Ohshima Nobuharu, Nagase Hiroyuki, Koshino Takeshi, Miyamasu Misato, Yamaguchi Masao, Hirai Koichi, Yamamoto Kazuhiko, Fujisawa Takao, Nakagawa Naoki, Kishikawa Katsuya, Morita Yutaka
Department of Respiratory Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan.
Int Arch Allergy Immunol. 2002 Sep;129(1):67-75. doi: 10.1159/000065175.
The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT(1) receptor and CysLT(2) receptor.
This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT(1).
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(1). Binding assays were performed with [(3)H]LTD(4). Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca(2+) influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD(4) at 10(-6) M for 60 min followed by incubation for 4 h at 37 degrees C in the presence or absence of IL-5 and eosinophil-derived neurotoxin (EDN) release was evaluated.
The expression of the mRNA and protein of CysLT(1) on eosinophils and [(3)H]LTD(4)-specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-gamma) nor Th2 cytokines (IL-4 or IL-5) affected CysLT(1) expression in eosinophils. CysLTs induced an increase in intracellular free Ca(2+) in eosinophils via CysLT(1), as suggested by the efficient inhibition by a CysLT(1) antagonist, pranlukast, in addition to the rank order of potency being LTD(4), LTC(4) and LTE(4). LTD(4) stimulated eosinophils to migrate at 10(-6) M via CysLT(1). LTE(4) also induced significant eosinophil migration at 10(-6) M. LTD(4) enhanced EDN release induced by IL-5 via CysLT(1).
CysLTs induce migration and enhance degranulation in eosinophils via CysLT(1). Accordingly, interaction of CysLTs and CysLT(1) on eosinophils has the potential to play a prominent role in the pathophysiology of asthma.
半胱氨酰白三烯（CysLTs）通过两种受体介导其生物学作用：CysLT(1)受体和CysLT(2)受体。
本研究旨在研究CysLTs对嗜酸性粒细胞的直接作用，如趋化性和脱颗粒作用，重点关注CysLT(1)。
从无过敏史的正常志愿者静脉血中分离嗜酸性粒细胞（纯度>99%）。对其进行逆转录-聚合酶链反应分析和CysLT(1)的流式细胞术分析。用[(3)H]LTD(4)进行结合试验。用Fura-2乙酰氧基甲酯负载的纯化嗜酸性粒细胞用CysLTs刺激，并测量Ca(2+)内流。使用96孔多通道博伊登小室测量嗜酸性粒细胞对CysLTs的迁移反应。嗜酸性粒细胞用10(-6)M LTD(4)处理60分钟，然后在37℃孵育4小时，评估有无白细胞介素-5（IL-5）时嗜酸性粒细胞衍生神经毒素（EDN）的释放。
观察到嗜酸性粒细胞上CysLT(1)的mRNA和蛋白表达以及[(3)H]LTD(4)与嗜酸性粒细胞的特异性结合。1型辅助性T细胞（Th1）细胞因子（干扰素-γ）和2型辅助性T细胞（Th2）细胞因子（IL-4或IL-5）均不影响嗜酸性粒细胞中CysLT(1)的表达。CysLT(1)拮抗剂普仑司特有效抑制表明，CysLTs通过CysLT(1)诱导嗜酸性粒细胞内游离Ca(2+)增加，此外，其效力顺序为LTD(4)、LTC(4)和LTE(4)。LTD(4)在10(-6)M时通过CysLT(1)刺激嗜酸性粒细胞迁移。LTE(4)在10(-6)M时也诱导显著的嗜酸性粒细胞迁移。LTD(4)通过CysLT(1)增强IL-5诱导的EDN释放。
CysLTs通过CysLT(1)诱导嗜酸性粒细胞迁移并增强其脱颗粒作用。因此，CysLTs与嗜酸性粒细胞上的CysLT(1)相互作用在哮喘病理生理学中可能起重要作用。
