Pannexin-1 Channels Are Essential for Mast Cell Degranulation Triggered During Type I Hypersensitivity Reactions.

Paloma A Harcha,Juan C Sáez,Ximena López,Paola Fernández,Agustín D Martínez,Iván Barría,Pablo J Sáez

doi:10.3389/fimmu.2019.02703

Abstract

Mast cells (MCs) release pro-inflammatory mediators through a process called degranulation response. The latter may be induced by several conditions, including antigen recognition through immunoglobulin E (IgE) or “cross-linking,” classically associated with Type I hypersensitivity reactions. Early in this reaction, Ca2+ influx and subsequent increase of intracellular free Ca2+ concentration are essential for MC degranulation. Several membrane channels that mediate Ca2+ influx have been proposed, but their role remains elusive. Here, we evaluated the possible contribution of pannexin-1 channels (Panx1 Chs), well-known as ATP-releasing channels, in the increase of intracellular Ca2+ triggered during cross-linking reaction of MCs. The contribution of Panx1 Chs in the degranulation response was evaluated in MCs from wild type (WT) and Panx1 knock out (Panx1−/−) mice after anti-ovalbumin (OVA) IgE sensitization. Notably, the degranulation response (toluidine blue and histamine release) was absent in Panx1−/− MCs. Moreover, WT MCs showed a rapid and transient increase in Ca2+ signal followed by a sustained increase after antigen stimulation. However, the sustained increase in Ca2+ signal triggered by OVA was absent in Panx1−/− MCs. Furthermore, OVA stimulation increased the membrane permeability assessed by dye uptake, a prevented response by Panx1 Ch but not by connexin hemichannel blockers and without effect on Panx1−/− MCs. Interestingly, the increase in membrane permeability of WT MCs was also prevented by suramin, a P2 purinergic inhibitor, suggesting that Panx1 Chs act as ATP-releasing channels impermeable to Ca2+. Accordingly, stimulation with exogenous ATP restored the degranulation response and sustained increase in Ca2+ signal of OVA stimulated Panx1−/− MCs. Moreover, opening of Panx1 Chs in Panx1 transfected HeLa cells increased dye uptake and ATP release but did not promote Ca2+ influx, confirming that Panx1 Chs permeable to ATP are not permeable to Ca2+. These data strongly suggest that during antigen recognition, Panx1 Chs contribute to the sustained Ca2+ signal increase via release of ATP that activates P2 receptors, playing a critical role in the sequential events that leads to degranulation response during Type I hypersensitivity reactions.

Highlights

Mast cells (MCs) originate from hematopoietic precursors, which migrate through the bloodstream and differentiate in diverse tissues
No increase was observed in IgEsensitized Panx1−/− MCs exposed to OVA [6 ± 1 after OVA stimulation vs. 6± 2 under basal conditions (n = 4), p > 0.05] (Figure 1A), suggesting that Panx1 is required for histamine release after OVA recognition in sensitized MCs
Basal toluidine blue (TB) loss from wild type (WT) and Panx1−/− immunoglobulin E (IgE)-sensitized MCs was recorded during 5 min and cells were exposed to 10 μM OVA (Figure 1B)

Summary

Introduction

Mast cells (MCs) originate from hematopoietic precursors, which migrate through the bloodstream and differentiate in diverse tissues. These areas include surfaces normally exposed to pathogens such as skin, gastrointestinal, and airway epithelium [1, 2]. The best-described MC responses occurs during Type I hypersensitivity reactions, which are manifested in various allergic diseases, such as allergic asthma, most types of sinusitis, allergic rhinitis, and food allergies Under these pathological conditions, the host produces large amounts of plasmatic immunoglobulin E (IgE) [3,4,5,6] that binds and stabilizes their receptors (FcεRI) [7], coating and sensitizing MCs to specific antigens. The mechanism that allows release of the granules content includes influx of calcium ions (Ca2+) [11,12,13,14,15]

Methods

Results

Conclusion