A Functional Study on CysLT1 Receptors in Human Eosinophils

Abstract

Background: The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT₁ receptor and CysLT₂ receptor. Objective: This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT₁. Methods: Eosinophils were isolated from venous blood from normal volunteers who had no history of allergy (purity >99%). They were subjected to reverse transcription-PCR analysis and flow-cytometric analysis for CysLT₁. Binding assays were performed with [³H]LTD₄. Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca²⁺ influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD₄ at 10^–6M for 60 min followed by incubation for 4 h at 37°C in the presence or absence of IL-5 and eosinophil-derived neurotoxin (EDN) release was evaluated. Results: The expression of the mRNA and protein of CysLT₁ on eosinophils and [³H]LTD₄-specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-γ) nor Th2 cytokines (IL-4 or IL-5) affected CysLT₁ expression in eosinophils. CysLTs induced an increase in intracellular free Ca²⁺ in eosinophils via CysLT₁, as suggested by the efficient inhibition by a CysLT₁ antagonist, pranlukast, in addition to the rank order of potency being LTD₄, LTC₄ and LTE₄. LTD₄ stimulated eosinophils to migrate at 10^–6M via CysLT₁. LTE₄ also induced significant eosinophil migration at 10^–6M. LTD₄ enhanced EDN release induced by IL-5 via CysLT₁. Conclusion: CysLTs induce migration and enhance degranulation in eosinophils via CysLT₁. Accordingly, interaction of CysLTs and CysLT₁ on eosinophils has the potential to play a prominent role in the pathophysiology of asthma.