Following local anesthesia with nebulized lidocaine and i.v. conscious sedation, subjects underwent flexible bronchoscopy. Briefly, a flexible fiberoptic bronchoscope was inserted transorally and advanced through the vocal cords. Bronchoalveolar lavage (BAL) fluid was obtained from tertiary airways in the right upper lobes (RUL) and right lower lobes (RLL). BAL was performed sequentially in the RUL and RLL with 20 ml of sterile saline followed by 10 ml of air, and this was repeated for a total of five times per airway. Lung macrophages were isolated, as previously described (13 (link), 14 (link)). BAL fluid was filtered through two-layer gauze, centrifuged, and washed twice in 0.9% NaCl. Cells were counted with a T10 automated cell counter (Bio-Rad, Hercules, CA). Cytospins were performed using a Shandon Cytospin 3 centrifuge (Thermo Fisher Scientific, Waltham, MA). Briefly, 75,000 cells resuspended in 200 μl of 0.9% NaCl were loaded into a cytology funnel (Fisher Scientific, Pittsburgh, PA) and centrifuged for 10 min. Cells were allowed to air dry and processed for viewing via Hema 3 Stat pack (Fisher). Imaging was done on an Olympus (Waltham, MA) BX41 microscope with DP2-BSW software (version 2007).