Fig. 4. ACE expression affects the p38-MAPK–mediated neutrophil oxidative response and bacterial killing in vitro.

(A) Phospho-PKC isoforms were measured by ELISA in ACE KO, WT, and NeuACE bone marrow neutrophils after stimulation with LPS for 30 min in vitro (n = 8 per group). (B) Phospho–p38-MAPK in bone marrow neutrophils was measured by ELISA after stimulation with LPS for 30 min in vitro (n = 8 per group). Mice were treated with ramipril or losartan for 1 week before neutrophil isolation. (C) Western blotting of ACE KO, WT, and NeuACE neutrophils for phospho–p38-MAPK is shown. β-Actin serves as the loading control. Numbers below bands indicate relative band density as determined by Image Studio Lite version 5.2 (LI-COR). (D) Superoxide (O ⁻₂) concentration in neutrophils after stimulation with LPS (1 μg/ml) for 10 min at room temperature was measured using a cytochrome C reduction assay (n = 8 per group). For p38-MAPK inhibition, neutrophils were pretreated with 500 nM SB203580 for 2 hours at room temperature. (E) Measurement of phospho–p47-phox in neutrophils treated with or without SB203580 and stimulated with LPS by Western blot. TAP2 serves as the loading control. Numbers below bands indicate relative band density. (F) Ex vivo MRSA clearance by blood from ACE KO, WT, and NeuACE mice after treatment with SB203580 is shown. Blood was drawn from mice and mixed with SB203580 for 1 hour. Then, after addition of MRSA (1.4 × 10⁶ CFU/ml), blood samples were assessed for their ability to eliminate MRSA after a 2-hour incubation (n = 5). (G) In vitro intracellular killing of MRSA in neutrophils purified from bone marrow (n = 6 per group). One-way (A) or two-way ANOVA (B, D, F, and G) with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.