Bhandaru Madhuri, Pasham Venkanna, Yang Wenting, Bobbala Diwakar, Rotte Anand, Lang Florian
Department of Physiology, University of Tübingen, Tübingen, Germany.
Cell Physiol Biochem. 2012;29(3-4):533-42. doi: 10.1159/000338507. Epub 2012 Apr 3.
Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.
硫唑嘌呤是一种强效免疫抑制药物，它通过干扰树突状细胞（DCs）的成熟和功能而部分发挥作用，DCs是连接固有免疫和适应性免疫的抗原呈递细胞。DCs受到细菌脂多糖（LPS）的刺激，LPS触发活性氧（ROS）的形成，同时伴随着Na(+)/H(+)交换体的激活。该载体参与细胞内pH值、细胞体积和迁移的调节。本研究探讨了硫唑嘌呤是否影响DCs中Na(+)/H(+)交换体的活性。从鼠骨髓中分离出DCs，利用2',7'-双（2-羧乙基）-5-（和-6）-羧基荧光素（BCECF-AM）荧光估计细胞内pH值（pH(i)），通过铵脉冲后Na(+)依赖性再碱化测定Na(+)/H(+)交换体活性，利用流式细胞术分析中的前向散射测定细胞体积，利用2',7'-二氯二氢荧光素二乙酸酯（DCFDA）荧光测定ROS产生，利用酶联免疫吸附测定法测定TNFα释放，利用Transwell迁移试验测定迁移。将DCs暴露于脂多糖（LPS，1μg/ml）导致Na(+)/H(+)交换体活性短暂增加，同时伴有ROS形成、细胞体积增加、TNFα产生和迁移受刺激。硫唑嘌呤（10μM）在LPS暴露前未显著改变Na(+)/H(+)交换体活性、细胞体积和ROS形成，但显著减弱了LPS诱导的Na(+)/H(+)交换体活性刺激、ROS形成、细胞肿胀、TNFα产生和细胞迁移。总之，硫唑嘌呤通过细菌脂多糖干扰树突状细胞Na(+)/H(+)交换体的激活，这一作用可能参与了该药物的抗炎作用。
