Neutrophils release Hp in response to activation. (A) Purified peripheral-blood neutrophils were stimulated by the calcium ionophore ionomycin (1 μM) and PMA (2.5 μg/mL) for 15 minutes, and the contents of exocytosed Hp and Lf in the supernatant were detected by Western blot analysis. Ionomycin- and PMA-activated neutrophils released Hp and Lf, whereas unstimulated neutrophils did not. (B) Purified neutrophils from one donor were stimulated for 15 or 30 minutes by PMA (2.5 μg/mL), fMLP (100 nM), TNF-α (50 ng/mL), and 10% autologous serum-opsonized E coli (multiplicity of infection, 10 bacteria per cell). Supernatants containing exocytosed granule proteins and cell pellets were subjected to ELISA analysis (MPO, Lf, Gel, top histogram) or Western blot analysis (Hp, lower histogram). The percentage of MPO, Lf, and Gel released by neutrophils was calculated as the amount of protein detected in the supernatant divided by the total amount of protein detected in the supernatant and pellet multiplied by 100.