overexpressing mice (). First, emphysema in adult βENaC-overexpressing mice may reflect failure of alveolar septation during development. Because overexpression of βENaC under control of the CCSP promoter is turned on several days before birth (43
), increased pulmonary Na+
absorption in the prenatal period may cause a slight deflation of the lungs with reduced transpulmonary pressure, which may reduce a stimulus for postnatal growth of alveolar walls. Second, the observation that lungs seem to be consistently hyperinflated even in the absence of constant pressure fixation indicates that early airway mucus plugging caused persistent air-trapping, which may result in mechanical overdistention of distal airspaces with irreversible disruption of alveolar septi, loss of pulmonary elastance, and alveolar remodeling. Third, it is possible that chronic pulmonary inflammation contributes to the development of emphysema in βENaC-overexpressing mice. Several leukocyte-derived proteases, including neutrophil elastase and macrophage elastase, have been shown to cause emphysema in mice (44
). Previous studies demonstrated that overexpression of several proinflammatory mediators, including TNF-α and IL-13, in genetically modified mice causes an imbalance in the pulmonary protease/antiprotease system and emphysema (47
) and indicated that proteases/antiproteases may play a key role during lung development (49
). Because TNF-α, IL-13, and neutrophils are increased and macrophages are morphologically activated in the lungs of βENaC-overexpressing mice, we speculate that disruption of the protease/antiprotease balance in the developing lung may cause ASL depletion–induced emphysema. Although further dissection of the relative roles of these mechanisms and their relationship to airway Na+
hyperabsorption is required, it is noteworthy that emphysema, together with mucus plugging, constitutes an early and invariable feature in the CF lung (27
). Recent studies indicated that improvement of ASL hydration by preventive treatment with the specific ENaC blocker amiloride can reduce mortality, airway inflammation, and airway mucus obstruction in βENaC-overexpressing mice (50
). Future studies are required to determine if improved hydration of airway surfaces can prevent emphysema formation in βENaC-overexpressing mice.
On the basis of the similarities in the pulmonary phenotype between adult βENaC-overexpressing mice and chronic bronchitis, including airway mucus obstruction, goblet cell metaplasia, chronic inflammation, and emphysema (51
), we speculate that ASL depletion may play a critical role in the pathogenesis of reduced mucus clearance observed in the airways of smokers and patients with chronic bronchitis (52
). In contrast to CF, no intrinsic ion transport abnormalities have been reported in airways from patients with chronic bronchitis. However, cigarette smoke has been shown to decrease CFTR expression and cAMP-dependant Cl−
secretion in airway epithelia in vitro
and in vivo
), providing a mechanism for ASL depletion. Furthermore, cigarette smoke induces hypersecretion of mucin macromolecules. In the presence of limited ion transport compensation (55
), the mucins secreted “dry” onto airway surfaces (56
) are not properly hydrated, producing “secondary” ASL depletion. The ASL depletion consequent to both mechanisms would be predicted to produce the slow mucus clearance and mucus adhesion characteristic of COPD (52
The observation that ASL depletion can cause concomitant neutrophilic and Th2-driven airway inflammation is remarkable in the context of the debate over whether COPD and asthma are distinct disease entities, as proposed by the “British hypothesis,” or whether they are based on a common etiologic background, as put forward by the “Dutch hypothesis” (59
). Our results point to the possibility of a single etiologic hypothesis for seemingly diverse chronic obstructive airway diseases, including CF, COPD, and asthma, in which a defect in mechanical clearance of inhaled particulates, allergens, or pathogens, caused by primary or secondary ASL depletion, plays a critical role in disease pathogenesis. In this context, the interplay between deficient mechanical lung clearance and the host immune response determines whether a stimulus triggers a neutrophil-dominated airway disease, a Th2-driven airway disease, or both.
In summary, the longitudinal evaluation of the spontaneous course of lung disease in βENaC-overexpressing mice provides novel insights into the in vivo pathogenesis of chronic obstructive lung disease. First, we show that ASL depletion is sufficient to initiate severe airway mucus obstruction in the absence of goblet cell metaplasia or mucus hypersecretion. Second, we show that airway mucus plugging/hypoxia is associated with epithelial necrosis, constituting a mechanism to initiate airway inflammation in the absence of infection. Third, we demonstrate that ASL depletion causes exaggerated eosinophilic airway inflammation in a Th2-biased host. Finally, we show that increased airway Na+ absorption can cause emphysema. Taken together, these results suggest that deficient airway surface hydration plays a critical role in the pathogenesis and serves as a novel therapeutic target in of chronic obstructive pulmonary diseases of different etiologies.