CC16 levels correlate with cigarette smoke exposure in bronchial epithelial cells and with lung function decline in smokers

Abstract

Background: Club cell protein-16 (CC16) expression has been associated with smoking-related lung function decline. The study hypothesis was that CC16 expression in both serum and bronchial epithelium is associated with lung function decline in smokers, and exposure to cigarette smoke will lead to reduction in CC16 expression in bronchial epithelial cells.

Methods: In a cohort of community-based male Chinese subjects recruited for lung function test in 2000, we reassessed their lung function ten years later and measured serum levels of CC16. CC16 expression was further assayed in bronchial epithelium from endobronchial biopsies taken from an independent cohort of subjects undergoing autofluorescence bronchoscopy, and tested for correlation between CC16 immunostaining intensity and lung function. In an in-vitro model, bronchial epithelial cells were exposed to cigarette smoke extract (CSE), and the expression levels of CC16 were measured in bronchial epithelial cells before and after exposure to CSE.

Results: There was a significant association between FEV₁ decline and serum CC16 levels in smokers. Expression of CC16 in bronchial epithelium showed significant correlation with FEV₁/FVC. Bronchial epithelial cells showed significant decrease in CC16 expression after exposure to CSE, followed by a subsequent rise in CC16 expression upon removal of CSE.

Conclusions: Results of these clinical and laboratory investigations suggested that low serum CC16 was associated with smoking-related decline in lung function, demonstrated the first time in a Chinese cohort. The data also lend support to the putative role of CC16 in protection against smoking-related bronchial epithelial damage. (Abstract word count: 243) US CLINICAL TRIAL REGISTRY: NCT01185652 , first posted 20 August, 2010.

Keywords: Biomarkers; Forced expiratory volume; Lung function; Smoking; Spirometry.

Fig. 1

A study flow chart to show the recruitment of subjects

Fig. 2

Immunostaining for CC16 expression in endobronchial biopsies showing (A) Negligible staining (scored as 0 with the same intensity in bronchial epithelium) in control and (B) Weak staining (scored as 1) and (C) Strong staining (scored as 2) of CC16 in the bronchial epithelium. Note: Immunostaining were scored with the bronchial epithelial cytoplasmic staining intensity, not the stromal or nuclear staining intensity

Fig. 3

Effects of cigarette smoke extract (CSE) exposure on the expression levels of CC16 mRNA. Eight immortalized normal human bronchial epithelial cell lines were treated with CSE for 96 h after overnight serum starvation. CSE was then removed and washed with PBS for further culture for another 96 h in the absence of CSE. Data were shown as mean +/− standard deviation of eight cell lines and were analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. The p values for comparison of group means at 0 h vs 96 h, and 96 h vs 192 h, are also listed. * p < 0.001 vs PBS vehicle control

Fig. 4

Cell viability monitoring with Trypan Blue and MTT assays for different cell lines during experiments