MUC5B is the major mucin in the gel phase of sputum in chronic obstructive pulmonary disease

Abstract

Rationale: Overproduction of mucus is a contributory factor in the progression of chronic obstructive pulmonary disease (COPD). The polymeric mucins are major macromolecules in the secretion. Therefore, we hypothesized that the polymeric mucin composition or properties may be different in the sputum from individuals with COPD and smokers without airflow obstruction.

Objectives: To determine the major polymeric mucins in COPD sputum and whether these are different in the sputum from individuals with COPD compared with that from smokers without airflow obstruction.

Methods: The polymeric mucin composition of sputum from patients with COPD and smokers without airflow obstruction was analyzed by Western blotting analysis. The tissue localization of the mucins was determined by immunohistochemistry, and their size distribution was analyzed by rate-zonal centrifugation.

Measurements and main results: MUC5AC and MUC5B were the major mucins. MUC5AC was the predominant mucin in the smoker group, whereas MUC5B was more abundant from the patients with COPD, with a significant difference in the ratio of MUC5B to MUC5AC (P = 0.004); this ratio was correlated with FEV(1) in the COPD group (r = 0.63; P = 0.01). The lower-charged glycosylated form of MUC5B was more predominant in COPD (P = 0.012). No significant associations were observed with respect to sex, age, or pack-year history. In both groups, MUC5AC was produced by surface epithelial cells and MUC5B by submucosal gland cells. Finally, there was a shift toward smaller mucins in the COPD group.

Conclusions: Our data indicate that there are differences in mucin amounts and properties between smokers with and without COPD. Further studies are needed to examine how this may impact disease progression.

Figures

Figure 1.

Amount of MUC5AC and MUC5B mucins in sputum and saliva collected from study subjects. The concentration of MUC5AC and MUC5B in the sol and gel phases of sputum, and saliva, were determined by quantitative Western blotting (see Methods). Data are presented as μg/g of total sputum or saliva for chronic obstructive pulmonary disease (COPD) (open bars) and smokers with no airflow obstruction (gray bars). The data are presented as box and whisker plots; the median (horizontal line inside bars) is shown, as well as a box showing the interquartile ranges (25th and 75th percentile); individual outlier points beyond the 10th and 90th percentile are indicated (open circles, only seen in patients with COPD).The asterisk and the solid diamond on the data shown for the sol indicate the median value for MUC5AC and MUC5B in the saliva from the two groups. Inset shows an example of agarose gel separation of MUC5AC and MUC5B mucins from gel (G) and sol (S) phases of sputum and saliva (sal) of a single individual. The different glycoforms of MUC5B are highlighted; low charge (L) and high charge (H). The gel shown was loaded with different amounts of sample to highlight the different mucins present and was not used for quantitative analysis.

Figure 2.

Amount of MUC5AC and MUC5B mucins in the gel phase of sputum collected from study subjects. The concentration of MUC5AC and MUC5B in the gel phase of sputum was determined by Western blotting (see Methods). (A) Data for each mucin are presented as μg/g of gel, and in (B) the ratio of the mucins (MUC5B/MUC5AC) is shown; COPD (open bar), smokers with no airflow obstruction (gray bar). The median (horizontal line inside bar) is shown, as well as a box showing the interquartile ranges (25th and 75th percentile); individual outlier points beyond the 10th and 90th percentile are indicated (open circles).

Figure 3.

Ratio of MUC5B to MUC5AC mucins in the gel phase as a function of FEV1. The ratio of the mucins (MUC5B/MUC5AC) in the gel phase of sputum is plotted against post-bronchodilator FEV1. The median (horizontal line inside bar) is shown as well as a box showing the interquartile ranges (25th and 75th percentile); individual outlier points beyond the 10th and 90th percentile are indicated (open circles).

Figure 4.

Ratio of the glycoforms of the MUC5B mucin in the gel phase of sputum collected from study subjects. The ratio of the concentration of the MUC5B glycoforms in the gel phase of sputum was determined by Western blotting (see Methods) for COPD (open bar) and smokers with no airflow obstruction (gray bar). The median (horizontal line inside bars) is shown, as well as a box indicating the interquartile ranges (25th and 75th percentile); individual outlier points beyond the 10th and 90th percentile are indicated (open circles).

Figure 5.

Comparison of mucin content in bronchial biopsies from a healthy smoker and a patient with COPD. Representative sections from a healthy smoker (A–H) and a patient with COPD (I–P). Staining with H&E, PAS-alcian blue (PAS-AB), EuMUC5B (MUC5B), and MAN-5ACI (MUC5AC) at the surface epithelium (A–D and I–L), and in the submucosal glands (E–H and M–P) are shown. Sections from two other patients with COPD and one healthy smoker gave similar staining patterns.

Figure 6.

Analysis of the size distributions of MUC5AC and MUC5B mucins in the gel phase of sputum collected from study subjects. (A) A typical example of the separation of the MUC5AC mucins in the sputum from an individual with COPD (filled circles) and a smoker (open circles) after rate–zonal centrifugation. (B) For each of the samples analyzed—smokers (n = 14) and those with COPD (n = 11)—the resultant MUC5AC and MUC5B distributions were normalized and the distribution split into four sections (fractions 1–6, section A; 7–12, section B; 13–18, section C; and 19–24, section D), representing different mucin sizes from smallest (section A) to largest (section D). The proportion of mucin in each fraction was then summed for all the smoker and COPD samples, and these data are shown for MUC5AC (left-hand bars) and MUC5B (right-hand bars). Gray bars represent smokers and open bars represent patients with COPD. Error bars represent ± 1 SEM.