Background 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.
Objective The present study was undertaken to investigate the role of superoxide anion in mediator release in rat and human cell systems.
Methods RBL-2H3 cells were directly stimulated with anti-rat FcεRI α-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 FcεRI 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).
Results Cross-linking of FcεRI on RBL-2H3 cells or on human leucocytes from healthy donors by the anti-FcεRI 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-FcεRI mAb or by allergen in parallel.
Conclusion These results indicate that both RBL-2H3 cells and human basophils generate superoxide anion upon FcεRI 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.