Yoshimaru T, Suzuki Y, Matsui T, Yamashita K, Ochiai T, Yamaki M, Shimizu K
Department of Immunology and Microbiology, Nihon University School of Medicine, Tokyo, Japan.
Clin Exp Allergy. 2002 Apr;32(4):612-8. doi: 10.1046/j.0954-7894.2002.01263.x.
Previous studies have shown that rat peritoneal mast cells and mast cell model rat basophilic leukaemia (RBL-2H3) cells generate intracellular reactive oxygen species (ROS) in response to antigen challenge. However, the physiological significance of the burst of ROS is poorly understood.
The present study was undertaken to investigate the role of superoxide anion in mediator release in rat and human cell systems.
RBL-2H3 cells were directly stimulated with anti-rat FcepsilonRI alpha-subunit monoclonal antibody (mAb). For the analysis of human cell system, leucocytes were isolated by dextran sedimentation from healthy volunteers or from patients, and challenged either with anti-human FcepsilonRI mAb or with the relevant antigens. Superoxide generation was determined by chemiluminescence-based methods. The releases of histamine and leukotrienes (LT)s were determined by enzyme-linked immunosorben assay (ELISA).
Cross-linking of FcepsilonRI on RBL-2H3 cells or on human leucocytes from healthy donors by the anti-FcepsilonRI mAb resulted in a rapid generation of superoxide anion, as determined by chemiluminescence using superoxide-specific probes. Similarly, leucocytes from patients generated superoxide anion in response to the challenge with the relevant allergen but not with the irrelevant allergen. Furthermore, diphenyleneiodonium (DPI), a well-known inhibitor of flavoenzymes suppressed the superoxide generation and the release of histamine and LTC4 induced by the anti-FcepsilonRI mAb or by allergen in parallel.
These results indicate that both RBL-2H3 cells and human basophils generate superoxide anion upon FcepsilonRI cross-linking either by antibody or by allergen challenge and that blockade of the generation prevents the release of allergic mediators. The findings strongly support the role of superoxide generation in the activation of mast cells and basophils under both physiological and pathological conditions. The findings suggest that drugs regulating the superoxide generation have potential therapeutic use for allergic disorders.
先前的研究表明，大鼠腹膜肥大细胞和肥大细胞模型大鼠嗜碱性粒细胞白血病（RBL - 2H3）细胞在抗原刺激下会产生细胞内活性氧（ROS）。然而，ROS爆发的生理意义却知之甚少。
本研究旨在探讨超氧阴离子在大鼠和人类细胞系统中介质释放中的作用。
用抗大鼠FcepsilonRIα亚基单克隆抗体（mAb）直接刺激RBL - 2H3细胞。对于人类细胞系统的分析，通过葡聚糖沉降从健康志愿者或患者中分离白细胞，并用抗人FcepsilonRI mAb或相关抗原进行刺激。通过基于化学发光的方法测定超氧阴离子的产生。通过酶联免疫吸附测定（ELISA）测定组胺和白三烯（LT）的释放。
使用超氧阴离子特异性探针通过化学发光测定，抗FcepsilonRI mAb使RBL - 2H3细胞或健康供体的人类白细胞上的FcepsilonRI交联导致超氧阴离子的快速产生。同样，患者的白细胞在受到相关过敏原而非无关过敏原刺激时会产生超氧阴离子。此外，一种著名的黄素酶抑制剂二苯基碘鎓（DPI）可同时抑制抗FcepsilonRI mAb或过敏原诱导的超氧阴离子产生以及组胺和LTC4的释放。
这些结果表明，RBL - 2H3细胞和人类嗜碱性粒细胞在FcepsilonRI通过抗体或过敏原刺激交联时都会产生超氧阴离子，并且超氧阴离子产生的阻断可防止过敏介质的释放。这些发现有力地支持了超氧阴离子产生在生理和病理条件下肥大细胞和嗜碱性粒细胞激活中的作用。这些发现表明，调节超氧阴离子产生的药物对过敏性疾病具有潜在的治疗用途。
