Ageing and long-term smoking affects KL-6 levels in the lung, induced sputum and plasma

Abstract

Background: KL-6 is a high-molecular-weight glycoprotein classified as a human MUC1 mucin. It was hypothesized that KL-6 could be detectable in the circulating blood and especially in airway secretions in lung diseases associated with mucus production such as chronic obstructive pulmonary disease (COPD). Additional aims of this study were to investigate whether the levels of KL-6 in plasma and sputum are related to ageing and smoking history.

Methods: The concentrations of KL-6 in plasma and induced sputum supernatants from young and/or middle aged/elderly non-smokers, smokers and patients with COPD were assayed by ELISA (n = 201). The subjects were classified into five groups according to age, smoking status and presence of COPD. In addition, KL-6 expression in control and diseased lung i.e. samples from patients with COPD (n = 28), were analyzed by immunohistochemistry and digital image analysis.

Results: The plasma levels of KL-6 increased with age both in non-smokers and smokers. Among middle aged/elderly subjects, plasma KL-6 levels in all smokers regardless of COPD were significantly higher than in non-smokers, whereas sputum levels of KL-6 were significantly higher in COPD compared not only to non-smokers but also to smokers. KL-6 was more prominently expressed in the bronchiolar/alveolar epithelium in COPD than in the control lungs. Plasma and sputum KL-6 levels correlated inversely with obstruction and positively with smoking history and ageing. The linear multiple regression analysis confirmed that age and cigarette smoking had independent effects on plasma KL-6.

Conclusions: KL-6 increases with ageing and chronic smoking history, but prospective studies will be needed to elucidate the significance of KL-6 in chronic airway diseases.

Figure 1

KL-6 levels in plasma. KL-6 levels in plasma obtained from young subjects (non-smokers; NS and smokers; S) and middle aged/elderly subjects (non-smokers, smokers and COPD) in (A) both males and females, and in (B) males only. The box represents the 25^th to 75^th percentiles, the solid lines within the boxes show the median values, the whiskers are the 10^th and 90^th percentiles, and the points represent outliers. Horizontal bars indicate mean values. ^†p < 0.05; ^††p < 0.01; ^†††p < 0.001 (young non-smokers vs young smokers, Mann-Whitney U test). *p < 0.05; **p < 0.01; ***p < 0.001 (middle aged/elderly non-smokers vs middle aged/elderly smokers or COPD, Mann-Whitney U test).

Figure 2

KL-6 levels in induced sputum. KL-6 levels in induced sputum obtained from middle aged/elderly subjects (non-smokers; NS, smokers; S and COPD) in (A) both males and females, and in (B) males only. The box represents the 25^th to 75^th percentiles, the solid lines within the boxes show the median values, the whiskers are the 10^th and 90^th percentiles, and the points represent outliers. Horizontal bars indicate mean values. *p < 0.05; **p < 0.01; ***p < 0.001 (middle aged/elderly non-smokers vs middle aged/elderly smokers or COPD, Mann-Whitney U test).

Figure 3

KL-6 expression and localization in diseased lung. (A) KL-6 expression and localization in representative sections of lung specimens from non-smoker, smoker, and patient with COPD. Positive KL-6 expression was seen mainly in type II pneumocytes as well as in macrophages in the lungs of non-smokers, smokers, and patients with COPD. The bronchial/alveolar epithelium of patients with COPD displayed highly positive areas of KL-6 staining in contrast to the situation in non-smokers and smokers. (B) Quantitative image analysis of KL-6 in the lung tissues of 7 non-smokers, 7 smokers and 14 patients with COPD. Three representative areas consisting of the parenchymal portion of the lung tissue were analyzed from all stained sections (sum of the bronchiolar/alveolar epithelium, interstitium and macrophages; Epi+Int+Mac). Quantitative image analysis of the immunoreactivity for KL-6 was also conducted separately in the bronchial/alveolar epithelium and interstitium (sum of the bronchiolar/alveolar epithelium and interstitium; Epi+Int; C) or the bronchial/alveolar epithelium (sum of the bronchiolar/alveolar epithelium; Epi; D). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (between all four groups, Kruskall-Wallis test). For patient characteristics see Table 3.