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

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.

Keywords: ATP; degranulation; histamine; immune cells; inflammation; ovalbumin; pannexon.