Figure 6.

Detection of HOCl production within neutrophils stimulated by zymosan or bacteria, and dependence on NOX2 activity and MPO. A, flow cytometry scattergrams from unstimulated neutrophils (polymorphonuclear (PMN), top) and neutrophils stimulated with 20:1 zymosan in HBSS containing 1 mm methionine, in the absence (middle) and presence (bottom) of DPI, recorded after 30 min. The gated neutrophil populations (left panels) were analyzed for R19 fluorescence (middle panels) and represented as histograms (right panels). B, corresponding merged R19 fluorescence (red) and bright field (Cy3/DIC) images of neutrophils treated for 30 min as in A and recorded by fluorescence microscopy. C, time course of mean fluorescence increase for the entire neutrophil population treated as in A for neutrophils that were unstimulated (○) or stimulated with zymosan in the absence (●) or presence (▴) of DPI or treated in chloride-free gluconate buffer (×). Results show mean ± S.E. from three independent experiments. Data points are normalized against the fluorescence (93 FU) of phagocytic neutrophils at 30 min. D, detection of R19 and R19S-Cl by LC-MS in neutrophils (10⁶) harvested and lysed following 30 min stimulation as in A. Data are means of duplicates ± S.D. from a representative of two independent experiments. E, R19-S fluorescence increase over time measured by flow cytometry as in A following addition of S. aureus (at 10:1) to normal neutrophils in the absence (●) or presence (▴) of MPO inhibitor TX1, and neutrophils from an individual with MPO deficiency (×), compared with normal neutrophils incubated without bacteria (○). Results show mean ± S.D., the mean maximum fluorescence of normal neutrophils after phagocytosing bacteria was 13 FU. F, inhibition of R19 fluorescence by MPO inhibitors. Neutrophils were incubated with S. aureus (at 10:1) in the presence of 1 mm methionine for 30 min with and without pre-exposure for 10 min to 10 μm TX1 or HX1. *, significance relative to S. aureus with no inhibitor as determined by one-way analysis of variance with Dunnett's post-test correction for multiple comparisons (p ≤ 0.0001).