Macrophage phagocytosis and killing.

Macrophage phagocytosis and bacterial killing were assessed by using an established in vitro assay (13, 14). Primary peritoneal macrophages were obtained from 6 ccl3^−/− and 6 WT mice as described previously (13, 14) and seeded into 48-well plates at 5 × 10⁵ cells per well in triplicate for each mouse and assay time point. Ten microliters of mid-exponential-phase NTHi bacteria (5 × 10⁷ bacteria), a titer that does not saturate the cells, as demonstrated previously (13), was added to each well. After incubation for 1 h, extracellular bacteria were removed and killed by washing, followed by the addition of fresh Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), macrophage colony-stimulating factor, and 50 μg/ml gentamicin. After the addition of gentamicin, incubation was continued for 1 or 3 h. The cells were then rinsed and lysed. Supernatants and cell lysates were plated onto chocolate agar in serial dilutions, incubated overnight at 37°C, and then evaluated by manually counting CFU. Six wells were used per time point and mouse strain. The recovery of bacteria after macrophage treatment with gentamicin for 1 h was used to represent phagocytosis. The ratio of the number of NTHi bacteria recovered after gentamicin treatment for 3 h compared to the number of bacteria recovered at 1 h posttreatment was considered to represent intracellular killing (14, 22). Percent bacterial killing was calculated as previously described (22), by using the following formula: % killing = 1 − (number of CFU in tested wells 3 h after cultivation with gentamicin/number of CFU in tested wells 1 h after cultivation) × 100.