A Functional Study on CysLT<sub>1</sub> Receptors in Human Eosinophils

Abstract

<i>Background:</i> The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT<sub>1</sub> receptor and CysLT<sub>2</sub> receptor. <i>Objective:</i> This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT<sub>1</sub>. <i>Methods:</i> 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 CysLT<sub>1</sub>. Binding assays were performed with [<sup>3</sup>H]LTD<sub>4</sub>. Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca<sup>2+</sup> influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD<sub>4</sub> at 10<sup>–6</sup><i>M</i> 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. <i>Results:</i> The expression of the mRNA and protein of CysLT<sub>1</sub> on eosinophils and [<sup>3</sup>H]LTD<sub>4</sub>-specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-γ) nor Th2 cytokines (IL-4 or IL-5) affected CysLT<sub>1</sub> expression in eosinophils. CysLTs induced an increase in intracellular free Ca<sup>2+</sup> in eosinophils via CysLT<sub>1</sub>, as suggested by the efficient inhibition by a CysLT<sub>1</sub> antagonist, pranlukast, in addition to the rank order of potency being LTD<sub>4</sub>, LTC<sub>4</sub> and LTE<sub>4</sub>. LTD<sub>4</sub> stimulated eosinophils to migrate at 10<sup>–6</sup><i>M</i> via CysLT<sub>1</sub>. LTE<sub>4</sub> also induced significant eosinophil migration at 10<sup>–6</sup><i>M</i>. LTD<sub>4</sub> enhanced EDN release induced by IL-5 via CysLT<sub>1</sub>. <i>Conclusion:</i> CysLTs induce migration and enhance degranulation in eosinophils via CysLT<sub>1</sub>. Accordingly, interaction of CysLTs and CysLT<sub>1</sub> on eosinophils has the potential to play a prominent role in the pathophysiology of asthma.