Expression and role of a2 vacuolar-ATPase (a2V) in trafficking of human neutrophil granules and exocytosis

Abstract

Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils.

The effect of Rho drugs on mast cell activation and degranulation

“…Stimulated cells were also examined by flow cytometry. When mast cells are stimulated, the release of granular content results in the exposure of phosphatidylserine on the external surface of the cell, which can be quantified by FITC-annexin V staining [33,34]. For RBL-2H3 cells, a P4 gate was set on resting cells that included 95% of cells and a P5 gate on stimulated cell that included 95% of cells not in P4 ( Fig.…”

The curious case of vacuolar ATPase: regulation of signaling pathways

“…Following these findings, it was further demonstrated that a 2V deficiency in tumor cells alters the resident macrophage population in the tumor microenvironment and affects in vivo tumor growth [ 34 ]. a 2V is expressed on the primary granules of neutrophils and helps maintain pH in exocytic pathway during neutrophil activation [ 35 ]. Treatment of human neutrophils with recombinant N terminal peptide of a2V (a2NTD) promoted neutrophil migration and polarization [ 36 ].…”

New Insights for RANKL as a Proinflammatory Modulator in Modeled Inflammatory Arthritis

“…The fact that Tg197/Tg5519 inflamed ankles overexpress various V-ATPase subunits either osteoclast specific such as ATP6V1B2, and TCIRG1 or ubiquitous ATP6V1A, ATP6V1E1, and ATPV0D1 indicates an overwhelming osteoclastic activity that causes massive joint destruction, which is also confirmed by the histological analysis. Upregulated V-ATPase subunits could also have an impact on inflammatory responses such as phagocytosis, cytokine secretion and exocytosis of neutrophil granules (57, 58). Notably, recent studies have shown that in inflammatory conditions, osteoclasts can differentiate from dendritic cells in the presence of RANKL and behave as antigen-presenting cells (59).…”