Epithelial clara cell injury occurs in bronchiolitis obliterans syndrome after human lung transplantation

Abstract

Bronchiolitis obliterans syndrome (BOS) is a condition of progressive airflow obstruction that affects a majority of lung transplant recipients and limits long-term posttransplant survival. Although epithelial injury appears central to the development of BOS, little is known regarding the specific epithelial cell types that are affected in this condition. We hypothesized that BOS would involve preferential injury to the secretory Clara cells that function in innate defense and epithelial repair. To test this hypothesis, we assessed tissue transcript, tissue protein and lung fluid protein expression of Clara cell secretory protein (CCSP), a marker for Clara cells, in lung transplant recipients with BOS, BOS-free patients and in donor controls. Our results demonstrate that CCSP tissue transcript and protein expression are significantly reduced in lung transplant recipients with BOS compared to BOS-free or donor controls. In addition, we demonstrate that CCSP protein levels are significantly reduced in the lung fluid of patients with BOS compared to BOS-free controls, in cross-sectional and longitudinal analysis. Collectively, these complementary results illustrate that BOS involves a selective alteration in the distribution and function of bronchiolar Clara cells.

Figure 1

H&E and MT staining of advanced BO airways. Tissue stained for H&E (A, C) and Masson Trichrome (B,D). Images are representative of BO-affected airways from explanted lungs. Images A and B show a concentric lesion with areas of damaged epithelium (A) with subepithelial fibrosis (B). Images C and D show diffuse fibrosis from an advanced lesion with epithelial remnants (C) in the center of the fibrosis (D). All images are presented at 200x.

Figure 2

CCSP expression is significantly affected by BOS. Relative fold change in mRNA from BOS explant lung tissue (n=5) compared to BOS-free control lung tissue (n=5) using taqman validated primers and probes. A. CCSP, a marker for Clara cells. B. FoxJ1, marker for ciliated cells. BO-affected tissue shows a significant decrease in CCSP transcript compared to normal BOS-free control tissue, while FoxJ1 transcript remains relatively unchanged.

Figure 3

Depletion of epithelial CCSP occurs in BOS but is preserved in BOS-free and normal donor tissues: Tissue is co-immuno-stained for CCSP (red) and AT (green) and nuclear counter stained with DAPI (blue). White arrow heads show areas of CCSP expression throughout an airway. CCSP and AT were assessed in the airways of BOS-free donor tissue (A, B), BOS-free transplant tissue (C, D), early BOS tissue (E, F), and advanced BOS tissue (G, H). Representative airways showing normal CCSP expression in BOS-free donor tissue (A, B) and BOS-free transplant tissue (C, D). Representative airways demonstrating the depletion of CCSP in BOS 1(E) and BOS 2 (F). Similar CCSP loss in the airways of advanced BOS 3 (G,H). CCSP expression in the BOS free donor tissue is very similar to BOS free transplant tissue, while early BOS tissue and advanced BOS tissue show significant CCSP loss. Images A–D, F are presented at 200x, image E is presented at 400X.

Figure 4

Semi-quantitative analysis demonstrates that CCSP expression is reduced per length of airway in patients with advanced BOS as compared to BOS-free donors. The analysis used representative terminal bronchioles from BOS-free donors (n=3) compared to patients with advanced BOS (n=3) and assessed a comparable length of airway in each sample.

Figure 5

Depleted CCSP levels in lung transplant patients with BOS. A. A cross-sectional sampling of lavage fluid from lung transplant patients with BOS (n = 26) compared to patients who remained BOS-free (n = 62), BOS-free with acute rejection (n = 25), BOS-free with acute infection (n = 18), or were in BOS 0p (n = 24). B. A longitudinal sampling of lavage fluid from lung transplant with BOS compared to antecedent samples from the same patients obtained prior to BOS diagnosis (n = 12 pairs).