Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT), which involves changes in cellular morphology of highly polarized epithelial cells and the gain of mesenchymal cell phenotype with migratory and invasive capacities, is implicated in smoking-related chronic obstructive pulmonary disease (COPD). However, the interactions of fibroblasts and epithelial cells and the participation of fibroblasts in the EMT processes in COPD are poorly understood. Here, we investigated the hypothesis that EMT is active in human bronchial epithelial (HBE) cells of COPD patients, and that mediators secreted by lung fibroblasts from COPD patients induce EMT. METHODS: Primary HBE cells from normal subjects and COPD patients were purchased from LONZA. HLFs were derived from resected lung obtained from normal (N) and COPD (D) subjects and their conditioned medium (CM) was collected after 2-day culture in serum-free medium. The expression of epithelial and mesenchymal markers as well as EMT-related transcription factors in lung biopsies, and in HBE cells following stimulation with CM from both normal human lung fibroblasts (NHLF) and COPD human lung fibroblasts (DHLF) was evaluated by immunohistochemistry, qRT-PCR and western blot. RESULTS: Basal mRNA expression of mesenchymal markers and EMT-related transcription factors were increased in DHBE cells compared to normal human bronchial epithelial cells (NHBE) cells as well as in COPD lungs. CM from NHLF significantly induced vimentin expression in both NHBE and COPD human bronchial epithelial cells (DHBE) cells, but only increased N-cadherin expression in DHBE cells. CM from NHLF significantly induced Twist1 and Twist2 expression in NHBE cells and increased Snai2 (Slug) expression in DHBE cells. While CM from NHLF had no effect on such EMT markers, CM from DHLF significantly increased the protein expression of E-cadherin and vimentin in NHBE cells compared to control. N-cadherin expression was upregulated to a greater degree in NHBE cells than DHBE cells. Only CM from DHLF significantly increased E-/N-cadherin ratio in DHBE cells. CONCLUSIONS: Our results suggest that DHBE cells have partially undergone EMT under baseline conditions. DHLF-CM promoted EMT in NHBE, suggesting that interactions between fibroblast and epithelial cells may play an important role in the EMT process in COPD.

IL-22 contributes to TGF-ß1-mediated epithelial-mesenchymal transition in asthmatic bronchial epithelial cells.

BACKGROUND: Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-ß1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects. METHODS: Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-ß1, IL-22 and TGF-ß1+IL-22. RESULTS: Primary bronchial epithelial cells stimulated with TGF-ß1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-ß1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics. CONCLUSION: The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-ß1 cooperativity in driving EMT in primary human bronchial epithelial cells.

Lipopolysaccharide induced connective tissue growth factor gene expression in human bronchial epithelial cells.

BACKGROUND AND OBJECTIVE: Connective tissue growth factor (CTGF) is up-regulated in the lungs of patients with chronic obstructive pulmonary disease (COPD). Cigarette smoke and repeated bacterial infections, both of which are rich sources of LPS, are major causes of COPD. The high levels of LPS in lung epithelial lining fluid also suggest that it may have a considerable impact on the airway epithelium, in terms of cytokine and growth factor production. The aim of this study was to clarify the mechanism of LPS-induced CTGF expression in bronchial epithelial cells. METHODS: The expression and transcriptional regulation of the CTGF gene were assessed using the cultured human bronchial epithelial cell line, BEAS-2B. RESULTS: LPS significantly up-regulated CTGF mRNA expression in a dose-dependent fashion, with 100 microg/mL LPS causing a twofold increase after 2 h. CTGF protein expression was also up-regulated by LPS after 8 h. Transforming growth factor-beta1 mRNA expression was not changed by LPS treatment. A pharmacological inhibitor of nuclear factor (NF)-kappaB, MG132, inhibited LPS-induced CTGF mRNA expression. Furthermore, luciferase assays demonstrated that deletion of base pairs -253 to -53 from the CTGF promoter, where the Smad and proximal NF-kappaB binding sites are located, decreased the induction of CTGF by LPS. After stimulation with LPS, the p65 subunit of NF-kappaB was shown to be bound to the CTGF promoter in vitro and in situ. CONCLUSIONS: LPS directly induced CTGF expression in bronchial epithelial cells, independently of transforming growth factor-beta1, suggesting a possible mechanism for airway remodelling in COPD that is induced by smoking and repeated bacterial infections.

Relationship between peripheral airway function and patient-reported outcomes in COPD: a cross-sectional study.

BACKGROUND: Health status, dyspnea and psychological status are important clinical outcomes in chronic obstructive pulmonary disease (COPD). However, forced expiratory volume in one second (FEV1) measured by spirometry, the standard measurement of airflow limitation, has only a weak relationship with these outcomes in COPD. Recently, in addition to spirometry, impulse oscillometry (IOS) measuring lung resistance (R) and reactance (X) is increasingly being used to assess pulmonary functional impairment. METHODS: We aimed to identify relationships between IOS measurements and patient-reported outcomes in 65 outpatients with stable COPD. We performed pulmonary function testing, IOS, high-resolution computed tomography (CT), and assessment of health status using the St. George's Respiratory Questionnaire (SGRQ), dyspnea using the Medical Research Council (MRC) scale and psychological status using the Hospital Anxiety and Depression Scale (HADS). We then investigated the relationships between these parameters. For the IOS measurements, we used lung resistance at 5 and 20 Hz (R5 and R20, respectively) and reactance at 5 Hz (X5). Because R5 and R20 are regarded as reflecting total and proximal airway resistance, respectively, the fall in resistance from R5 to R20 (R5-R20) was used as a surrogate for the resistance of peripheral airways. X5 was also considered to represent peripheral airway abnormalities. RESULTS: R5-R20 and X5 were significantly correlated with the SGRQ and the MRC. These correlation coefficients were greater than when using other objective measurements of pulmonary function, R20 on the IOS and CT instead of R5-R20 and X5. Multiple regression analyses showed that R5-R20 or X5 most significantly accounted for the SGRQ and MRC scores. CONCLUSIONS: IOS measurements, especially indices of peripheral airway function, are significantly correlated with health status and dyspnea in patients with COPD. Therefore, in addition to its simplicity and non-invasiveness, IOS may be a useful clinical tool not only for detecting pulmonary functional impairment, but also to some extent at least estimating the patient's quality of daily life and well-being.

Longitudinal study of airway dimensions in chronic obstructive pulmonary disease using computed tomography.

BACKGROUND AND OBJECTIVE: Chest CT has been widely used for the evaluation of structural changes in lung parenchyma and airways in cross-sectional studies. There has been no report on the annual changes in airway dimensions as assessed by CT in COPD patients. The objective of this study was to investigate the annual changes in airway dimensions and lung attenuation using CT in patients with COPD and to evaluate the correlations among annual changes in CT measurements and pulmonary function. METHODS: Eighty-three men with COPD had completed five annual assessments of CT scans and pulmonary function tests over 4 years. Airway dimensions of the basal segment bronchi and lung attenuation on CT images were analysed in 38 subjects in whom the same airway could be measured at least three times, including at entry and at the end of the study. RESULTS: Mean annual decline in FEV(1) was 21 mL/year. Annual changes in the percentage of low attenuation areas were not significantly correlated with decline in FEV(1). On the other hand, annual changes in the percentage of wall area (WA%/year) were significantly inversely correlated with annual changes in FEV(1) (r = -0.363, P = 0.025), whereas WA%/year did not differ among severity stages at entry and did not correlate with baseline FEV(1). CONCLUSIONS: The results showing that annual changes in airway thickening correlated with annual decline in air flow limitation suggests the importance of treatment of airway inflammation in COPD. CT is a useful tool for quantitative estimation not only of emphysema but also of airway lesions in longitudinal studies.

Comparison of airway dimensions in different anatomic locations on chest CT in patients with COPD.

OBJECTIVE: It is not well known whether there is heterogeneity in the airway dimensions at different anatomic locations in individual patients with COPD. The first objective was to compare airway dimensions of the basal segment bronchus between COPD patients and healthy controls. The second and third objectives were to compare the airway dimensions in two anatomic locations, and to investigate the relationship between CT measurements and pulmonary function among COPD patients. METHODS: Thirty males with COPD (aged 68.7 +/- 8.1 years) and 18 healthy males (aged 64.9 +/- 14.0 years) were enrolled in the study. COPD was diagnosed according to the criteria of the Global Initiative for Obstructive Lung Disease Workshop Report. Pulmonary function tests and CT scans were performed on all subjects. Airway dimensions and lung attenuation were automatically determined using methods that were validated with a phantom. RESULTS: Age, smoking index and height did not significantly differ between the COPD patients and healthy controls. The COPD patients had a significantly thicker airway wall than healthy controls. Among the COPD patients, there were no significant differences in the airway dimensions of bronchi in different segments; however, the airway and lung attenuation measurements of the lower lung field were more strongly correlated with FEV(1) than those of the upper lung field. CONCLUSION: Patients with COPD had no significant heterogeneity in airway dimensions at different anatomic locations. The airway and lung attenuation measurements of the lower lung field were more strongly correlated with airflow limitation than those of the upper lung field.