Methods to track leukocyte and erythrocyte transit through the bronchial vasculature in sheep

Study of the underlying mechanisms of leukocyte recruitment in the airway circulation is a crucial aspect of understanding the pathology of inflammatory airways disease. However, few in vivo studies have focused on leukocyte kinetics through the systemic airway vasculature. Because the bronchial vasculature of the sheep shows anatomical similarity to that of the human as well as being easily accessible for perfusion, we developed methods to study leukocyte transit through the sheep bronchial circulation. Leukocytes were isolated from whole blood after hypotonic lysis of red cells and labeled with 5-(and 6) carboxyfluorescein succinimidylester (CFSE; 0.1 microM), a green fluorescent dye. Red blood cells were labeled using PKH26-GL Red Fluorescent Cell Linker and served as a marker for blood flow and volume. Labeled leukocytes were tested for activation during the labeling process by monitoring surface levels of leukocyte adhesion molecules CD11b and L-selectin. When activated with phorbol myristate acetate (10 ng/ml), sheep leukocytes showed a marked increase in CD11b expression and a decrease in L-selectin. However, sheep leukocytes labeled with CFSE showed no significant increase of CD11b or shedding of L-selectin, suggesting that the labeling process did not significantly activate the adhesion properties of the cells. Aliquots containing both labeled erythrocytes and leukocytes were infused into the bronchial vasculature of the sheep through the cannulated bronchial artery at normal bronchial flow (0.6 ml/kg). Serial blood samples were withdrawn from the outflow of the bronchial vasculature (the left atrium) and analyzed using conventional flow cytometry. Retention of leukocytes and transit relative to red cell transit could then be evaluated with this technique. In addition, since cells are stably labeled, airway tissue samples can be removed and analyzed histologically to determine sites of extravasation. These methods offer an approach for examining leukocyte kinetics in situ in the airways of a relevant animal model.