A small volume technique to examine and compare alveolar macrophage phagocytosis of apoptotic cells and non typeable Haemophilus influenzae (NTHi)

A defective ability of alveolar macrophages to phagocytose both apoptotic airway epithelial cells and bacteria in chronic lung diseases potentially associated with inflammation and bacterial colonisation of the lower airways, often with non-typeable Haemophilus influenzae (NTHi), has been shown. We routinely assess phagocytosis in the airway of children: however, the small volume of BAL obtained usually precludes the investigation of phagocytosis of both (potentially equally relevant) apoptotic cells and NTHi. Methods: We established a 'one-tube, dual target' flow-cytometric assay for investigating alveolar macrophage phagocytosis of both apoptotic cells and NTHi. The effect of bacterial presence on phagocytosis of apoptotic cells was assessed by comparing results using this technique to standard 'two tube, single target' methods. The comparative ability of alveolar macrophages to phagocytose NTHi or apoptotic cells was assessed in 10/group of healthy adult controls and patients with COPD, 12 children with bronchiectasis, and 10 children controls. We then assessed the influence of increasing concentrations of NTHi targets on the ability of THP-1 macrophages to simultaneously phagocytose apoptotic cells. Results: Alveolar macrophages phagocytosed NTHi more avidly than apoptotic cells (mean +/- SEM: apoptotic cells 15.4% +/- 0.5 vs. NTHi 17.2% +/- 0.7, p < 0.05). The presence of NTHi targets (ratio of 1:100 macrophage: NTHi; 2 x 10(7) CFU routinely applied in our assay) had no effect on the ability of macrophages to simultaneously phagocytose apoptotic cells. However, when bacterial numbers were increased (up to 4-fold) there was a small but significant suppressive effect on the ability of macrophages to phagocytose apoptotic cells. Conclusions: We describe a small volume 'one tube, dual target' technique to measure phagocytosis of apoptotic cells and NTHi. We show that alveolar macrophages phagocytose NTHi more avidly than apoptotic cells, and that an increased presence of NTHi in the airway may reduce the ability of alveolar macrophages to phagocytose apoptotic cells. (C) 2015 Elsevier B.V. All rights reserved.