Previous studies have investigated whether there is a difference in the amounts of the polymeric mucins, MUC5AC and MUC5B, in the airways of individuals with COPD and those from smokers without airway obstruction (15
). To address this issue, these studies analyzed mucin gene expression of airways epithelial cells or the mucin content of epithelial goblet cells. No quantitative analysis of the mucins in the secreted mucus gel that may obstruct the airways was reported, nor was any information presented on the other source of mucin production: the submucosal gland mucous cells. Here, for the first time we have compared the polymeric mucins present in the sputum from individuals with COPD with those from smokers without airway obstruction. MUC5AC and MUC5B are the major polymeric mucins in these sputum samples and their relative levels showed a significant difference between the two groups. MUC5B was more prominent in the COPD sputum, and increasing levels of this mucin correlated with a lower level of lung function. We have previously reported that MUC5B was the major polymeric mucin in purified mucin preparations from COPD sputum (11
). However, in that study, we did not control for salivary contamination (see
subsequent text), and there were no patient details, such as FEV1
, age, pack-years, or current medication.
At first glance, our compositional data are at odds with the mucin gene expression and immunohistochemical data that have indicated an increase in MUC5AC (15
) and a decrease in MUC5B (16
) in the epithelial cells in COPD airways. However, these studies did not analyze mucin production by submucosal glands, which have been shown to undergo hypertrophy in COPD. Our immunohistochemical data indicate that MUC5B is mainly being produced by submucosal glands, and thus we suggest that, with increasing severity of disease, the glandular secretions are making a greater contribution to the mucin composition of the gel. In support of this, immunoreactivity of intraluminal mucus in patients with COPD suggested that MUC5B was the predominant mucin (15
In the two groups, smokers without airway obstruction and COPD, MUC5AC is present at levels above that found previously in induced sputum from healthy individuals (11
). These data are consistent with the reported increase in MUC5AC mRNA expression and stored mucin in the airways of smokers with airflow obstruction, and increased MUC5AC correlated with FEV1
). Here we show that increasing amounts of MUC5B in sputum, in conjunction with already high levels of MUC5AC, correlates with level of FEV1
. Moreover, the lower-charged glycoform of MUC5B is increased in the COPD sputum. This is in agreement with previous analyses of MUC5B isolated from COPD sputum, which demonstrated a decrease in “acidity” of the mucin compared with that from normal subjects (11
). Whether this is due to a gross change in glycosylation machinery of MUC5B-secreting cells or production from a specific population of MUC5B-secreting cells is not currently known. However, immunohistochemical data using a carbohydrate-specific probe suggest that the different MUC5B glycoforms may be the products of different cells (21
How the changes in mucin composition of sputum relate to the pathobiology of COPD is not known. One might speculate that the increased levels of mucin alone might alter the transport properties of the mucus gel, resulting in reduced lung function and increased incidence of infection. On top of this, the change in glycosylation (increased amounts of low-charge MUC5B) could also affect the protective properties of the gel. For example, low–charge density MUC5B mucins might form a tighter, less expanded gel network that is less efficiently transported. In this regard, mucus plugging the airways of an individual in status asthmaticus
was enriched in the low-charged form of MUC5B (22
). Altered glycosylation of mucins has previously been reported in airways disease (11
). Because mucin glycans are known receptors for microorganisms, changes in glycosylation might favor bacterial colonization, increasing the risk of exacerbation and infection (24
Entanglement of polymeric mucin chains is an important mechanism for mucus gel formation (5
), and the extent of mucin polymerization might be expected to impact the rheological properties of the mucus gel. Solubilization of the mucins in the highly denaturing solvent, 6 M GdmCl, and subsequent analysis of their sedimentation behavior revealed a shift toward smaller mucins in COPD, which was more pronounced for MUC5AC. Whether this difference arises as part of the synthetic process, or after secretion in the mucus, is not known. However, MUC5AC and MUC5B have been shown to be degraded in cystic fibrosis mucus, and it was suggested that MUC5AC may be more susceptible to proteolytic degradation than MUC5B (26
). Although the size of the polymeric mucins is important, one cannot rule out that the amounts of other components of the mucus gel that might influence either the physical or antibacterial properties of the gel are altered. For example, the trefoil peptide, TFF3, has been shown to be raised in COPD (27
), and other studies have suggested that these peptides can interact with mucins to alter the rheological properties of mucus (28
Initial analysis of the data referenced mucin content to total sputum weight (sol plus gel) and showed no overall significant change in total mucin between the two clinical groups. However, we could not rule out that mucin present in saliva, a MUC5B-containing secretion (30
), was present in the expectorated sputum samples. If saliva were present, then one would reasonably expect that this watery secretion would most likely contaminate the sol samples. Indeed, the MUC5B levels in the sol samples were very similar to those of saliva. Further evidence to indicate that expectorated sputum contains saliva was recently demonstrated in a proteomic analysis of induced sputum (31
). Therefore, in subsequent analysis, we only looked at the gel component of the sputum, as it was more solid-like in nature and could be removed from the liquid-like sol, and was thus unlikely to suffer major contamination by saliva.
It is important to consider some of the limitations of the methods used here. First, there are a number of potential pitfalls associated with using Western blotting to determine the amounts of MUC5AC and MUC5B, and these arise due to the complex nature of these glycoproteins. For example, the antisera used are raised against peptidic epitopes in cysteine regions of the polypeptides that are supposed to be nonglycosylated. However, this has not been proven, and glycosylation could therefore affect antibody binding. Furthermore, proteolytic cleavage of the mucins could destroy epitopes. In addition, the mucin standards used were purified from nonrespiratory secretions, and could be modified differently to their airways counterparts. However, even after taking these potential problems into account, this is currently the best method available to analyze specific mucin gene products from sputum. Another issue to consider is whether there is a difference between induced and spontaneous mucus in terms of mucin composition, as we were unable to obtain spontaneous sputum from all subjects. Inspection of the data showed that there was no obvious difference in the nine samples produced by induction, and these data points were effectively indistinguishable from the spontaneous samples. Moreover, there are no published reports of differences between spontaneous and induced sputum regarding mucin composition.
In conclusion, we have found differences in mucin properties that could contribute to progression of COPD. Further studies are needed to see if altered mucus transport or binding properties contribute to the bacterial colonization and infection of the lower airways often seen in more severe disease.