Evaluation of epithelial mesenchymal transition in patients with chronic obstructive pulmonary disease

Abstract

Background: The reticular basement membrane (Rbm) in smokers and especially smokers with COPD is fragmented with "clefts" containing cells staining for the collagenase matrix-metalloproteinase-9 (MMP-9) and fibroblast protein, S100A4. These cells are also present in the basal epithelium. Such changes are likely hallmarks of epithelial mesenchymal transition (EMT). We aimed to confirm the epithelial origin of these Rbm cells, and to exclude potential confounding by infiltrating inflammatory cells.

Methods: Endobronchial biopsy sections from 17 COPD current smokers, with documented Rbm splitting and cellularity were stained for neutrophil elastase (neutrophil marker), CD68 (macrophage/mature fibroblasts), CD4+/CD8+ T lymphocytes, CD19 (B-cells), CD11c (dendritic cells/inflammatory cells), and S100 (Langerhans cells). The number of cells in the Rbm and epithelium staining for these "inflammatory" cell markers were then compared to numbers staining for S100A4, "a documented EMT epitope". Slides were double stained for S100A4 and cytokeratin(s).

Results: In the basal epithelium significantly more cells stained for S100A4 compared to infiltrating macrophages, fibroblasts or immune cells: median, 26 (21.3 - 37.3) versus 0 (0 - 9.6) per mm, p < 0.003. Markedly more S100A4 staining cells were also observed in the Rbm compared to infiltrating macrophages, neutrophils, fibroblasts or immune cells or any sub-type: 58 (37.3 - 92.6) versus 0 (0 - 4.8) cells/mm Rbm, p < 0.003. Cells in the basal epithelium 26 (21.3 - 37.3) per mm) and Rbm (5.9 (2.3 - 13.8) per mm) frequently double stained for both cytokeratin and S100A4.

Conclusions: These data provide additional support for active EMT in COPD airways.

Figure 1

Bronchial biopsy specimen from a COPD current smoker double-stained for both S100A4 (green) and cytokeratin (red). Black arrows showing cells in the basal epithelium and reticular basement membrane (Rbm) and also within the superficial lamina propria, double-stained for both cytokeratin (red, anti-pan-cytokeratin monoclonal antibody, epithelial marker) and S100A4 (green, anti-S100A4 polyclonal antibody, mesenchymal marker). Overall, there are fewer double-stained cells in the Rbm than in the basal epithelium; consistent with loss of epithelial markers as these cells gain mesenchymal markers. Original magnifications 100 ×/1.30 Oil. Scale bar = 50 μm.

Figure 2

Bronchial biopsy specimen from a COPD current smoker stained for immune and inflammatory cell markers compared to S100A4. Black arrows showing cells positive for: (A) CD4 (anti-CD4 monoclonal anti-body); (B) CD8 (anti-CD8 monoclonal antibody); (C) CD68 (anti-CD68 monoclonal antibody; macrophage and mature fibroblast marker); (D) neutrophil elastase (anti-neutrophil elastase monoclonal antibody; neutrophil marker); (E) CD11c (anti-CD11c monoclonal antibody; dendritic cell/inflammatory cell marker); compared to (F) S100A4 (anti-S100A4 polyclonal antibody; mesenchymal marker) stained cells, in the basal epithelium and reticular basement membrane (Rbm). There are many more S100A4 positive cells in the basal epithelium and Rbm compared to cells stained for any inflammatory cell marker. Most of the cells positive for inflammatory cell markers are in the lamina propria below the Rbm. Original magnifications, × 630. Scale bar = 50 μm.

Figure 3

Bronchial biopsy specimen from a COPD current smoker stained for S100 compared to S100A4. Black arrows showing cells stained for: (A) S100 (anti-S100 polyclonal antibody; Langerhans cell marker); compared to (B) S100A4 (anti-S100A4 polyclonal antibody; mesenchymal marker). Original magnifications, × 400. Scale bar = 50 μm.