Figure 2. Leishmania amazonensis (La) promastigotes activate ROS production in human neutrophils.

(A) H₂O₂ production was measured with Amplex Red (5 μM) after neutrophils (NØ; 2 × 10⁶) were stimulated with PMA (100 nM) or fixed promastigotes (1 NØ: 5 La ratio), and the fluorescence recorded over 25 min of incubation, as shown on inset. Data shown as mean ± SEM of 9 independent experiments were expressed as the amount of H₂O₂ produced in pmol/s/2 × 10⁶ neutrophils and as Amplex Red arbitrary units (AU, Inset). (B–G) Neutrophils (10⁶) were incubated with DPI (32 μM) for 30 min, and then stimulated with live (1NØ: 0.1La ratio) or fixed promastigotes (1NØ: 1 La ratio), incubated with DHR 123 (1.2 μM) for 20 min and analyzed by flow cytometry. Data are expressed as the percentage of DHR positive cells (B,D,F), and as the mean fluorescence intensity (MFI) (C,E,G) are shown as mean ± SEM of 23 (B,C), 4 (D,E) and 6 (F,G) different donors. Neutrophils (10⁶) were incubated with MitoSox Red probe (2.5 μM) for 30 min at 35 °C, 5% CO₂, cells were washed and stimulated with fixed promastigotes (1NØ: 1 La ratio, H,I). Neutrophils (10⁶) were also treated with MitoSox Red (2.5 μM) and MitoTEMPO (100 μM, (J,K)). In both cases cells were then incubated for 30 min at 35 °C, 5% CO₂ and analyzed by flow cytometer. Data are expressed as the mean ± SEM fluorescence intensity (MFI) of 20 (H) and 12 (J) donors. Data are expressed as the mean ± SEM percentage of MitoSox positive cells and (I,K) are shown as mean ± SEM of 11 donors. *p < 0.0001 and **p < 0.05.