Recent studies have identified HVCN1 as the proton channel responsible for charge compensation during NADPH oxidase activity in neutrophils [10
]. Additionally, unexpected roles for HVCN1 were found in neutrophils and B cells [12
]. In this report, we used WT and HVCN1-deficient eosinophils to determine the function of HVCN1 in this cell type. Our studies revealed several novel findings. First, we demonstrate that Hvcn1
mRNA expression is increased in allergic lung and is at a high basal level in eosinophils. Second, we show that unlike mouse neutrophils, eosinophils do not require HVCN1 for chemotaxis. Finally, we demonstrate that HVCN1 is required for prevention of activation-induced eosinophil cell death, likely because of the membrane depolarization and cytosolic acidification that occurs following PMA stimulation in the absence of HVCN1.
In the present study, HVCN1-deficient eosinophils demonstrated no migration defect, which is in contrast to the results of HVCN1-deficient neutrophils. This can be due to the different mechanism involving calcium mobilization which controls numerous cellular functions including cell migration. In neutrophils, Ca2+
entry occurs largely across store-operated Ca2+
channels from the extracellular environment [34
]. Thus, the increased membrane depolarization can reduce the driving force for extracellular Ca2+
into the cells and as a result impair migration ability, which has been shown in fMIVIL-activated HVCN1-deficient neutrophils [12
]. However, in human eosinophils, several studies showed that pre-incubation of eosinophils by intracellular calcium chelator 1,2-bis-(o
-tetraacetic acid acetoxy-methyl ester (BAPTA-AM) dose-dependently prevented calcium flux and the chemotactic response to platelet activating factor (PAF) and complement fragment 5a (C5a), but the depletion of extracellular calcium had no effect [35
], suggesting that intracellular calcium plays a more important role in regulating eosinophil migration. The other potential explanation (not mutually exclusive) for the lack of effect of HVCN1 deficiency on eosinophil migration comes from the finding that mEotaxin-1 does not induce significant ROS generation by mouse eosinophils [37
], which suggests that mEotaxin-1 does not activate the NADPH oxidase and thus induce the membrane depolarization in HVCN1-deficient eosinophils. Together, HVCN1 deficiency does not affect mouse eosinophil migration.
In our study, we found that HVCN1 was required for optimal ROS production. However, HVCN1-deficient eosinophils still had ~50% ROS production retained (Figure A-D). Similarly, HVCN1-deficient neutrophils and B cells had ~30% ROS production retained [10
], suggesting that proton channels are not indispensible even though they provide the bulk of compensating charge in phagocytes [38
] and that other channels might facilitate charge compensation during ROS generation. For instance, ClC-3 Cl-
], CLIC-1 Cl channels [41
], TRPV1 nonselective cation channels [42
], SK2 and SK4 Ca2+
], and Kv1.3 delayed rectifier K+
] have been proposed to contribute to charge compensation in leukocytes. However, while these channels might provide charge compensation, they would not alleviate the acidification. In contrast, the sodium-hydrogen exchanger could extrude the extra acid but could not compensate the extra charge as this exchanger is not electrogenic [45
]. Thus, HVCN1 is ideally suited to facilitate charge compensation during ROS generation as it provides both charge and acidity compensation.
HVCN1 was required for optimal ROS production by eosinophils. Our study suggests two likely reasons for this finding: (1) HVCN1-deficient eosinophils were more depolarized than WT eosinophils after PMA stimulation (Figure B), which was most likely caused by the lack of compensating charge provided by proton channels. As a result, the membrane depolarization hinders the flow of electrons across the voltage-dependent flavocytochrome [5
], which is needed to reduce oxygen to superoxide. (2) The deficiency of HVCN1 was also associated with a substantial cytosolic acidification, which happened as quickly as within 30 minutes after PMA stimulation (Figure C). The activity of NADPH oxidase responsible for ROS production, being optimal at intracellular pH 7.0-7.5, could be decreased in this resulting acidic cytosol [47
In addition to inhibiting NADPH oxidase activity, membrane depolarization and cytosolic acidification may be responsible for the observed activation-induced cell death. However, we cannot exclude other possible reasons. For instance, imbalance of osmolarity across the membrane (presumably caused by K+ efflux) might also account for the increased cell death of HVCN1-deficient eosinophils following PMA stimulation.
In summary, our study identifies cell-specific roles for HVCN1 in eosinophil respiratory burst and prevention of activation-induced cell death but not eosinophil migration. These findings have implications for our understanding of the basic mechanism of eosinophil function, as well as for targeting of eosinophils in eosinophil-associated diseases.