Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes

Abstract

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was ∼1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only ∼1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface.

Keywords::

• healthy individuals
• oxygen consumption
• reactive oxygen species
• NADPH oxidase
• Hv1 channel

Acknowledgements

The excellent technical assistance of Ms Beáta Molnár, Mrs Eszter Halász and Mrs Anikó Rajki is highly appreciated. We sincerely thank Dr. Katalin Német (Budapest) for the anti-67^phox antibody and for the 7D5 supernatant.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by grants from the Hungarian Research Fund (OTKA K106138 and F-68023) and from the National Office for Research and Technology (OMFB-01680/2009) and by “Lendület” grant from the Hungarian Academy of Sciences. The authors declare that no competing interests exist.