Clinical Features and Airways Inflammation in Never Smokers and Smokers With COPD

Induced sputum was collected and analysed in the three arms (Healthy subjects, Never smokers with COPD and smokers with COPD) The volume of the sputum sample, was treated with an equal volume of 0.1% dithiothreitol. The samples were then vortexed and placed in a shaking water bath at 37°C for 15 min to ensure complete homogeneisation. The suspensions were centrifuged at 400× g and the sputum supernatant stored at -80°C until cytokines analysis. The cell pellet was resuspended in 0.5% bovine serum albumin in phosphate buffered saline and cytospin were made by putting 100 µL of the cell suspension in the funnels. Slides for differential cell counts were stained with May-Grünwald Giemsa.

All the subjects underwent pulmonary function tests (Healthy subjects, Never smokers with COPD and smokers with COPD). Pulmonary function parameters were measured using a portable spirometer (Easy One ndd. Medizintechnik; Zurich, Switzerland) according to the ATS criteria (American Thoracic Society). Reversibility test was performed 15 min after inhalation of 400 µg of Salbutamol (Ventolin, GlaxoSmithKline, Middlesex, UK). All spirometry data were graded for quality and only tests that met high quality scores were used for the final analysis.

IL-8 and TNF-α were measured in the sputum supernatant of all the subjects (Healthy subjects, Never smokers with COPD and smokers with COPD). Commercially available kits were used to detect IL-8 (Human IL-8 Immuno-Biological Laboratories ELISA Kit) and TNF-α (Human TNF-alpha Sigma-Aldrich ELISA Kit) concentrations in the sputum supernatants. The absorbance was measured at 450 nm. The lower limits detection were 2 pg/mL for IL-8 and 5 pg/mL for TNF-α. The intra assay coefficients of variability were 8.7% for IL-8 and 7.7% for TNF- α.