Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa

Abstract

Airway inflammation represents a hallmark of the cystic fibrosis (CF) disease. However, the mucosal distribution of immune cells along the CF airways has not been clearly defined, particularly in intermediate bronchi and distal bronchioles. We analysed lung tissues collected at the time of transplantation from homozygous DeltaF508+/+CF patients versus non-CF donors. Using immunohistochemistry, the distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, polymorphonuclear neutrophils (PMN), mast cells, CD3+ T cells, including the CD4+ and CD8+ subsets, CD20+ B cells, CD38+ plasma cells and CD68+ macrophages, was analysed at lobar, segmental and distal levels of the bronchial tree. Using image cytometry, the number of cells per mm2 was assessed in the depth of the bronchial wall. In CF airways, alterations mainly consisted in lesions of the surface epithelium. Numerous immune cells were heterogeneously distributed all along the bronchial tree and mainly located in the mucosa, beneath the surface epithelium. Compared to non-CF donors, the lymphoid aggregates formed by B cells were significantly larger all along the CF airways (P = 0.001). The number of T lymphocytes was higher at the CF distal level (P = 0.035), where we observed an intense tissue damage. PMN preferentially accumulated (P = 0.033) in the CF surface epithelium, which overexpressed ICAM-1 but not VCAM-1 and E-selectin. These results highlight the nature of the inflammatory infiltrate in the CF airway mucosa and emphasize a prominent implication of PMN, B and T lymphocytes in the CF disease.

Fig. 1

a–d, Histological sections of HES-stained bronchi and bronchioles. Microscopic observation of CF (a) and non-CF (b) lobar bronchi and of CF (c) and non-CF (d) segmental bronchi. The CF surface epithelium is pseudostratified, with focally denuded areas, particularly at the segmental level (arrows). Inflammatory cells are mainly observed in the lamina propria and in the lumen (lu, airway lumen; ep, surface epithelium; lp, lamina propria; cd, ciliated duct; sm, submucosa). e–f, microscopic observation of CF (e) and non-CF (f) bronchioles. Large aggregates of mononucleated cells surround the CF bronchioles (arrows). The lumen is filled with mucus, cell debris and cells with morphological characteristics of macrophages and PMN. The alveolar walls are thickened (lu: airway lumen; ep: bronchiolar epithelium; pa: parenchyma; al: alveoli; ve: vessel). Original magnification ×10 (a–d) and ×20 (e–f).

Fig. 2

Quantitative analysis of immune cells in 9 CF patients versus 3 non-CF donors. (a) Comparison of the number of PMN in the surface epithelium along the bronchial tree. (b) Distribution of PMN in bronchial walls at the segmental level. (c) Mean size of B lymphocytes aggregates along the airways. (d) Distribution of T lymphocytes in bronchial walls at the distal level. ▪ CF and □ non-CF. Results are given as mean ±SEM. Significant differences (CF versus non-CF) are indicated as a P-value obtained by a two-way anova.

Fig. 3

Immunohistochemical staining of immune cells in histological sections. PMN (elastase) in (a) CF and (b) non-CF segmental bronchi (lu: airway lumen; ep: surface epithelium; lp: lamina propria; sm: submucosa). Note the high number of neutrophils in the airway lumen and close to the basement membrane (arrows). (c) Large clusters of B lymphocytes (CD20⁺) surrounding a CF bronchiole (arrows). (d) In non-CF bronchioles, B cells are very rare (lu: airway lumen; ep: bronchiolar epithelium; pa: parenchyma; al: alveoli; ve: vessel). T lymphocytes (CD3⁺) in (e) CF and (f) non-CF distal bronchioles. Original magnification a–b ×10 and c–f ×20.