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. 2013 May 24:14:24.
doi: 10.1186/1471-2172-14-24.

Proton channel HVCN1 is required for effector functions of mouse eosinophils

Xiang Zhu  1 , Eucabeth Mose, Nives Zimmermann
Affiliations

Proton channel HVCN1 is required for effector functions of mouse eosinophils

Xiang Zhu et al. BMC Immunol. .

Abstract

Background: Proton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood.

Results: In the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation.

Conclusions: Eosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death.

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Figures

Figure 1
Figure 1
HVCN1 mRNA and protein expression levels in lung tissue and eosinophils. (A-B) The microarray (A) and real-time RT-PCR (B) analyses show increased Hvcn1 levels in the lung tissue of the allergen (Aspergillus)-challenged mice compared to saline-challenged controls. (C) Real-time RT-PCR analysis of Hvcn1 mRNA expression levels relative to housekeeping gene Actb (β-actin) in WT and HVCN1-deficient eosinophils and neutrophils from the different source (seen in “Methods”). Data are expressed as mean ± SD of 4–6 mice per group. **, P < 0.01. (D) Western blotting of HVCN1 in BM-derived eosinophils from WT and HVCN1-deficient mice, the representative of 4 experiments.
Figure 2
Figure 2
ROS production from WT and HVCN1-deficient eosinophils. (A) Time-dependent H2O2 production by WT and HVCN1-deficient (KO) BM-derived eosinophils was measured using HBSS solution containing 25 μM Amplex Red and 0.05 U/ml HRP. Eosinophils were stimulated by PMA (50 ng/ml) in the presence or absence of Zn2+ or DPI at the indicated concentrations. Data are representative of 4 separate experiments and expressed as mean ± SD. The arrow indicates the addition of PMA at Time = 0. (B) Mean H2O2 production under the same conditions as (A) at 60 minutes following PMA stimulation. Data are expressed as the mean ± SD of 4 separate experiments done in triplicate. (C) Time-dependent superoxide production by WT and HVCN1-deficient BM-derived eosinophils was determined using HBSS solution containing lucigenin (200 μM). Eosinophils were stimulated by PMA (50 ng/ml) in the presence or absence of SOD (1000 U/ml). Data are representative of 4 separate experiments and expressed as mean ± SD. The arrow indicates the addition of PMA at Time = 0. (D) Mean superoxide production under the same conditions as (C) at 30 minutes after PMA stimulation. Data are expressed as the mean ± SD of 4 separate experiments done in triplicate. *, P < 0.05; **, P < 0.01; n.s., not significant.
Figure 3
Figure 3
Chemotaxis and intracellular calcium flux in WT and HVCN1-deficient eosinophils. (A) The in vivo migration of neutrophils (left; n = 3 experiments) and eosinophils (right; n = 4 experiments) was determined by morphologically counting the cells collected from the peritoneal cavity of WT and HVCN1-deficient mice at the indicated time points following intraperitoneal injection of thioglycollate medium. Data are expressed as the mean ± SD with mice from each experiment indicated by different symbols. (B) Transwell migration assay was performed to compare the in vitro migration of WT and HVCN1-deficient eosinophils subjected to mEotaxin-1 at the indicated concentrations. The results are expressed as migratory index by determining the ratio of total cell number under mEotaxin-1 attraction to total cell number without mEotaxin-1. Data are representative of 3 experiments and expressed as mean ± SD. (C) Cytosolic Ca2+ response in WT and HVCN1-deficient eosinophils was determined with intracellular fluorescence dye Fluo-4 AM in the presence of 1 mM extracellular Ca2+. Shown are the representative overlaid traces out of 5 experiments for mEotaxin-1 (1-300ng/ml). The arrow indicates the time of mEotaxin-1 addition. The trace (blue: WT eosinophils; red: HVCN1-deficient eosinophils) represents the average fluorescence intensity from cultured BM-derived eosinophils at the collection speed of approximately 150 cells per second by flow cytometer Canto II. *, P < 0.05; **, P < 0.01; n.s., not significant.
Figure 4
Figure 4
Enhanced cell death in HVCN1-deficient eosinophils following PMA stimulation. (A) The viable eosinophils at the indicated time points following PMA (50 ng/ml) stimulation were stained with trypan blue and counted. Data are expressed as the mean ± SD of 3 separate experiments performed in duplicate. (B) Analysis of eosinophil apoptosis / cell death after four-hour PMA (50 ng/ml) stimulation by flow cytometer Calibur I. Data are expressed as the mean ± SD of 4 separate experiments. (C) Kinetic analysis of eosinophil apoptosis / cell death following PMA (50 ng/ml) stimulation. (D)Analysis of eosinophil apoptosis / cell death after four-hour anisomycin (10 μM) or anti-Fas antibody (1 μg/ml) stimulation by flow cytometer Canto II. Data are expressed as the mean ± SD of 3 separate experiments. *, P < 0.05; **, P < 0.01; n.s., not significant.
Figure 5
Figure 5
Increased membrane depolarization and cytosolic acidification in activated HVCN1-deficient eosinophils. (A) Flow cytometric analysis of eosinophil cell death (7-AAD positive cells) after four-hour PMA (50 ng/ml) stimulation in the presence or absence of ZnCl2 (1 mM) or DPI (10 μM). Data are expressed as the mean ± SD of 3 separate experiments. (B) Mean fluorescence intensity (MFI) of DiBAC4(3) in WT and HVCN1-deficient BM-derived eosinophils. PMA evoked a larger depolarization in HVCN1-deficient than WT eosinophils. The vertical arrow indicates the direction of depolarization. Data are representative of 5 separate experiments. (C) Intracellular pH in WT and HVCN1-deficient BM-derived eosinophils was measured using the pH-sensitive dye SNARF-4 AM (5 μM) at the indicated time points after PMA (50 ng/ml) stimulation. Dead cells were excluded by gating out cells that failed to retain SNARF-4 AM fluorescence. Data are expressed as the mean ± SD of 4 separate experiments. (D) Intracellular pH in WT and HVCN1-deficient BM-derived eosinophils was measured 30 minutes after PMA (50 ng/ml) stimulation in the presence or absence of ZnCl2 at the indicated concentrations. Data are expressed as the mean ± SD of 3 separate experiments. *, P < 0.05; **, P < 0.01; n.s., not significant.
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