ACE increases neutrophil NET formation and IL-1β production. (A) NET formation was measured by fluorescent microscopy. Neutrophils were purified to 98% using magnetic beads and a kit from Stem Cell. They were then cultured and stimulated with PMA (50 nM) for 3 hours. After staining with Cytox green, pictures were taken at ×20 using fluorescent microscope. (B) Elastase release by WT and Neu^ACE neutrophils. WT and Neu^ACE neutrophils were purified from blood by gradient centrifugation. Some mice were treated with DPI (3 mg/kg/single dose) for 1 day before neutrophil collection and the purified cells were treated again for 1 hour (10 µM) before MRSA challenge. In all groups, cells were treated with MRSA for 4 hours before NET-associated neutrophil elastase was measured by ELISA. n = 5/group. (C) Following ACE inhibition by ramipril, NET formation was determined by elastase-based ELISA, as described previously. (D) Following 3 days of subcutaneous skin infection with MRSA, Neu^ACE mice had increased levels of IL-1β in skin lesions compared with WT; n = 8/group. The difference in IL-1β production was abolished by treatment with DPI (3 mg/kg per day, i.p.) beginning 1 day before infection and continuing throughout the experiment; n = 8/group. (E) To assess IL-1β production in vitro, bone marrow neutrophils from WT and Neu^ACE mice were cultured with MRSA (5 × 10⁶ neutrophils/well, 10 CFU MRSA/neutrophil) for 12 hours. Total IL-1β (cells plus supernatant) was determined by ELISA; n = 5/group. *P ≤ .05, **P ≤ .005, ***P ≤ .0005.