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.
Highlights
Neutrophils that encounter a bacterium or fungus engulf it into a phagocytic vacuole of invaginated plasma membrane, into which cytoplasmic granules release their contents of potentially lethal enzymes (Fig 1)
The respiratory burst is impaired in Hvcn1-/- neutrophils whilst extracellular acid release is normal In wild type (WT) neutrophils, oxygen consumption was increased tenfold by phorbol myristate acetate (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
Summary
Neutrophils that encounter a bacterium or fungus engulf it into a phagocytic vacuole of invaginated plasma membrane, into which cytoplasmic granules release their contents of potentially lethal enzymes (Fig 1). Inhibition or deletion of the HVCN1 channel has been shown to result in exaggerated acidification of the cytosol after phagocytosis of zymosan [17] or stimulation of the oxidase with phorbol myristate acetate (PMA) [18] which led to the suggestion that this channel might be important for the expulsion of protons from the neutrophil cytosol [17,18] this was not measured directly in either of these studies Those observations raise the possibility that the depressant effect of the loss of the HVCN1 channel on the NADPH oxidase might be due to the development of an excessively acidic cytosol, which inhibits the oxidase [19], rather than as a consequence of impaired charge compensation. To determine which of these mechanisms is the more feasible within the environment of the phagocytic vacuole, we measured the pH dependence of the activity of MPO and that of the two major neutral proteases, cathepsin G and elastase, and related these to the observed phagocytic vacuolar pH in normal neutrophils
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