Characterization of Neu^ACEneutrophils. (A) FCM analysis of ACE expression by blood neutrophils. (i) Dot plots show the approach for the profiling of neutrophils. Neutrophils were defined as CD11b⁺Ly6G⁺. FSC, forward scatter; SSC, side scatter. (ii) Histogram of flow cytometry results using anti-ACE antibody and neutrophils from ACE KO (peak 1), WT (2), ACE 10/10 (3), and Neu^ACE mice (4). (iii) Analysis of ACE expression by ACE KO, WT, ACE 10/10, and Neu^ACE neutrophils. Data are presented as mean fluorescence intensity (MFI) (n = 4/group). (B) ACE protein expression in neutrophils. Neutrophils were purified from bone marrow using Percoll gradient centrifugation. Flow cytometric analysis after staining cells with CD11b and Ly6G showed that approximately 90% of the cells were neutrophils. Purified neutrophils from ACE KO, WT, Neu^ACE, and ACE 10/10 mice were lysed in ACE assay buffer. ACE expression was determined by western blot analysis (ii) and by ACE catalytic activity (iii). ACE 10/10 mice were previously created by gene targeting of the ACE gene.¹⁵  These mice overexpress ACE in myelomonocytic cells. By enzyme assay, Neu^ACE neutrophils make 3.7-fold more ACE than ACE 10/10 neutrophils. **P ≤ .005, ***P ≤ .0005, KO-ACE KO, 10/10-ACE10/10.