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Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

Levine AP, Duchen MR, de Villiers S, Rich PR, Segal AW - PLoS ONE (2015)

Bottom Line: Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced.In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly.Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, University College London, London, United Kingdom.

ABSTRACT
The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0125906.g003: 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).

Mentions: The fluorescence ratio of SNARF coupled to heat killed Candida showed a sigmoidal relationship with pH and was approximately linear with pH 7–10 (Fig 3D). We tracked changes in pH in the phagocytic vacuoles of individual neutrophils from the time that labelled Candida were engulfed. Shortly following engulfment, the vacuole underwent a significant alkalinisation (Fig 3). In human neutrophils, this started almost immediately (Fig 3E and S1 Video). The mean maximum pH reached post-phagocytosis was 9.0 (SD 8.3–10.2) (Fig 3E) and this elevated pH was maintained for 20–30 minutes. When the NADPH oxidase was inhibited by DPI, the vacuole acidified to about 6.3 (SD 6.1–6.56) (Fig 3B and 3E), although SNARF determinations are not very accurate at this pH, these results are similar to previous findings [27] and when added following phagocytosis, DPI rapidly reduced the vacuolar pH (Fig 4C and S2 Video) [20]. These elevations in vacuolar pH occurred despite the buffering capacity of the heat-killed Candida (S1 Fig). The maximum difference in the vacuolar pH in human neutrophils in the presence or absence of DPI (~6.5 and ~9, respectively (Fig 3E)) required 1.9 fmols of OH- per Candida. Assuming this pH rise was entirely due to non-proton charge compensation, it would amount to ~8% of the total ~24 fmol compensating charge, close to the ~5% previously estimated [23].


Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

Levine AP, Duchen MR, de Villiers S, Rich PR, Segal AW - PLoS ONE (2015)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401748&req=5

pone.0125906.g003: 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).
Mentions: The fluorescence ratio of SNARF coupled to heat killed Candida showed a sigmoidal relationship with pH and was approximately linear with pH 7–10 (Fig 3D). We tracked changes in pH in the phagocytic vacuoles of individual neutrophils from the time that labelled Candida were engulfed. Shortly following engulfment, the vacuole underwent a significant alkalinisation (Fig 3). In human neutrophils, this started almost immediately (Fig 3E and S1 Video). The mean maximum pH reached post-phagocytosis was 9.0 (SD 8.3–10.2) (Fig 3E) and this elevated pH was maintained for 20–30 minutes. When the NADPH oxidase was inhibited by DPI, the vacuole acidified to about 6.3 (SD 6.1–6.56) (Fig 3B and 3E), although SNARF determinations are not very accurate at this pH, these results are similar to previous findings [27] and when added following phagocytosis, DPI rapidly reduced the vacuolar pH (Fig 4C and S2 Video) [20]. These elevations in vacuolar pH occurred despite the buffering capacity of the heat-killed Candida (S1 Fig). The maximum difference in the vacuolar pH in human neutrophils in the presence or absence of DPI (~6.5 and ~9, respectively (Fig 3E)) required 1.9 fmols of OH- per Candida. Assuming this pH rise was entirely due to non-proton charge compensation, it would amount to ~8% of the total ~24 fmol compensating charge, close to the ~5% previously estimated [23].

Bottom Line: Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced.In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly.Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicine, University College London, London, United Kingdom.

ABSTRACT
The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.

No MeSH data available.


Related in: MedlinePlus