S87 Differential Inflammatory Responses of Primary Bronchial Epithelial Cells from Subjects with COPD, Healthy Smokers and Never Smokers

Introduction We investigated the responses of primary bronchial epithelial cells (PBECs) obtained from subjects with COPD, healthy smokers (HS), and non-smokers (NS) to cigarette smoke extract (CSE) treatment. We hypothesised that PBECs from subjects with COPD respond differently to CSE and Pseudomonas aeruginosa lipopolysaccharide (PA LPS) stimulation than PBECs obtained from HS and NS. Methods PBECs from 16 COPD subjects, 11 HS and 10 NS were obtained at fibreoptic bronchoscopy and cultured up to air-liquid interface and stimulated with PA LPS, either with or without pre-treatment with CSE for 24 h. COPD patients and HS were similar for smoking history in pack years and all 3 groups were matched for age. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labelling (TUNEL) method. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 expression by flow cytometry. CSE was prepared by combusting 1 Marlboro cigarette through 25 ml of media. Activation of NF-κB, mitogen-activated protein kinase (MAPK), and caspase-3 were determined by western blotting. Results Constitutive release of IL-8 and IL-6 was greatest from the COPD cultures. 5% CSE pre-treatment followed by PA LPS stimulation reduced cytokine release from COPD PBECs, but increased the release from HS and NS cultures. Constitutive TLR-4 expression, MAPK and NF-κB activation were reduced only in COPD cultures after 5% CSE treatment. After treatment with CSE for 24 h, 44% of the COPD cells were apoptotic and 9% necrotic, whereas only 18% of the healthy smoker’s cells and 6% of the non-smokers cells were apoptotic, with no cells in the latter 2 groups becoming necrotic. COPD cultures had the highest levels of cleaved caspase-3 after CSE treatment. Conclusions 5% CSE attenuates inflammatory responses to LPS in cells from people with COPD but not from NS or HS. COPD epithelial cells have an increased susceptibility to apoptosis. Research funded by NI RDO.