Epithelial cell proliferation contributes to airway remodeling in severe asthma

Abstract

Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction.

Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling.

Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis.

Measurements and main results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p=0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p=0.002) and Ki67 was increased (p<0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p<0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p=0.002), suggesting increased cell death.

Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe asthma.

Figure 1.

Increased epithelial and lamina reticularis (LR) thickness in bronchial biopsies in subjects with severe asthma. (A) Endobronchial biopsies were obtained from subjects with severe or mild persistent asthma, subjects with chronic bronchitis, and normal subjects, and subjected to hematoxylin–eosin staining. Representative photomicrographs are demonstrated for each condition. Bar = 20 μm. (B) Corresponding quantitative analysis of results in (A). Values for epithelial and LR thickness represent mean of at least three measured sections from each individual divided by the area by the length of the corresponding basement membrane (BM). (C) Quantitative analysis of epithelial desquamation from subjects with severe asthma, subjects with chronic bronchitis, and normal subjects. Values represent the percentage of BM covered by intact epithelium (Intact), a single layer of basal cells (Basal), or completely denuded (Denuded). Values represent the mean ± SD of at least three measured sections from each individual. A significant difference (p < 0.05) for the severe asthma group compared with the mild persistent asthma, normal, or chronic bronchitis groups is indicated by an asterisk.

Figure 2.

Increased epithelial cell proliferation and decreased cell death suppression in bronchial biopsies in subjects with severe asthma. (A) Endobronchial biopsies were obtained from subjects with severe or mild persistent asthma, subjects with chronic bronchitis, and normal subjects, and were immunostained with anti-retinoblastoma (anti-Rb), anti–Bcl-2, or anti-Ki67 monoclonal antibody. Representative photomicrographs are shown for each condition. Control staining with nonimmune IgG gave no detectable signal above background (data not shown). Bar = 20 μm. (B) Corresponding quantitative analysis of changes in (A). Values for Rb and Bcl-2 immunostaining were derived by quantitating the brown intensity of epithelial (epi) immunostaining using an image analysis system. Values for Ki67 immunostaining were derived by quantitating the positive cells over the area of the corresponding tissue in mm². Values represent mean ± SEM. A significant difference (p < 0.05) for the severe asthma group compared with the mild persistent asthma, normal, or chronic bronchitis groups is indicated by an asterisk.

Figure 3.

Increased epithelial cell proliferation and decreased cell death suppression in bronchial epithelial brushings in subjects with severe asthma. Representative immunoblot results for Rb and Bcl-2 or control β-tubulin monoclonal antibody from endobronchial brushings obtained from subjects with severe asthma and normal subjects. (B) Corresponding quantitative analysis of changes in (A). Values represent the mean percent ± SEM of Rb or Bcl-2:β-tubulin ratio in 12 subjects with severe asthma compared with Rb or Bcl-2:β-tubulin ratio in 12 normal subjects. *p < 0.05

Figure 4.

Increased cellular apoptosis in bronchial biopsies in subjects with severe asthma. Endobronchial biopsies were obtained from subjects with severe asthma and normal subjects and were immunostained with anti-Fas monoclonal antibody or TUNEL (terminal deoxynucleotidyl-mediated dUTP nick end labeling) immunofluorescence (reflecting cellular apoptosis by DNA fragmentation). Representative photomicrographs are shown for each condition. Control staining with nonimmune IgG gave no detectable signal above background (data not shown). Bar = 20 μm. (B) Corresponding quantitative analysis of changes in (A). Values for Fas immunostaining were derived by quantitating the brown intensity of epithelial immunostaining using an image analysis system. Values for TUNEL positivity were derived by quantitating the positive cells over the area of the corresponding tissue in mm². Values represent mean ± SEM. A significant difference (p < 0.05) for the severe asthma group compared with the normal subjects group is indicated by an asterisk.