Abstract
Rationale: Cystic fibrosis (CF) is believed to be associated with mucus hypersecretion; thus, the principal airway gel-forming mucins, MUC5AC and MUC5B, are also expected to be increased relative to non-CF secretions. However, we have shown that these mucins are decreased during stable CF disease.
Objectives: In this study, we determine if these mucins increase during a pulmonary exacerbation of CF.
Methods: Expectorated sputum was collected from 11 adults with CF during stable disease and then during a pulmonary exacerbation and from 12 healthy control subjects. MUC5AC and MUC5B proteins were measured by Western blot. DNA content was measured using microfluorimetry.
Results: MUC5AC protein increased by 908% and MUC5B by 59% (p < 0.05 for both) during an exacerbation compared with periods of stable disease. During stable disease, the vol/vol quantity of MUC5AC protein was 89% less than normal mucus, and the mucin-associated sugars, measured using a lectin binding assay, were 46% less compared with normal mucus. The concentration of DNA in CF sputum did not increase during an exacerbation.
Conclusions: During a CF exacerbation, concentration of secreted mucin increased to the amount found in mucus from normal subjects, suggesting that the capacity to secrete mucin in response to an infection or inflammatory stimulus is preserved in CF airways. This might help to protect the airway from injury.
Composition of sputum in cystic fibrosis (CF) during a pulmonary exacerbation compared with that of normal mucus provides information on the pathophysiological influence of recurrent airway inflammation on the progression of the lung destruction in CF. Mucin is the major component of mucus in healthy airways, and is responsible for protection of the epithelium.
In CF, mucin is decreased during stable disease but increases during a pulmonary exacerbation; however, it still does not exceed the concentration of normal, noninflamed airways.
Mucus is a protective airway coating secreted in the healthy airway, whereas sputum is the product of airway inflammation and usually contains cells, inflammatory mediators, bacteria, and polymerized DNA resulting from inflammatory cell necrosis (3, 4). Mucus is composed mainly of water and ions, with approximately 5% of the content due to proteins secreted by airway cells and lipids (5–7). In health, the mucin glycoproteins are the major macromolecular component of the mucus gel (8). The mucins are responsible for the rheologic, protective, and clearance properties of the mucus (3, 9–12). There are two major classes of mucins: the secreted and the membrane-tethered mucins (8, 13, 14). Several secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6) have genes that are clustered on chromosome 11p15 and contain domains with significant homology to the von Willebrand factor D domains that are sites for oligomerization (15). In sputum, MUC5AC and MUC5B are the major oligomeric mucins, with only trace amounts of MUC2 (16). MUC5AC appears to be produced primarily by the goblet cells in the tracheobronchial surface epithelium, whereas MUC5B is secreted primarily by the submucosal glands (17). At least 12 mucin genes (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC11, MUC13, MUC15, MUC19, and MUC20) have been observed at the mRNA level in tissues of the lower respiratory tract from healthy individuals (18–26).
Goblet cell hyperplasia in the CF airway (27) suggests that, with increased airway secretions, there is also increased mucin content. Nevertheless, we showed that, in sputum from adult subjects with CF without a pulmonary exacerbation, the concentration of secreted mucins, in particular MUC5AC, were markedly decreased compared with that in normal subjects (28). This is consistent with the finding by Rose and colleagues that mucins were present at lower concentrations in three subjects with CF compared with two normal volunteers (29).
Because mucin was decreased during stable periods, in these studies we evaluated if gel-forming mucins or DNA polymers increase in sputum during a CF pulmonary exacerbation.
Some of the results of these studies have been previously reported in the form of an abstract (30).
We collected two sputum specimens, 2 to 12 months apart from 11 subjects with CF. Sputum was collected by expectoration after pulmonary function testing without the use of saline induction. One specimen was collected during a period of disease stability and one during a pulmonary exacerbation. The exacerbation sputa were collected within 7 days of the onset of clinical symptoms of an exacerbation. The stable sputa were collected at least 30 days after the onset of the symptoms of an exacerbation were diagnosed and antibiotic therapy was started; there had not been symptoms of worsening disease or another exacerbation during this time.
A pulmonary exacerbation was defined by an increased cough or sputum, hypoxemia, or a decrease in weight or exercise tolerance, together with a documented decrease in FEV1 of at least 5% from the previous clinic visit (2). In all cases, a new course of oral or intravenous antibiotics was started when an exacerbation was diagnosed, but macrolides were not administered because they may alter the mucin output (31). The clinical characteristics and demographics of the subjects with CF are given in the online supplement.
For use as healthy control samples, mucus was collected from the end of endotracheal tubes (ETTs) from 12 subjects who had no lung disease and who required nonthoracic surgery under general anesthesia (32, 33). See the online supplement for subject demographics.
Protease inhibitors (Set I; Calbiochem, San Diego, CA) were added in equal volumes to the sample during thawing. The samples were homogenized by aspirating several times through progressively smaller needles (final needle size, 28 G), diluted 1:10 with phosphate-buffered saline, and homogenized again with a 28-G needle.
Synthetic peptides with sequences RNQDQQGPFKMC of the MUC5AC apoprotein, and RNREQVGKFKMC of the MUC5B apoprotein, were used to raise antibodies in rabbits (17, 28, 34). For details, see online supplement.
Agarose gel electrophoresis and membranes were probed as per established protocol (28). Quantitative measurements were obtained directly from images using CanoScan software (Version 4.0, Canon, Germany) on the band density and area. Each band was analyzed by using a selection area of the same size. The mean surface area covered was calculated using National Institutes of Health imaging software (Scion Image 4.0.2; National Institutes of Health, Bethesda, MD).
To compare different blots, we established an internal mucin control. The mucin control was collected from a voluminous sputum sample from a single patient undergoing lung transplantation for non-CF bronchiectasis. The test samples were compared with this control to allow comparison of the samples on different blots. Mucin and DNA quantities in CF sputum and normal control mucus were normalized to this internal control (= 100%).
DNA was measured using microfluorimetry (35) and compared with calf thymus DNA standard. Samples were stained using 33258 Hoechst (Sigma Chemical Co., St. Louis, MO) and fluorescence was measured by spectrophotofluorimetry.
Samples were stained with L4889 lectin from Ulex europaeus agglutinin (UEA) conjugated with tetramethyl rhodamine isothiocyanate (TRITC) for carbohydrate epitopes. Samples were also stained with YoYo-1 for DNA. Fluorescence was measured by laser scanning confocal microscopy. Mucinlike glycoprotein and DNA filaments were quantified using the PolyFiberQuant software (36).
Statistical analysis was performed using SPSS 11.5 for Windows (SPSS, Inc., Chicago, IL). Results are given as means ± SE.
We had the opportunity to serially collect sputum on seven occasions from a subject with CF during stable disease and over the course of a pulmonary exacerbation. During the exacerbation, the two gel-forming mucins increased but at different rates (Figure 1). MUC5AC slowly increased over 2 weeks after the onset of the exacerbation, whereas MUC5B was at maximum on the day the exacerbation was first clinically recognized, and the increased concentration of MUC5B was then sustained. Both mucins returned to preexacerbation levels by 9 weeks after the exacerbation onset.
Figure 1. Sputum analysis from a 27-year-old subject with cystic fibrosis during a pulmonary exacerbation. On the first day of the exacerbation (arrow = Day 0), MUC5B was elevated compared with visits 1 and 2, and 8 weeks before the exacerbation (= Day −7, −14, −56). MUC5AC was slightly increased on the day of exacerbation, but increased over the next 2 weeks (= +14 d). Both mucins returned to baseline by 9 weeks (= +63 d) after the exacerbation.
[More] [Minimize]We compared the mucin concentration in paired sputa from 11 subjects with CF when clinically stable and during a pulmonary exacerbation (Figure 2). In two patients, MUC5B decreased. There was a significant increase in both MUC5AC and MUC5B during the exacerbation (Wilcoxon test, p < 0.05). MUC5AC increased during the exacerbation by 908% and MUC5B by 59% compared with the period of stable disease.
Figure 2. Gel electrophoresis analysis of sputum from 11 subjects with cystic fibrosis (CF). Sputum was collected from each subject during stable disease and during a pulmonary exacerbation. The samples were loaded as volume equivalents from the sputum. The results are shown as mean density of the sample related to the internal control (= 100% relative concentration). The blots were probed with specific MUC5AC and MUC5B antibodies.
[More] [Minimize]We compared the CF data with data from mucus samples from normal control subjects (n = 11) (Figure 3). All samples were loaded on the gel as volume equivalents from the sputum (Figures 1–3). Because the avidity of the antibodies for MUC5AC and MUC5B was different, these separate results cannot be directly compared.
Figure 3. Sputum samples during stable disease and during pulmonary exacerbation from 11 subjects with cystic fibrosis (CF) and mucus from normal 11 control subjects were analyzed by gel electrophoresis probed with specific MUC5AC and MUC5B antibodies. The results are shown as mean density of the samples related to the internal control (= 100% relative concentration). *Significant to CF stable disease (p < 0.05). ETT = endotracheal tube, control.
[More] [Minimize]In ETT mucus from normal subjects, there was 89% more MUC5AC and 40% more MUC5B compared with the mucin in sputum from subjects with CF when stable (Mann-Whitney U test, p < 0.05 for both) and 14% less MUC5AC and 5% more MUC5B compared with the sputum from subjects with CF during an exacerbation (Mann-Whitney U test, p = not significant). These results are similar to previously published results (28).
Over the last 9 years, we have collected expectorated sputa from a cohort of CF clinic patients for prospective studies of sputum structure as part of an ongoing evaluation (n = 115 sputa from 60 subjects). We measured the mucinlike glycoconjugate content from these sputa and compared these with ETT mucus using laser scanning confocal microscopy imaging of fluorescent-conjugated UEA lectin. Mucin and DNA polymers were analyzed by the PolyFiberQuant image analysis program (36). The number of mucin pixels per image was significantly greater in normal mucus (= 100%) compared with CF samples (= 46%) (n = 115; unpaired two-tailed t test, p = 0.04). However, there was no significant difference in the total mucinlike glycoconjugates in comparing CF sputum obtained when stable and during a pulmonary exacerbation.
When CF sputum was held at 37°C with no protease inhibitors added, the mucin content was reduced by half after 3 hours. Mucin did not decrease during incubation if the samples were supplemented with protease inhibitors (details are available in the online supplement).
In CF, the relative DNA quantity in sputum was no greater during an exacerbation (5.20 mg/ml) compared with stable disease (6.67 mg/ml) despite an increase in inflammation and, presumably, neutrophils (Wilcoxon test, p = not significant). The DNA content of CF sputum, both during stability and pulmonary exacerbation, was significantly greater than that of normal mucus (0.96 mg/ml) (Mann-Whitney U test, p < 0.05) (Figure 4).
Figure 4. Total DNA concentration in sputum from 11 subjects with cystic fibrosis (CF) during periods of stable disease and during a pulmonary exacerbation, compared with mucus from 11 normal control subjects. *Significant to endotracheal tube (ETT) (p < 0.05).
[More] [Minimize]During a CF pulmonary exacerbation, the MUC5B concentration was inversely correlated with DNA concentration (r = −0.60, p = 0.03) (Figure 5).
Figure 5. MUC5B and DNA concentrations were inversely correlated (R2 = 0.365, p < 0.05) in the sputum (n = 22) of 11 subjects with cystic fibrosis.
[More] [Minimize]The DNA concentration in sputum from subjects with CF during stable disease was inversely correlated with FEV1% predicted (correlation coefficient = −0.43, p = 0.043), but not FVC% (correlation coefficient = −0.345, p = 0.116) (Figure 6). This is similar to published data (37). The DNA concentration was not significantly correlated with pulmonary function during a pulmonary exacerbation.
Figure 6. Relationship of the DNA concentration in sputum of 11 subjects with cystic fibrosis during stable disease with FEV1% (p = 0.043) and FVC% (not significant, p = 0.116).
[More] [Minimize]Persons with CF have extensive secretion plugging of their airways leading to increased susceptibility of infection and decreased pulmonary function. Contrary to the hypothesis that CF secretions contains more mucin, we have reported that the concentration of MUC5AC and MUC5B are very significantly decreased in the CF airways relative to normal mucus (28). Airway defense against infection may be compromised when there is decreased mucin. Therefore, we wished to determine if the CF airway is able to increase mucin secretion in response to a pulmonary exacerbation.
We first confirmed that, during stable CF disease, MUC5AC protein assayed by Western blotting was 89% less than in normal mucus, and MUC5B was decreased by 40%. These results are similar to our previous study (28). We confirmed these results using a different mucin staining technique with UEA lectin, which binds to mucinlike glycoprotein but does not distinguish mucin subtypes. Using this assay, mucinlike glycoconjugates were decreased in CF sputum during stable disease by 46% compared with normal mucus.
We then demonstrated that, during a CF pulmonary exacerbation, the mucin protein concentration dramatically increases as follows: MUC5AC increases by 908%, and MUC5B increases by 59% relative to a period of stable disease; although the total per solids composition was greater in CF secretions than in normal mucus, the percentage of solid composition was similar during stable periods and during an exacerbation of airway disease. This suggests that, in the CF airway, inflammatory or immune mediators can stimulate mucin production and/or secretion (8, 38). This may be a protective response.
Interestingly, the temporal increase in these mucins appeared to be different. We observed in a single patient that, during an exacerbation, MUC5AC increased for up to 2 weeks after the onset of the exacerbation, whereas MUC5B was at maximum concentration on the day the exacerbation was first diagnosed, and this increased MUC5B was sustained for 2 weeks. Both mucins returned to preexacerbation baseline by 9 weeks after the exacerbation. Although taken from a temporally collected series of sputa from a single subject, these data suggest that MUC5AC secretion was induced over time, whereas MUC5B was released from stores and sustained production. They further suggest that, during a pulmonary exacerbation, it takes weeks after the onset of the exacerbation until the mucin concentration returns to the amount seen in stable disease. This variability in mucin concentration may in part account for inconsistencies in the reported analysis of mucin in CF secretions.
No differences in mucin pixels with lectin binding were observed between CF stable and exacerbated samples, although decreased levels of MUC5AC mucin were seen by Western analysis. There are several possible explanations for this apparent discrepancy. It may be that the mucin glycopeptides contribute to the mucin pixels in the stable CF samples and that epitopes recognized by the antibodies were proteolytically fragmented. However, we have shown that, when sputum is quickly processed in the presence of protease inhibitors, as we have done, there is minimal fragmentation, and fragments were not detected in the low-molecular-weight portion of overexposed Western gels (see the online supplement for details). Differences in UEA lectin staining may also be due to differences in mucin glycosylation between normal and CF mucins. A further possibility might be that because UEA lectin nonspecifically binds to carbohydrates on all mucin subtypes, a differentiation between MUC5AC and MUC5B cannot be made and the lectin identifies “total” mucin. Because there are no standard controls for MUC5AC and MUC5B, we do not know which is the predominant mucin in CF sputum. If it is MUC5B, a decrease in MUC5AC may not contribute much to the total mucin mixture and therefore UEA lectin may not show a significant difference in total mucin between stable and exacerbation.
Because of these specific exacerbation-related changes in MUC5AC, it is possible that the concentration of this mucin might be useful as a diagnostic marker for evaluating and monitoring a pulmonary exacerbation in patients with CF. Further studies with careful documentation of clinical symptoms and disease severity are necessary to confirm this speculation.
There are several possible reasons why mucins are decreased in CF sputum. One possibility might be that this decrease is a result of increased mucin degradation (29, 39) due to protease activity from inflammatory cells. We analyzed the rate of mucin degradation by CF sputum proteases and found that the mucin amount was reduced by half after 3 hours. During a pulmonary exacerbation, mucins may be secreted and cleared more quickly and thus have a shorter exposure to endogenous airway proteases. However, we believe that it is unlikely that there is increased sputum clearance during a pulmonary exacerbation of CF, thus it appears more likely that mucin secretion is preserved as a protective response to inflammation during an exacerbation, even in the chronically infected CF airway. The finding that the lectin-associated mucin glycoproteins in CF sputa are decreased compared with normal mucus does not support the hypothesis that mucins are degraded by proteases, because even small mucin fragments would be visualized and included in quantification of the total mucin concentration. Furthermore, we did not find evidence of mucin fragmentation on our Western gels.
In nasal polyps, the MUC5AC mRNA in CF is decreased compared with that in healthy nasal mucosa (40). Voynow and colleagues also reported that CF nasal epithelial cells in culture express less MUC5AC mRNA compared with normal cells (41). These findings suggest that, in CF, the MUC5AC gene expression in the epithelium is decreased.
Although there is plugging and chronic expectoration of sputum in persons with CF, mucin appears to be only a minor component of these secretions and DNA is the dominant polymer seen. In our previous study, we showed that the amount of DNA in CF sputum was greatly increased compared with chronic bronchitis sputum (28), most likely as a result of leukocyte necrosis (42, 43). If increased inflammation during a pulmonary exacerbation increases both the recruitment of neutrophils into the airway and neutrophil degradation, we would expect that there would be increased sputum DNA during an exacerbation (44). We confirmed that, in sputum of subjects with CF, DNA was significantly greater than that from non-CF mucus and that the concentration of DNA in our CF samples (1–11 mg/ml) was consistent with previously reported studies (3–14 mg/ml) (45–47). However, we found that the concentration of DNA did not differ significantly in subjects with CF during stable disease and pulmonary exacerbation. These data are consistent with previous studies reporting that sputum IL-8 and neutrophil counts do not significantly increase during an exacerbation of CF (37). This may be due to the intense neutrophilic inflammation that is constantly present in the CF airway, giving very limited opportunity for increasing IL-8, neutrophils, and DNA to be detected.
It may be that decreased mucin secretion increases susceptibility to airway infection in the CF airway. Pseudomonas aeruginosa has been shown to bind to airway mucin (48, 49). The dense DNA–mucin polymer network might be a barrier to bacterial attachment to the epithelium, and thus adequate mucin secretion is probably critical for the initial clearance of airway bacteria. Speculating beyond this, the mucin gel may inhibit bacterial communication either by binding virulence factors and quorum sensing proteins or by impeding their diffusion to adjacent organisms. Thus, the mucin gel may prevent airway bacteria from forming the biofilms characteristic of CF airway disease.
During periods of disease stability, mucins, in particular MUC5AC, may be sequestered intracellularly, and do not contribute to superficial airway fluid mucin concentration. Mucin exocytosis takes place at the cell wall, and it appears that many transport processes across the airway epithelial cell membrane are regulated, at least in part, by CFTR (CF transmembrane ion regulator protein). Frustrated exocytosis might explain the abundant intracellular mucin staining that has been reported in CF, failure to secrete mucins might induce secretory cell hyperplasia, and frustrated exocytosis could exacerbate cellular dysfunction in the CF airway. Perhaps during exacerbations, other factors intervene to augment the release of these stored mucins or wash them out into the airways.
The authors thank Dr. Joseph Tobin, Wake Forest University, Winston-Salem, North Carolina, for collecting the ETT samples; L. Vannoy, K. Haus, and Dr. Samir Shah for technical assistance; and T. Ploch for help with the statistical analysis.
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