In Esophageal Squamous Cells From Eosinophilic Esophagitis Patients, Th2 Cytokines Increase Eotaxin-3 Secretion Through Effects on Intracellular Calcium and a Non-Gastric Proton Pump

Abstract

Background & Aims: In upper airway cells, T helper 2 cytokines that signal through interleukin-4 (IL-4) receptor-α have been shown to stimulate eotaxin-3 secretion via a nongastric proton pump (ngH+,K+ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH+,K+ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL-4 receptor-α signaling. Methods: ngH+,K+ATPase expression in EoE cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting. IL-4–stimulated eotaxin-3 secretion was measured by enzyme-linked immunosorbent assay after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), ethylene glycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetoxymethyl ester (calcium chelator), 2-aminoethoxydiphenyl borate (inhibitor of endoplasmic reticulum calcium release), verapamil, and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of patients with EoE and controls. Results: EoE cells expressed ngH+,K+ATPase messenger RNA and protein. Omeprazole and SCH 28080 decreased IL-4–stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4–stimulated eotaxin-3 secretion was blocked by ethylene glycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetoxymethyl ester, 2-aminoethoxydiphenyl borate, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4–stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls. Conclusions: EoE cells express a nongastric proton pump that mediates T helper 2 cytokine–stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block T helper 2 cytokine–stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment.