Abstract
Cystic fibrosis (CF) is characterized by progressive airway obstruction. Although it has been postulated that this is due in part to mucus hypersecretion, there are no published data showing an increase in the gel-forming mucins MUC5AC or MUC5B in CF secretions. We used confocal microscopy to assess the amount of mucin-like glycoprotein and DNA in CF sputum and found more mucin in bronchitis sputum and a much greater amount of DNA in CF sputum. We then used antibodies to MUC5AC and MUC5B with Western gels and dot-blot to quantify mucin in sputum from 12 patients with CF and 11 subjects without lung disease. There was a 70% decrease in MUC5B and a 93% decrease in MUC5AC in CF sputum (P < 0.005 for both). We conclude that the vol/vol concentration of MUC5AC and MUC5B are decreased in the CF airways relative to normal mucus. This may be due to a relative increase in other components of sputum in the CF airway or to a primary defect in mucin secretion in CF.
In health, the mucin glycoproteins are the major macromolecular component of the mucous gel, responsible for the rheologic properties of mucus (1). Gel-forming mucins influence the physical and clearance properties of mucus (2). Mucus is a protective coating secreted in the healthy airway, whereas sputum is the product of airway inflammation and usually contains cells, inflammatory mediators, bacteria, highly polymerized DNA inflammatory cell necrosis, and mucin polymers (2, 3). Chronic airway diseases, such as asthma, chronic bronchitis (CB), and cystic fibrosis (CF), are thought to be associated with mucus hypersecretion. Mucins from the airway mucus of “normal” subjects and from CF sputum have similar molecular mass (10–30 × 106 D) (1, 4), but mucins are present at higher concentrations (wt/wt) in normal mucus than CF sputum (5), and DNA is often present at high levels in CF sputum (3).
Respiratory mucins are polydisperse in mass (2–40 × 106 D) and length (0.5–10 μm) (4) and appear as filaments under electron microscopy. Northern blot and in situ hybridization have shown that at least eight mucin genes (MUC) are expressed as messenger RNA in the respiratory tract (6). Mucins are classified as secreted or membrane-tethered. Three secreted, gel-forming mucins, MUC2, MUC5AC, and MUC5B, have been reported to be expressed by airway epithelium, but only MUC5AC and MUC5B have been convincingly demonstrated to be major gel-forming mucins in normal or pathologic airway secretions (7, 8). MUC5AC appears to be produced primarily by the goblet cells in the tracheobronchial surface epithelium (7), whereas MUC5B is secreted primarily by the submucosal glands (9).
There are few published data regarding specific MUC mucin protein composition of airway secretions in health or disease (6, 10, 11). The current paradigm is that MUC5AC and MUC5B are predominant mucins in airway secretions in normal subjects and in patients with CF, asthma, and CB, and that mucin levels are variable (11). Although patients with CF have poor mucus clearance and increased airway secretions, there are no published data that demonstrate increased mucin content in the CF airway. Immunohistochemistry studies show that MUC5AC and MUC5B mucins are expressed in the same histologic pattern in CF compared with normal tissues with an increase of MUC5AC-positive cells due to goblet cell hyperplasia and metaplasia in CF tissues (12). The marked remodeling in the CF airway suggests that with increased secretions, there may be increased mucin content. To test the general hypothesis that CF mucus contains increased mucin content, we examined sputa from patients with CF and those with CB by confocal microscopy and studied CF sputum and mucus from normal subjects by gel electrophoresis and dot-blot using specific antibodies to differentiate between the major gel-forming mucins MUC5AC and MUC5B.
Sputum was collected from patients with CF (n = 15) and from patients with bronchitis (n = 2) who routinely attend the Wake Forest University clinics. Sputum was collected over 30 min during pulmonary function testing by direct expectoration into a sterile cup. Salivary contamination was minimized by having the subject swallow saliva before expectorating and by separating sputum from saliva by visual inspection at the time of transfer into cryovials for preservation at −70°C until further processing. The clinical characteristics and demographics of the subjects with CF are given in Table 1
Lane No. | Age (Yr) | Height (Percentile) | Weight (Percentile) | Sex | FVC% | FEV1% | Genotype |
|---|---|---|---|---|---|---|---|
| 12 | 9 | 50 | 25 | Female | 49 | 32 | Homozygous dF508 |
| 13 | 10 | 50 | 25 | Female | 22 | 19 | Homozygous dF508 |
| 14 | 21 | 1 | 1 | Male | 36 | 19 | Homozygous dF508 |
| 15 | 18 | 50 | 50 | Male | 88 | 87 | Homozygous dF508 |
| 16 | 10 | 50 | 75 | Female | 83 | 65 | Homozygous dF508 |
| 17 | 19 | 25 | 10 | Female | 98 | 87 | Homozygous dF508 |
| 18 | 31 | 5 | 5 | Male | 56 | 31 | Homozygous dF508 |
| 19 | 31 | 5 | 5 | Male | 57 | 32 | Homozygous dF508 |
| 20 | 36 | 50 | 75 | Female | 73 | 52 | dF508/unknown |
| 21 | 38 | 25 | 5 | Female | 33 | 24 | Homozygous dF508 |
| 22 | 12 | 10 | 5 | Female | 79 | 43 | Homozygous dF508 |
| 23 | 16 | 10 | 1 | Female | 62 | 44 | Homozygous dF508 |
Mucus was collected from the end of noncuffed endotracheal tubes (ETT) of 11 subjects who had no lung disease but required nonthoracic surgery under general anesthesia. At the time the subject was extubated, the ETT was removed from the airway and mucus coating the tube was removed by scraping the ETT (13, 14).
Sputum and ETT mucus collection were approved by the Wake Forest University Institutional Review Board.
We used laser scanning confocal microscopy (LSCM) to examine sputum components from three patients with CF and two with CB. Sputa were dual-labeled using 10 μg/ml fluorescent Texas Red-conjugated Ulex europaeus agglutinin (UEA) lectin (Sigma, St. Louis, MO) for mucin-like glycoproteins and 1μM YOYO-1 (Molecular Probes, Eugene, OR) for DNA. A Carl Zeiss LSM 510 (Carl Zeiss, Jena, Germany) and a Leica laser scanning confocal microscope (Leica CLSM; Leica, Lasertechnik GmbH, Heidelberg, Germany) were used to collect images of the stained sputum. Dual excitation wavelengths of 488λ and 568λ were employed to visualize the DNA in combination with mucin. Images were recorded in a planar matrix (X, Y) using the 40× oil objective. Optical sections in the Z-axis were recorded by adjusting the stage height by stepper motors. Quantitative measurements of fluorescence intensity and area were obtained directly from images using VoxelView software (Vital Images, Fairfield, IA). Representative fields of interest were visually selected and random coordinates within the field were imaged and analyzed to give mean fluorescent intensities. Serial images for each specimen were analyzed and the mean surface area covered by Texas Red–UEA and YOYO-1 was calculated using NIH Image imaging software (National Institutes of Health, Bethesda, MD; available online at http://rsb.info.nih.gov/nih-image).
Synthetic peptides with sequences RNQDQQGPFKMC, present in the C-terminal portion and in the two stretches flanking a tandem repeat region of the MUC5AC apoprotein, and RNREQVGKFKMC, present in the cysteine-rich domains of the super-repeats within the central exon of the MUC5B apoprotein, were conjugated to keyhole-limpet hemocyanin (KLH) and used to raise antibodies in rabbits (Genemed Synthesis, San Francisco, CA). The antibodies were characterized and specificity was ascertained by preabsorption studies using increasing concentrations of the antigenic peptides as previously done for the original LUM5–1 and LUM5B-2 antibodies (7, 9). Rabbits were bled 4 wk later for antisera, named LUM5-1–WFU for the MUC5AC antibody and LUM5B-2–WFU for the MUC5B antibody. Each antiserum was evaluated for specificity using enzyme-linked immunosorbent assay for the peptides and by Western blot for the proteins in human mucus. To verify the specificity of our antibodies, we performed a polyacrylamide gel electrophoresis with cell lysates, secretions from normal human tracheobronchial epithelial cells (passage 2) (Clonetics Corp., La Jolla, CA), and human mucus. The blots were analyzed with antisera for MUC5AC and MUC5B and the preimmune sera of the same rabbit. We found one well-defined band of high molecular weight with the antisera. To increase the specificity of the antibodies and reduce nonspecific binding, we affinity-purified the antipeptide antibody from the whole serum using the immobilized amino acid sequences of interest (SulfoLink-Kit, Pierce Chemical Co., Rockford, IL).
Protease inhibitors (Protease Inhibitor set I, Calbiochem, La Jolla, CA) were added in equal volumes to the sample during thawing. The samples were homogenized by aspirating several times through progressively smaller needles (final size 28G), diluted 1/10 with phosphate-buffered saline (PBS), and homogenized again with a 28G needle. Total protein concentration was measured using a BCA-Kit (Pierce).
Sputum and ETT samples were applied in Laemmli buffer (250 mM Tris, pH 6.8; 4% sodium dodecyl sulfate; 20% glycerol; 0.001% bromophenol blue, 20 mM dithiothreitol [DTT]) and electrophoresed in 1% agarose gels (15 × 15 cm), prepared in running buffer (25 mM Tris, 250 mM glycine, 0.1% sodium dodecyl sulfate). Electrophoresis was performed in a horizontal gel apparatus at 100 V at room temperature. To identify small proteins that remained in the gel, the gel was stopped when the dye front was two-thirds of the distance from the wells. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrical transfer (30V) for 18 h at 4°C.
Samples were applied to the top row of a 96-well plate in a potassium thiocyanate (KSCN)-DTT solution (KSCN 0.6 M, DTT 20 mM). The samples were diluted (1:1) from row to row with the KSCN-DTT solution, transferred to a 96-well acrylic Dot-Blot System (Schleicher and Schuell, Dassel, Germany), and blotted to a nitrocellulose membrane while applying a vacuum for 1 min. The blots were then probed with anti-MUC5AC or MUC5B and detected with chemiluminescence to determine the limiting detectable dilution of each sample, reported as the relative concentration. Dot-blots were analyzed by counting the number of visible dots per sample, equivalent to the number of serial dilutions required to reach limiting detectable titer. The sum of the dots of each patient sample was used as an exponent of 2 and the mean was used to compare the relative concentrations of mucins among patient samples, using the equation (2x1 + 2x2)/n . For example, for the ETT mucus concentration of MUC5B, four dots were counted for sample 1, two for sample 2, six for sample 3, and five for sample 4. The calculation (24 + 22 + 26 + 25)/4 was used to determine the mean of 28.2 shown in Figure 3.
The membranes were blocked with 5% nonfat skimmed milk in PBS for 30 min at room temperature. They were incubated with primary antibodies (1:100 MUC5AC; 1:250 MUC5B) for 1 h in 1% nonfat skimmed milk in PBS, washed 3 times in PBS for 10 min, and incubated with the secondary horse radish peroxidase–labeled goat–anti-rabbit antibody (1:1000) (Jackson-Immuno, West Grove, PA) in 1% nonfat skimmed milk in PBS for 1 h. Finally, they were washed 3 times in PBS for 10 min. Membranes were developed using the Pico-Developer Kit (Pierce). Exposures were taken on X-Omat Blue XB-1 film (Kodak, Rochester, NY) at equal times. The membranes of the gel electrophoresis/dot-blot were first probed with affinity-purified MUC5AC, stripped, and probed again with affinity-purified MUC5B antibody. We then repeated the gel electrophoresis/dot-blot using new samples and the membranes were then probed first with affinity-purified MUC5B, stripped, and then probed with affinity-purified MUC5AC antibody with equivalent results.
The respiratory mucins were electrophoretically separated by size in a 1% agarose gel and immobilized onto a polyvinylidene-fluoride membrane by an 18 h electrical transfer. The membrane was washed in PBS, incubated at 37°C for 30 min in neuraminidase (in 50 mM sodium acetate, 150 mM sodium chloride, 100 mM calcium chloride, pH 5.5) to remove sialic acid, washed and followed by an incubation in a glass bottle at 4°C for 15 min with trifluoromethanesulfonic acid to remove core sugars (15). The membrane was then thoroughly washed in PBS, blocked, and probed with anti-mucin antibodies.
As a test of our initial hypothesis that mucin concentration was increased in CF sputum, we used LSCM to visualize polymer components sputum from three patients with CF and two with CB (16). Specimens were dual-labeled using fluorescent Texas Red–conjugated UEA lectin for mucin glycoprotein and YOYO-1 for DNA (Figure 1)
Figure 1. Confocal micrograph showing the mucin polymers (Texas Red–UEA) and DNA polymers (Green-YoYo1) in bronchitis and CF sputum. Sputum was collected at the start of a pulmonary exacerbation of each disease. Note the increased green staining DNA polymers and decreased mucin stain (red) in the CF sputum when compared with CB sputum. The punctate YoYo-1 staining of the CB sputa suggests that there was a greater amount of DNA in intact inflammatory cell nuclei. There was 45% less mucin and 416% more DNA by area in CF sputum than in CB sputum.
[More] [Minimize]We measured the gel forming mucin protein content of sputum from patients with CF (n = 12) and mucus from normal control subjects (n = 11) using gel electrophoresis and dot-blots probed with specific MUC5AC (LUM5-1–WFU) and MUC5B (LUM5B-2–WFU) antibodies. The CF samples were obtained from patients routinely attending the CF clinic at Wake Forest University. The age of the patients with CF was 20.92 ± 3.04 (mean ± SEM) years. Clinical and demographic data are summarized in Table 1. For comparison, control mucus was collected from the endotracheal tube (ETT) of surgical patients with no lung disease (age 18.5 ± 8.03 y). The Western blots were probed with anti-peptide antibodies for both MUC5AC and MUC5B and showed significantly decreased content of both mucins that was most striking for MUC5AC, where the protein was not detectable in many samples even after 5 min of film exposure (Figure 2)
Figure 2. ETT samples from normal control subjects (details in the text) and sputum from patients with CF was examined on 1% agarose gel electrophoresis, blotted to nitrocellulose membranes, and probed with affinity-purified MUC5AC and MUC5B antibodies. Equal volumes of sputum were loaded in each lane and all blots were incubated and exposed together under equal conditions.
[More] [Minimize]Since the oligomerization of mucins results in broad banding of high molecular weight polymers, it was difficult to quantitate changes in mucin content. Therefore, we used dot-blot to determine the relative concentration of each mucin. The blots revealed a quantitative decrease in both MUC5AC and MUC5B in CF sputum relative to normal mucus (Figure 3)
Figure 3. Serial diluted dot-blot analysis of ETT (dotted bars) mucus from normal control subjects and sputum from patients with CF (hatched bars). The samples were loaded as volume equivalents from the sputum. The blot was probed with affinity-purified antibodies for MUC5AC and MUC5B. The results for MUC5AC and MUC5B cannot be directly compared with one another because antibody affinity is probably different. (*Students t test P < 0.005).
[More] [Minimize]The membranes of the Western and dot-blot were first probed with affinity-purified MUC5AC, then stripped and probed with affinity-purified MUC5B antibody. We repeated the Westerns and dot-blots using new samples probed first with affinity-purified MUC5B, stripped and then probed with affinity-purified MUC5AC antibody. The results were identical (data not shown).
All samples were loaded on the gel as volume equivalents from the sputum (Figures 2 and 3). Since dilution of the sputa could affect the appearance of the Western blots, samples were also loaded onto a gel as total protein equivalents (not shown). The total protein of the volume equivalent samples was 3,809 ± 1,179 μg/ml in the CF samples and 6,296 ± 1,584 μg/ml in the normal mucus. Total protein was normalized to 1,000 μg/ml diluting the samples with PBS prior to repeating the Westerns. The appearance of MUC5AC and MUC5B in the Western gels was similar in both the volume and protein equivalent loaded blots.
To determine if changes in glycosylation would affect antibody binding, we repeated the gel electrophoresis, deglycosylated the mucin bound to the membrane, and probed the membrane with MUC5AC and MUC5B antibodies. We compared the deglycosylated samples, directly with untreated samples on a different membrane under equivalent antibody and exposure conditions. We found that there was no difference in MUC5AC nor MUC5B comparing the deglycosylated to the native mucins (not shown), so the decreased mucin content we observed in CF sputum samples was not a result of changes in glycosylation.
To determine whether the mucins were physically sheared by aspiration through the 28G needles, we aspirated three salivary samples and compared them with native salivary samples in a Western blot. We found no significant differences in the MUC5B content of the sheared and control samples. As expected, MUC5AC could not be detected in any of these samples.
Patients with CF and CB have extensive mucus plugging in their airways leading to increased susceptibility to infection and decreased pulmonary function. Although it has been speculated that in CF this is due to mucus hypersecretion, there are no published data supporting this hypothesis. Therefore we wished to determine if there is an altered mucin concentration in CF airway secretions by measuring mucin in expectorated sputum.
In our initial small study (n = 2 CB and 3 CF) using LSCM we found that in CF sputum the mucin-like glycoprotein was reduced compared with CB sputum. We also found that the amount of DNA in CF sputum was greatly increased compared with CB sputum, most likely as a result of polymorphonuclear leukocyte necrosis (3, 17, 18). Thus DNA probably has a much greater effect on CF sputum properties and volume than mucins. These samples were completely consumed by LSCM analysis precluding further testing.
Given the limitations of lectin binding assays for determining changes in individual mucin species, specific antibodies were raised against MUC5AC and MUC5B. Both the Western blots and the dot-blots revealed a significant reduction of MUC5AC and MUC5B in CF airway sputum compared with normal airway mucus. This was most dramatic for MUC5AC, which required detection by titration with dot-blots to detect any mucin in 8 of 12 patient samples. Consistent with these findings, Rose obtained mucus by hypertonic saline induction and showed that mucins were present at lower concentrations in three patients with CF compared with two normal volunteers (5). It was speculated that this was due to increased proteinase activity in vivo. However, our results show that MUC5AC and MUC5B mucins from patients with CF and normal control subjects have the same apparent size (Figure 2), thus suggesting that these mucins were not proteolytically digested in our CF samples.
The ratio of MUC5B to MUC5AC in the normal control mucus was 0.5, while in CF sputum the ratio increased to 2.4, reflecting a larger decrease in MUC5AC in the CF samples. This shift in mucin expression was also seen by Davies and coworkers, who reported an increased ratio of MUC5B to MUC5AC in both CF and CB sputum compared with that from healthy control subjects (19). Kirkham and colleagues also found an increase in the relative amount of the MUC5B mucin to MUC5AC mucin in both CF and COPD expectorated sputum in comparison with asthma mucin (P < 0.05), but not in comparison to saline-induced mucus from healthy control subjects (11). We have previously shown that biophysical properties of ETT mucus are not significantly affected by general anesthesia (13). As opposed to saline-induced sputum, ETT mucus represents proximal airway secretions, making this an ideal comparator to expectorated sputa.
It is possible that increased proliferation of the airway epithelium, resulting directly from CF transmembrane ion regulator protein (CFTR) abnormalities or indirectly from infection, injury, and repair, could lead to a generally less mature (differentiated) epithelium and that this, in turn, might result in decreased mucin biosynthesis and secretion. CFTR plays a crucial role in the differentiation of the respiratory epithelium during ovine (20) and murine (21) fetal lung development, and may play a regulatory role in the development of the secretory epithelium (21). Differences in intracellular pH might enhance proliferation of CFTR−/− cells. Cytosolic pH is thought to be one of the factors that control the rate of proliferation (22), with cytoplasmatic alkalanization constituting a signal for mitogenesis (23). Cells expressing a mutated CFTR have been reported (24) to have an elevated cytosolic pH, and an overexpression of wild-type CFTR, but not a mutant CFTR, results in growth arrest (25). Thus, a mutation in the CFTR may result in an elevated intracellular pH, which stimulates the cell to undergo proliferation more readily and thus increase the rate at which epithelial cells proliferate and differentiate.
Increased proliferation of CF airway epithelial cells (25–27) might also be due to chronic inflammation (26). Airway inflammation in CF is associated with an increased number of bacteria and increased neutrophilic response, including human neutrophil elastase (HNE) release (28). HNE has been linked to increased cell turnover and proliferation (27, 29, 30). Chronic exposure to HNE induces secretory cell hypertrophy and hyperplasia (29, 31). Gallagher and Gottlieb (32) also showed a significant increase in regenerating CF intestinal epithelial cells due to an increased proliferation rate both in CFTR-null mice and in cell culture.
The decreased mucin concentration could also result from reduced synthesis. A defect in Golgi pH in CF cells may also affect mucin biosynthesis, assembly, and secretion (33). Barasch and colleagues suggested that a defect in Golgi pH in CF cells would decrease the activity of pH-sensitive enzymes, which leads to a defect of intracellular glycoprotein processing (22, 33). Intracellular mucins have a broad distribution of molecular mass (2 × 106 – 15 × 106 D). Analogous to the multisubunit glycoprotein, von Willebrand factor, it can be speculated that mucins are slowly polymerized within storage granules. There is evidence that while in these granules, the molecules can further be oligomerized to form large linear complexes with a relative molecular mass in excess of 10 – 40 × 106 D (34). Altered secretion of the mucin oligomers has also been observed in vitro. Extracellular ATP rapidly increased mucin protein secretion by normal pancreatic cell lines but was not able to induce mucin secretion by the corresponding CF cell line, suggesting that CFTR reduces ATP-dependent mucin secretion (35). There may also be transcriptional regulation of mucin genes. MUC5AC gene expression was decreased relative to MUC2 messenger RNA in patients with CF compared with normal control subjects (36). Thus, there are several steps in the complex process of mucin synthesis, storage, and secretion in which the CFTR defect could decrease the mucin content of airway secretions. These observations prompt the hypothesis that CF lung epithelial cells may fail to fully differentiate as a result of chronic inflammation, an increased cell turnover or a defective CFTR leading to a decreased secretion of large oligomeric mucin.
Decreased mucin concentration could also result from increased mucin degradation (5, 37) due to proteinase activity from inflammatory cells (38). The patients with CF in this study were all infected with Pseudomonas aeruginosa, making it likely that their airway secretions contained increased bacteria- and neutrophil-derived proteinases (39). To minimize the impact of continued proteolysis after sample collection, we froze aliquots as quickly as possible, treated all sputum samples with protease inhibitors while thawing, and performed all analyses on freshly thawed aliquots.
Although there is mucus plugging and chronic expectoration of sputum in persons with CF, mucin does not appear to be a major component of these secretions. At least during periods of relative clinical stability, there appears to be more DNA in CF secretions than mucin. The CF protein, CFTR, acts as a cAMP-regulated chloride channel (40), but it is not clear how CFTR dysfunction leads to the clinical manifestations of CF lung disease. It may be that mucin secretion is linked to Cl− and water secretion in the airway, increased turnover in secretory cells, or that decreased mucin secretion might be another manifestation of the CFTR defect.
It could also be that decreased mucin secretion increases susceptibility to airway infection in the CF airway. Pseudomonas aeruginosa has been shown to bind to airway mucin (41). The dense 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 inhibit the formation of the bacterial biofilms characteristic of CF airway disease.
The authors thank Dr. J. S. Koo at the M. D. Anderson Cancer Center, Houston, for his helpful suggestions; Dr. I. Carlstedt at Lund University, Sweden, for providing us with antibodies; Dr. J. Tobin, Wake Forest University, Winston-Salem, North Carolina for collecting the ETT samples; and Dr. V. J. Kempf, J. Gyves, L. Clarkson, and H. Easterling for technical assistance. This work was supported by Christiane Herzog Stiftung, Germany, and by the U.S. Cystic Fibrosis Foundation (CFFTI Grant #Rubin00A0).
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