P2X4 receptor stimulation enhances MrgprB2-mediated mast cell activation and pseudoallergic reactions in mice.

Pseudoallergies caused by drugs make disease treatment difficult. Mas-relate G protein-coupled receptor X2 (MRGPRX2), which is specifically expressed in mast cells (MCs), has been implicated in pseudoallergies. High concentrations of therapeutic agents are typically required to stimulate MRGPRX2. Although regulatory mechanisms may enhance this response, the factors involved in this regulation are not well-understood. In this study, the effects of extracellular ATP on MC activation induced by MrgprB2, the mouse ortholog of human MRGPRX2, were examined in mouse peritoneal MCs (PMCs). ATP alone induced minimal PMC degranulation but markedly enhanced degranulation induced by the MrgprB2 agonist compound 48/80 (CP48/80), substance P, PAMP-12, and vancomycin. ATP promoted CP48/80-induced increase in intracellular Ca2+ in PMCs. This enhancement effect of ATP was absent in PMCs prepared from P2X4 receptor (P2X4R)-deficient mice and inhibited by the PI3K inhibitor wortmannin. In addition, P2X4R deficiency reduced the skin-specific and systemic anaphylactic responses to CP48/80 in vivo. In MC-deficient KitW-sh/W-sh mice, reconstitution with MCs obtained from wild-type mice led to a more severe anaphylactic response to CP48/80 compared to that from P2X4R-deficient mice. P2X4R-mediated effect may be involved in MrgprB2-mediated MC activation in vivo and is a potential target for alleviating pseudoallergic reactions.

Synergistic Cytokine Production by ATP and PGE2 via P2X4 and EP3 Receptors in Mouse Bone-Marrow-Derived Mast Cells.

ATP is an important intercellular messenger in the extracellular space. In mast cells (MCs), ATP stimulates the ionotropic P2X4 receptor (P2X4R), resulting in enhanced degranulation and exacerbation of acute allergic reactions. In this study, we investigate whether ATP regulates inflammatory cytokine production in MCs. Gene expression was analyzed by quantitative RT-PCR, and cytokine production was measured using ELISA. The stimulation of mouse bone-marrow-derived MCs (BMMCs) with ATP alone had little effect on cytokine secretion. However, the co-stimulation with prostaglandin (PG) E2 resulted in a marked increase in the secretion of various cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-13, accompanied by an increase in their mRNA levels. The effects of ATP were inhibited by P2X4R antagonists and diminished in BMMCs derived from P2X4R-deficient mice, suggesting that P2X4R mediated the reaction. The effects of PGE2 were mimicked by an EP3 receptor (EP3R) agonist and blocked by an EP3R antagonist. The synergistic cytokine mRNA elevations induced by ATP and PGE2 were blocked by nuclear factor-κB and Ca2+-calcineurin signaling inhibitors. Altogether, these results suggest that combining P2X4R and EP3R signaling enhances acute degranulation and the subsequent cytokine secretion, exacerbating allergic inflammation.

Extracellular ATP Augments Antigen-Induced Murine Mast Cell Degranulation and Allergic Responses via P2X4 Receptor Activation.

Extracellular ATP released from stimulated and/or damaged cells modulates physiological responses via stimulation of various purinoceptors. We previously showed that ATP potentiated the Ag-induced mast cell (MC) degranulation via purinoceptors pharmacologically similar to the ionotropic P2X4 receptor. In this study, we investigated the role of P2X4 receptor in MC degranulation induced by stimulation of IgE-FcεRI complex with Ag, using bone marrow-derived MCs (BMMCs) prepared from wild type and P2X4 receptor-deficient (P2rx4-/- ) mice. ATP significantly increased Ag-induced degranulation in BMMCs prepared from wild type mice. This effect of ATP was reduced in BMMCs prepared from P2rx4-/- mice. The potentiating effect of ATP was restored by expressing P2X4 receptor in P2rx4-/- BMMCs. The P2X4 receptor-mediated effects were maintained even after differentiating into the connective tissue-type MCs. P2X4 receptor stimulation did not affect the Ag-induced Ca2+ response but enhanced Ag-induced early signals, such as tyrosine phosphorylation of Syk and phospholipase C-γ. Interestingly, these effects of ATP on Syk phosphorylation were not impaired by pretreatment with Cu2+, an inhibitor of the P2X4 receptor channel, or removal of external Ca2+, suggesting that a mechanisms other than Ca2+ influx through ion channel activity may be involved. In vivo experiments revealed that systemic and intradermal passive anaphylaxis responses were significantly alleviated in P2rx4-/- mice. Taken together, the present data suggest that the P2X4 receptor plays an essential role in ATP-induced upregulation of MC degranulation in response to Ag, and also contributes to the Ag-induced allergic response in vivo.

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.