Elastase activity on sputum neutrophils correlates with severity of lung disease in cystic fibrosis

Discussion

This is the first study to examine the role of membrane-bound NE activity on the surface of airway neutrophils in CF lung disease. Chronic neutrophilic airway inflammation in CF is associated with increased levels of free NE activity in the extracellular compartment of the airway lumen, as well as increased expression of membrane-bound NE on the surface of activated airway neutrophils [2, 3, 22, 29, 30]. Despite detection of NE activity in these two distinct compartments, previous studies examining the role of NE in CF lung disease were limited to measurements of free NE activity in airway secretions and the pathogenetic relevance of surface-bound NE activity remains unknown [2–4]. In this study, we employed NE-specific FRET reporters designed to localise and quantify NE activity on cell membranes [21, 31] and measured surface-bound NE activity on sputum neutrophils from clinically stable adult CF patients with a broad spectrum of severity of lung disease (table 1). We found that surface-bound NE activity was consistently increased on sputum neutrophils from patients with CF compared to healthy controls, even in patients with low levels of free NE activity in sputum supernatant (figure 1). Both free and surface-bound NE activity was associated with other markers of airway inflammation, such as sputum neutrophil counts, IL-8, MPO or AAT–NE in our study population (figures 2 and 3) and correlated inversely with FEV₁ % pred in patients with CF (figure 4). In addition, surface-bound NE, but not free NE activity, correlated with FRC_pleth % pred (figure 4). These results suggest that increased membrane-bound NE activity on the surface of viable and activated airway neutrophils is associated with airway inflammation, lung function decline and air trapping, and may thus play an important and previously unrecognised role in CF lung disease.

Our study provides novel information on the source of elevated NE activity in CF lung disease. So far, rapid cell death and lysis of neutrophils upon recruitment into CF airways, leading to uncontrolled passive release of NE into the extracellular compartment, has been considered the main mechanism underlying the increased NE burden in CF lung disease [32]. However, a previous study identified a subset of viable neutrophils in CF airways [29, 32]. During homing to the airways, these neutrophils acquired a phenotype of conventional activation and, unexpectedly, showed evidence of mobilisation of NE-rich granula to the cell surface [29, 32]. Our results obtained with FRET sensors designed to detect membrane-bound NE activity confirm increased expression of NE on the surface of CF sputum neutrophils and demonstrate that this pool of NE has proteolytic activity in CF airways (figure 1). Of note, surface-bound NE activity was consistently increased on CF neutrophils, even when free NE activity in sputum supernatant was low (figure 1). These results suggest that in the microenvironment of CF airways, neutrophils acquire this activated configuration with increased surface-bound NE activity even at low levels of airway inflammation, when free NE activity is still contained by endogenous antiproteases such as α₁-antitrypsin and SLPI in the extracellular compartment [12, 27].

Of note, surface-bound NE was shown to be largely resistant to inhibition by endogenous inhibitors including α₁-antitrypsin and SLPI [33]. This suggests that this pool of surface-bound NE activity may be of particular relevance in early lung disease in young children with CF who, probably due to a still-intact antiprotease shield, often show no detectable free NE activity in BAL fluid despite evidence of structural lung disease upon chest CT [1, 2]. The pathophysiological relevance of increased surface-bound NE activity in the context of early CF lung disease is supported by previous studies in βENaC-overexpressing mice featuring CF-like lung disease with early-onset mucus plugging, airway inflammation and structural lung damage [22, 34, 35]. In this murine model, the level of airway neutrophilia is in the range of ∼5–30% of total cells, thus resembling that of infants and young children with CF and much lower than in adult CF patients, who typically exhibit ≥90% neutrophils [1, 2, 22, 24]. We showed that the antiprotease shield remains intact and no free NE activity is detectable in BAL fluid from βENaC-overexpressing mice, whereas surface-bound NE activity is consistently increased on airway neutrophils, similar to our findings in CF patients [22]. Furthermore, we demonstrated that genetic deletion of NE reduced airway inflammation, mucus hypersecretion and structural lung damage in βENaC-overexpressing mice [22, 30]. Collectively, these studies suggest that surface-bound NE activity may be an important player in the pathogenesis of early CF lung disease, and may explain why a subgroup of infants and young children with CF show lung abnormalities by chest CT, even in the absence of free NE activity in BAL fluid [1, 2].

Importantly, our data demonstrate that elevated surface-bound NE activity is associated with lung disease severity in adult CF patients who exhibit high levels of neutrophilic airway inflammation and free NE activity. As expected [4, 24, 36], neutrophil counts, IL-8 and MPO as markers of neutrophilic airway inflammation were markedly elevated and free NE activity was readily detectable in sputum samples of our adult patient cohort (figure 2 and table 2). Increased free NE activity was associated with increased levels of NE bound to α₁-antitrypsin and with reduced levels of SLPI (table 2). Because SLPI is cleaved and inactivated by free NE [12], this pattern is indicative of breakage of the antiprotease shield. In this context of chronic CF airway inflammation, we found that both free and surface-bound NE activity correlated inversely with FEV₁ % pred, the most widely used pulmonary function test to assess airflow limitation and disease progression in CF [2–4] (figure 4). In addition, our data demonstrate that surface-bound NE activity, but not free NE activity, showed a direct correlation with FRC_pleth % pred as a surrogate measure of pulmonary hyperinflation (figure 4). This indicates that surface-bound NE activity may contribute to the severity of CF lung disease independent of free NE in the extracellular compartment. We speculate that this difference may be related to direct effects of surface-bound NE activity that are probably mediated in a focused fashion via direct contact of activated neutrophils with airway epithelial cells, other structural or immune cells, or the extracellular matrix. Pulmonary hyperinflation, as determined from elevated FRC_pleth % pred, may reflect air trapping due to airflow obstruction in the small conducting airways, or hyperinflation due to emphysema related to structural lung damage of distal airspaces. Previous studies in βENaC-overexpressing mice demonstrated a role of surface-bound NE activity in the in vivo pathogenesis of mucus hypersecretion, airway inflammation and emphysema-like structural lung damage [22], and recent chest CT studies detected emphysema formation in adult CF patients [37, 38]. Based on these studies, we speculate that increased surface-bound NE activity on CF airway neutrophils may have disease-promoting effects in small airways and lung parenchyma. Furthermore, our present data suggest that this cell-bound pool of NE activity may be a promising biomarker of disease severity and an important target for therapeutic strategies designed to inhibit NE activity in the lungs of patients with CF.

Our study has several limitations. First, this cross-sectional study does not provide information on the long-term relationship between surface-bound NE activity and lung function decline in patients with CF. Therefore, future longitudinal studies in larger patient cohorts will be required to determine the predictive value of surface-bound NE activity as a biomarker of disease severity and progression. To determine whether surface-bound NE activity predicts pulmonary exacerbations and response to therapy, such studies should include measurements before, at onset and after treatment for pulmonary exacerbation. Second, this study is limited by the lack of chest imaging studies. Future studies including chest CT or magnetic resonance imaging outcome measures [39, 40] will be critical to specify and localise the structural abnormalities associated with surface-bound NE that determine lung function decline and pulmonary hyperinflation. Third, established surveillance programmes in infants and young children with CF that include annual BAL will provide opportunities to determine the role of this cell-bound pool of NE in early CF lung disease, where structural lung disease can occur even in the absence of free NE activity in BAL fluid [1, 2]. We expect that the protocols to capture surface-bound NE activity on airway neutrophils established and employed in the present study will be instrumental for the implementation of these future studies.

In summary, this study demonstrates for the first time that NE activity on sputum neutrophils is associated with severity of lung disease in patients with CF. We found that NE activity is consistently increased on the surface of CF sputum neutrophils, even at low levels of free NE activity in sputum supernatant. Furthermore, we demonstrate that surface-bound NE activity correlates with pulmonary function indices of airflow limitation and pulmonary hyperinflation. These results suggest that surface-bound NE activity, probably reflecting a freshly secreted pool of NE that is not inhibited by endogenous antiproteases, may play an important role in the pathogenesis and serve as a novel biomarker of disease severity in patients with CF and potentially other chronic neutrophilic lung diseases.