Ex vivo sputum analysis reveals impairment of protease-dependent mucus degradation by plasma proteins in acute asthma

Abstract

Rationale: Airway mucus plugs, composed of mucin glycoproteins mixed with plasma proteins, are an important cause of airway obstruction in acute severe asthma, and they are poorly treated with current therapies.

Objectives: To investigate mechanisms of airway mucus clearance in health and in acute severe asthma.

Methods: We collected airway mucus from patients with asthma and nonasthmatic control subjects, using sputum induction or tracheal aspiration. We used rheological methods complemented by centrifugation-based mucin size profiling and immunoblotting to characterize the physical properties of the mucus gel, the size profiles of mucins, and the degradation products of albumin in airway mucus.

Measurements and main results: Repeated ex vivo measures of size and entanglement of mucin polymers in airway mucus from nonasthmatic control subjects showed that the mucus gel is normally degraded by proteases and that albumin inhibits this degradation. In airway mucus collected from patients with asthma at various time points during acute asthma exacerbation, protease-driven mucus degradation was inhibited at the height of exacerbation but was restored during recovery. In immunoblots of human serum albumin digested by neutrophil elastase and in immunoblots of airway mucus, we found that albumin was a substrate of neutrophil elastase and that products of albumin degradation were abundant in airway mucus during acute asthma exacerbation.

Conclusions: Rheological methods complemented by centrifugation-based mucin size profiling of airway mucins in health and acute asthma reveal that mucin degradation is inhibited in acute asthma, and that an excess of plasma proteins present in acute asthma inhibits the degradation of mucins in a protease-dependent manner. These findings identify a novel mechanism whereby plasma exudation may impair airway mucus clearance.

Figure 1.

Healthy airway mucus is a cross-linked gel. The elastic modulus (G′) predominates over the viscous modulus (G″) across a broad range of frequencies in this typical frequency sweep from healthy induced sputum. The G′ predominance and plateau as well as the identical dependence of G′ and G″ on frequency (G′ and G″ are parallel lines) are hallmarks of a cross-linked gel. We used G′ and G″ at 0.8 Hz (arrow, vertical line) to facilitate comparison of these moduli between samples for all experiments. For calculations of the cough clearance index (CCI), we used G′ and G″ at high frequency (16.0 Hz); for calculations of the mucociliary clearance index (MCI), we used G′ and G″ at low frequency (0.2 Hz) (vertical lines). These high and low frequencies approximate the frequencies in the airways due to cough and ciliary beat, respectively. The frequency sweep plateau in this rheological signature of healthy sputum also enables direct determination of entanglement density and entanglement molecular weight from G_p, the plateau modulus.

Figure 2.

Airway mucus from patients with asthma in acute exacerbation has abnormal rheological properties. (A) Elastic and viscous moduli of acute asthmatic mucus (solid boxes) are greater than those of healthy mucus (open boxes). The greater increase in elastic modulus compared with the viscous modulus indicates that the predominant abnormality in asthmatic mucus is increased cross-linking and entanglement of mucins, not increased concentration of mucins. (B) Entanglement density is significantly greater in asthmatic than in healthy mucus. (C) The cough clearance index (CCI) is decreased in acute asthma, but the mucociliary clearance index (MCI) is not. Data are from eight healthy subjects and five subjects with asthma and are summarized as box plots with medians, range, and outliers. *P < 0.05 versus healthy controls by the rank-sum test.

Figure 3.

Healthy airway mucus is degraded by proteases. (A and B) Time- and temperature-dependent changes in the rheological properties of sputum from healthy subjects. (A) Sputum incubated at 37°C for 24 hours has markedly lower elastic (G′) and viscous (G″) moduli and (B) entanglement density than sputum incubated at 4°C. Data are from five healthy subjects. *P < 0.05 versus 4°C. (C) Time- and temperature-dependent changes in a representative size profile of mucin polymers in healthy sputum. After incubation at 4°C and 37°C, sputum samples were subjected to rate zonal centrifugation and fractions were transferred to nitrocellulose, followed by staining with MAN5ACI (MUC5AC polyclonal antiserum) (37). The size profiles of the 4°C and 37°C samples are markedly different, with the 37°C sample having predominantly smaller mucins. (D–F) Effect of protease inhibition on the rheological properties and mucin size profiles of healthy sputum. (D) Unlike sputum incubated with 0.9% NaCl (control), sputum incubated with protease inhibitors (PI) does not significantly decline in G′ or G″ over 24 hours and (E) does not change in entanglement density or (F) mucin size. Data for (D) and (E) are from three healthy subjects. **P < 0.01 versus saline control. (G–I) Effect of neutrophil elastase on the rheological properties and mucin size profiles of healthy sputum. (G) Unlike sputum incubated with 0.9% NaCl, sputum incubated with human neutrophil elastase (HNE) for 4 hours at 37°C significantly declines in G′ and G″ and (H) entanglement density and (I) shows a predominance of smaller mucins after rate zonal centrifugation. Data for (G) and (H) are from four healthy subjects and are presented as means ± SEM. *P < 0.05 versus saline control. The size profiles shown in (F) and (I) are from the induced sputum of two separate healthy subjects. The t test using log-transformed data was performed for all between-group comparisons.

Figure 4.

Asthmatic airway mucus resists proteolytic degradation. (A and B) Time- and temperature-dependent changes in the rheological properties of airway mucus from patients with asthma in the acute and recovery phases of exacerbation. (A) Airway mucus collected from patients with asthma early in the course of an exacerbation is markedly resistant to degradation when incubated at 37°C for 24 hours compared with tracheal aspirates from healthy subjects. Data are from four healthy subjects and five patients with asthma. ***P < 0.001 and **P < 0.01 versus healthy control subjects. (B) Comparison of airway mucus collected early in the hospital course of asthma exacerbation (acute asthma) and later in the hospital course (recovery asthma) shows that in the asthma recovery samples, elastic (G′) and viscous (G″) moduli decline to levels similar to those measured in healthy subjects. Data are from paired samples from three patients with asthma. *P < 0.05 versus acute asthma. (C) Size profile of mucin polymers in sputum samples from a subject with asthma during the acute and recovery phases of asthma exacerbation. Both sputum samples were subjected to rate zonal centrifugation and fractions were transferred to nitrocellulose, followed by staining with MUC5AC polyclonal antiserum. The size profiles of the acute and recovery samples are markedly different, with the recovery sample having predominantly smaller mucins. (D) Effects of albumin on the rheological properties of healthy sputum. Unlike sputum incubated with 0.9% NaCl, sputum incubated with human serum albumin does not significantly decline in G′ or G″ over 24 hours. Data are from five healthy subjects and are presented as means ± SEM. ***P < 0.001 and **P < 0.01 versus saline control. The t test using log-transformed data was performed for all between-group comparisons.

Figure 5.

Albumin is a substrate of neutrophil elastase, and albumin degradation products are abundant in sputum samples in acute asthma. (A) Degradation of albumin by elastase. Chromatographically purified human serum albumin was incubated for 120 and 360 minutes (undiluted control) and with purified human neutrophil elastase (1:1 molar ratio) for 30, 120, and 360 minutes at 37°C in phosphate-buffered saline. Degradation products were subjected to nonreducing (data not shown) and reducing SDS–PAGE. (B) Abundant albumin degradation products in airway mucus from patients with asthma in acute exacerbation but not in healthy sputum. The immunoblot shows equal volume loading (4 μl each) of sputum samples that had been diluted fourfold during processing.

Figure 6.

Schematic diagram illustrating a proposed mechanism of mucus degradation in health and in acute asthma. (A) Healthy airway mucus lines the patent airway and has optimal clearance via the mucociliary escalator. The mucus gel is formed mainly by mucins, and the sufficient neutrophil protease activity optimizes its rheological properties to enable effective transport. (B) Mucus clearance is reduced in acute asthma, and mucus plugs occlude the airway. Protease-dependent degradation of mucins is inhibited by high concentrations of albumin and other plasma proteins, which function as alternative substrates for neutrophil proteases. (C) Mucus clearance improves during asthma recovery, a necessary mechanism to restore airway patency. Neutrophils, increased in number, secrete proteases that overcome the inhibition of mucin degradation imposed by high concentrations of plasma proteins.