Exhaustion of Airway Basal Progenitor Cells in Early and Established Chronic Obstructive Pulmonary Disease

Abstract

Rationale: Up to 40% of smokers develop chronic obstructive pulmonary disease (COPD) over a period that spans decades. Despite the importance of COPD, much remains to be learned about susceptibility and pathogenesis, especially during early, prediagnostic stages of disease. Airway basal progenitor cells are crucial for lung health and resilience because of their ability to repair injured airways. In COPD, the normal airway epithelium is replaced with increased basal and secretory (mucous) cells and decreased ciliated cells, suggesting that progenitors are impaired.

Objectives: To examine airway basal progenitor cells and lung function in smokers with and without COPD.

Methods: Bronchial biopsies taken from smokers at risk for COPD and lung cancer were used to acquire airway basal progenitor cells. They were evaluated for count, self-renewal, and multipotentiality (ability to differentiate to basal, mucous, and ciliated cells), and progenitor count was examined for its relationship with lung function.

Measurements and main results: Basal progenitor count, self-renewal, and multipotentiality were all reduced in COPD versus non-COPD. COPD progenitors produced an epithelium with increased basal and mucous cells and decreased ciliated cells, replicating the COPD phenotype. Progenitor depletion correlated with lung function and identified a subset of subjects without COPD with lung function that was midway between non-COPD with high progenitor counts and those with COPD.

Conclusions: Basal progenitor dysfunction relates to the histologic and physiologic manifestations of COPD and identifies a subset that may represent an early, prediagnostic stage of COPD, indicating that progenitor exhaustion is involved in COPD pathogenesis.

Keywords: cell self-renewal; disease susceptibility; early diagnosis; multipotentiality; stem cells.

Figure 1.

Airway progenitor counts in non–chronic obstructive pulmonary disease (COPD) and COPD airways. (A) Steps to grow and quantify clone-forming basal progenitors from airway biopsies. (B) Representative image of an endobronchial biopsy (arrow). (C) Cytospin preparation of biopsy digest stained with antibodies to K5 (keratin 5) (green), K14 (keratin 14) (red), and DAPI (blue). K5/K14 dual positive cells are yellow. (D) Cytospin stained with antibodies to Muc5b (mucin 5B) (green), FoxJ1 (Forkhead box protein J1) (red), and DAPI (blue). Scale bars are shown and colored arrowheads point to each cell type in C and D. (E–H) Pictures of clones generated by human airway basal progenitor cells. (E) Bright-field image, (F) K5 (green), (G) K14 (red), and (H) p63 (transformation protein 63) (red) and DAPI (blue, in panels F–H). Scale bars (70 μm) are shown in E–H. (I) Progenitor counts (number of clones/10³ epithelial cells) in non-COPD (n = 31) and COPD (n = 19). Horizontal lines show means ± SE. P value indicates results of Mann-Whitney test.

Figure 2.

Self-renewal and multipotentiality of airway progenitor cells in non–chronic obstructive pulmonary disease (COPD) versus COPD. (A) Airway progenitor cell self-renewal in non-COPD (n = 18) and COPD (n = 13). (B and C) Representative images of basal cell clones made by non-COPD (B) and COPD (C) progenitors. Multipotentiality of progenitors was determined by measuring differentiation during air–liquid interface culture. Scale bars (70 μm) are shown in B and C. (D–G) En face images of differentiated membranes from non-COPD progenitors (D and E) and COPD progenitors (F and G). In D and F, immunostaining was performed for K5 (keratin 5) (green) and DAPI (blue). In E and G, immunostaining was performed for Muc5b (mucin 5B) (red), acetylated tubulin (ACT) (green), and DAPI (blue). Arrowheads in G show mucus accumulation in the membranes from COPD progenitors. The scale bar in D applies to D–G. (H) Quantification of differentiated cell types using morphometry (n = 10 of each for non-COPD and COPD). Data presented as means ± SE. P values indicate results of Man-Whitney test.

Figure 3.

Spatial distribution of cells within cross-sections of differentiated epithelium. Differentiated membranes from air–liquid interface cultures were paraffin-embedded, sectioned, and stained. (A and B) Hematoxylin and eosin staining shows the histology of differentiated epithelia generated by non–chronic obstructive pulmonary disease (COPD) and COPD progenitors. Insets in A and B show magnified areas to demonstrate the presence or absence of cilia in non-COPD versus COPD epithelia. (C–K) Immunostaining and quantification of each cell type using morphometry. Name and color of antigens used are shown in respective images. Representative images from 10 non-COPD and COPD cultures are shown here. Data in E, H, and K are presented as means ± SE. All P values indicate results of Mann-Whitney test. Scale bars are shown in each image. K5 = keratin 5; Ki67 = marker of proliferation, MKi67; p63 = transformation protein 63; Scgb1a1 = secretoglobin family 1A member 1.

Figure 4.

Airway progenitor counts and measurements of lung function in the combined and current smoker cohorts. (A–D) The combined cohort of current/ex-smokers with and without chronic obstructive pulmonary disease were examined by Pearson correlations to determine whether airway progenitor counts were related to measures of airflow, including FEV₁% predicted (A), FEV₁/FVC ratio (B), forced expiratory flow, midexpiratory phase (FEF_25–75) predicted (C), and FEF_25–75/FVC ratio (D). (E) Current and former smokers were examined for differences in airway progenitor counts. Data presented as means ± SE. P value indicates results of Mann-Whitney test. (F–L) Pearson correlations were performed to examine the relationship between airway progenitor counts and measures of lung function in current smokers, including FEV₁% predicted (F), FEV₁/FVC ratio (G), FEF_25–75% predicted (H), FEF_25–75/FVC ratio (I), RV/TLC ratio (J), Dl_CO % predicted (K), Dl_CO/Va ratio (L), and pack-years of cigarette smoke exposure (M). RV = residual volume.

Figure 5.

Relationship between progenitor counts and airflows in subjects with and without chronic obstructive pulmonary disease (COPD). (A–D) Current/ex-smoker subjects without COPD (non-COPD) were segregated into high and low progenitor groups and compared with COPD. All three groups were plotted and analyzed for differences based on (A) FEV₁% predicted, (B) FEV₁/FVC ratio, (C) forced expiratory flow, midexpiratory phase (FEF_25–75) predicted, and (D) FEF_25–75/FVC ratio. One-way ANOVA P < 0.001 for A–D. Results of a Tukey-Kramer post hoc analysis are shown in each panel.