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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

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.
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pone.0125906.g002: 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.

Mentions: In WT neutrophils, oxygen consumption was increased tenfold by PMA and to a similar level by the addition of opsonised Candida. PMA stimulated oxygen consumption was reduced to 73% of normal in cells isolated from Hvcn1-/- mice (p = 0.035), consistent with previous observations [15,16,18] was abolished by the oxidase inhibitor DPI (p<0.001), and was absent in neutrophils lacking gp91phox (p<0.001) (Fig 2A). Oxygen consumption by Hvcn1-/- cells phagocytosing opsonised Candida was 50% of that by WT mouse neutrophils (p<0.001), and significantly lower than in the same cells after PMA stimulation (p<0.001) but higher than the 20% measured by others [20]. Using Amplex Red to measure H2O2 generated by PMA stimulated cells, oxidase activity was decreased to similar levels in Hvcn1-/- at 58.4% ± 18.1 (SD) (n = 5) of that by WT cells.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401748&req=5

pone.0125906.g002: 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.
Mentions: In WT neutrophils, oxygen consumption was increased tenfold by PMA and to a similar level by the addition of opsonised Candida. PMA stimulated oxygen consumption was reduced to 73% of normal in cells isolated from Hvcn1-/- mice (p = 0.035), consistent with previous observations [15,16,18] was abolished by the oxidase inhibitor DPI (p<0.001), and was absent in neutrophils lacking gp91phox (p<0.001) (Fig 2A). Oxygen consumption by Hvcn1-/- cells phagocytosing opsonised Candida was 50% of that by WT mouse neutrophils (p<0.001), and significantly lower than in the same cells after PMA stimulation (p<0.001) but higher than the 20% measured by others [20]. Using Amplex Red to measure H2O2 generated by PMA stimulated cells, oxidase activity was decreased to similar levels in Hvcn1-/- at 58.4% ± 18.1 (SD) (n = 5) of that by WT cells.

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