Enumeration of Inflammatory Cells.

A bronchoalveolar lavage (BAL) was performed following completion of lung function in anesthetized rabbits. A cannula was inserted within the endotracheal tube, and 5 ml of sterile saline was administered into the lung followed by recovery of the fluid under gentle vacuum suction. The fluid was left in the lung for no more than a few seconds, and the amount of fluid recovered was noted (approximately 60% recovery). An aliquot of the lavage fluid (50 μl) was fixed with 50 μl of Turk’s solution (catalog number 109277; Merck KGaA, Darmstadt, Germany). Total cell counts were performed for this solution using a Neubauer hemocytometer. Two 100 μl samples of neat BAL fluid were centrifuged (Cytospin 3 Centrifuge; Thermo Scientific Shandon, Waltham, MA) onto a glass slide. The slides were left to dry, fixed (Reastain Quick-Diff Kit; Reagena, Toivala, Finland), and then mounted in DPX Mountant (Sigma-Aldrich). Differential cell counts were performed using confocal microscopy with oil at a 40× magnification.