Fig 1.
Schematic representation of the neutrophil phagocytic vacuole showing the consequences of electron transport by NOX2 onto oxygen.
The proposed ion fluxes that might be required to compensate the movement of charge across the phagocytic membrane together with modulators of ion fluxes are shown.
Fig 2.
Oxygen consumption and extracellular acidification rate by neutrophils.
(A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) by neutrophils from WT, Hvcn1-/- and gp91phox-/- mice in response to stimulation with PMA (with and without DPI) or opsonised Candida. The numbers of independent experiments is shown over the total number of measurements. Statistical significance: *** p<0.001, ** p<0.01 and * p<0.05. Differences between PMA stimulated WT and gp91phox-/- were p<0.001 in (A) and p<0.05 in (B). There were no significant differences in (B) between PMA and Candida or between WT and Hvcn1-/- cells. Median, quartiles and 95% centiles are shown.
Fig 3.
Time courses of changes in pH in the vacuole and cytoplasm of phagocytosing neutrophils.
Representative images of Candida phagocytosed by human neutrophils (A), with DPI (B), and by Hvcn1-/- neutrophils (C). Standard curves for the relationship between SNARF ratio and pH of organisms and cytoplasm are shown in (D). Candida alone were added to two different buffer systems, labelled Tris or Barbital, and intracellular organisms were exposed to the Barbital buffers after permeabilisation of neutrophils with saponin. Panels E-L show time courses of the pH changes of phagocytosed Candida and cytoplasm of human (E, I), mouse WT (F, J) and Hvcn1-/- (G, K) neutrophils synchronised to the time of particle uptake (0 minutes). In E-G and I-K, each individual black line represents serial measurements of the SNARF ratio of a single phagocytosed Candida or neutrophil cytoplasm, respectively. Mean ± SD (shaded areas) are shown. In the composite panels H and L the mean data have been smoothed. Data are plotted according to SNARF ratio with the approximate corresponding pH shown on the right y-axis. The number of independent experiments over the total number of individual cells examined is shown. The effect of DPI on vacuolar pH in human neutrophils is shown in E (pink and dashed lines, 12 cells).
Fig 4.
The effect of DPI, azide, 4ABH, KCN, zinc, and CCCP on vacuolar pH.
(A) The effect of 300 μM Zn2+, DPI and 60 μM CCCP on vacuolar pH in WT and Hvcn1-/- neutrophils at ~30 minutes following the addition of Candida without synchronisation to particle uptake. (B) The effect of 5 μM DPI, 5 mM sodium azide, 50 μM 4ABH and 1mM KCN, on vacuolar pH in human neutrophils at ~30 minutes following the addition of Candida without synchronisation to particle uptake. The effect of the addition of azide (C) or DPI (D) on vacuolar pH in human neutrophils synchronised to time of addition (red arrows). The numbers of independent experiments is shown over the total number of measurements. Median, quartiles and 95% centiles are shown. Statistical significance: *** p<0.001.
Fig 5.
Vacuolar size in WT and Hvcn1-/- neutrophils containing a single latex particle.
The cross-sectional area of a latex particle is ~7 μm2. Representative images of a WT (A) and Hvcn1-/- neutrophil containing a single latex particle are shown in (A) and (B), respectively. (C) Quantitation of vacuolar swelling in Hvcn1-/- neutrophils compared with WT, and the effects of 5 μM DPI, 60 μM CCCP and 3 μM valinomycin. The numbers of independent experiments is shown over the total number of measurements. Median, quartiles and 95% centiles are shown. Statistical significance: *** p < 0.001 and ** p<0.01.
Fig 6.
Effects of pH on enzymatic activity.
The effect of variations in pH on peroxidatic and chlorinating activities of MPO and on the protease activities of cathepsin G and of elastase are shown. Results shown are the mean + SD of at least three separate assays and are expressed as a percentage of the maximal observed activity.