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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

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.
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pone.0125906.g005: 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.

Mentions: The vacuoles of Hvcn1-/- neutrophils containing Candida underwent a profound swelling, which was much more obvious than in WT mouse or human cells (S1 Video and S3 Video). To determine the extent of this swelling and to distinguish it from that induced by the osmotic effects of the products of digestion of the Candida, we measured the cross-sectional areas of vacuoles containing a single indigestible latex particle with a diameter of 3 μm (a cross-sectional area of 7.1 μm2), similar to that of Candida. In neutrophils from Hvcn1-/- mice, the vacuoles swelled to a median cross-sectional area of 11.2 μm2 (quartiles 8.5–16.6) compared with 7.9 μm2 (6.8–9.1) in neutrophils from WT mice (p<0.001) or Hvcn1-/- treated with 5 μM DPI (7.9 μm2) (6.8–8.8) or CCCP (7.9 μm2) (6.9–9.0) (Fig 5C) after 30 minutes. These results indicate that osmotically active ions are driven into the vacuole by the oxidase in the absence of the proton channel. This swelling was reduced significantly (p<0.001) by 3 μM valinomycin to a median area of 9.9 μm2 (7.1–12.8). The estimated volumes utilising the radius calculated from the median cross-sectional area and assuming spherical vacuoles were: latex particles (14.2 μm3); WT vacuoles (16.7 μm3) and Hvcn1-/- (28.2 μm3) without and (23.4 μm3) with valinomycin, respectively.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401748&req=5

pone.0125906.g005: 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.
Mentions: The vacuoles of Hvcn1-/- neutrophils containing Candida underwent a profound swelling, which was much more obvious than in WT mouse or human cells (S1 Video and S3 Video). To determine the extent of this swelling and to distinguish it from that induced by the osmotic effects of the products of digestion of the Candida, we measured the cross-sectional areas of vacuoles containing a single indigestible latex particle with a diameter of 3 μm (a cross-sectional area of 7.1 μm2), similar to that of Candida. In neutrophils from Hvcn1-/- mice, the vacuoles swelled to a median cross-sectional area of 11.2 μm2 (quartiles 8.5–16.6) compared with 7.9 μm2 (6.8–9.1) in neutrophils from WT mice (p<0.001) or Hvcn1-/- treated with 5 μM DPI (7.9 μm2) (6.8–8.8) or CCCP (7.9 μm2) (6.9–9.0) (Fig 5C) after 30 minutes. These results indicate that osmotically active ions are driven into the vacuole by the oxidase in the absence of the proton channel. This swelling was reduced significantly (p<0.001) by 3 μM valinomycin to a median area of 9.9 μm2 (7.1–12.8). The estimated volumes utilising the radius calculated from the median cross-sectional area and assuming spherical vacuoles were: latex particles (14.2 μm3); WT vacuoles (16.7 μm3) and Hvcn1-/- (28.2 μm3) without and (23.4 μm3) with valinomycin, respectively.

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