Rapid Inhibitory Effect Of Glucocorticoids On The Degranulation Of Mast Cells And Its Nongenomic Mechanism

Glucocorticoids (GCs) are bioactive steroid molecules, which regulate growth-developmental, metabolic, and immune functions in body and play a pivotal role in stress. The mechanism of GCs' action has been well studied in life science with military significance and practical value. Over the passed decades, it was widely assumed that GCs work solely through regulating gene expression and synthesis of protein, which needs several hours or days to take its biological effect. In addition to the classical "genomic" mechanism, there is increasing recognition of alternative modalities of GCs' action that is independent from modulating gene expression and for this reason defined as "nongenomic mechanism". These are characterized by a rapid response (seconds to minutes) and insensitivity to inhibitors of gene transcription and protein synthesis.Nongenomic effects of GCs are well studied in neuroendocrinology, though GCs have been employed mainly in anti-allergic, anti-inflammatory and immunosuppressive conditions. In the treatment of allergic diseases, GCs are ones of the most potent agents available. So do GCs exert rapid effect through nongenomic mechanism on immune cell in regulation of immune reaction? Antigen-induced degranulation of mast cells plays a crucial role in the attack of allergic reaction. Cross-linking of the high affinity IgE receptor by antigen induces mast cells exocytosis, finally resulting in a cascade of morphological and biochemical reactions, and attack of allergic reaction. It is unclear whether GCs could exert rapid effects on them, or which of these signaling pathways would be modulated by GCs. So we studied GCs' rapid action and its nongenomic mechanism on antigen-induced degranulation of mast cells in the allergic reaction.In vivo, we studied GCs' rapid effect on the allergic reaction by examining the changes of pulmonary function in the allergic asthma model of guinea pigs. And GCs' rapid action on airway mast cells degranulations was evaluated by the computer-assisted morphometry. In vitro, we established the model of antigen-induced mast degranulation in rat basophilic leukemia (RBL-2H3) cells. Using whole-cell patch clamp and fluorometric assay, we examined GCs' nongenomic effect on antigen-induced exocytosis and histamine release of RBL-2H3 mast cells. Employing the flash photolysis technique, we studied the role of Ca2+ signal in the GCs' nongenomic effect on exocytosis of mast cells. And the activation of PKC in mast cells was detected by western blot analysis to get more information about the molecular mechanism involved in.The results were as follows:1. Inhaled GCs could rapidly inhibit the change of lung resistance (F?l) and dynamic lung compliance (Cdyn) in allergic asthma model of guinea pigs within 10 min, which inferred the possible involvement of nongenomic mechanism;2. Our morphological studies showed that inhaled GCs significantly inhibited airway mast cells degranulation in the allergic reaction rapidly, which suggested that mast cells might be one of the targets in GCs' rapid action;3. In vitro, GCs could dose-dependently inhibit antigen-induced exocytosis of mast cells, which could be mimicked by the GC-BSA, and neither glucocorticoid nuclear receptor antagonist (RU38486) nor protein synthesis inhibitor (actidione, ATI) could block the rapid action, which confirmed the existence of nongenomic mechanism;Histamine is thought to be a major mediator released from actived mast cells in the allergic reaction, and our result showed that GCs could dose-dependently inhibit antigen-induced histamine release of mast cells with time-course character, which could be mimicked by the GC-BSA, and neither GCs nuclear receptor antagonist nor protein synthesis inhibitor could block the rapid action;4. GCs could significantly reduce antigen-induced the increase in [Ca2+]j, with its rapid inhibitory effect on exocytosis. And under the condition of well-controlled [Ca2+]j , Neither the number of the granules in neither the readily releasable state nor the Ca2+- dependence of exocytosis is found to be rapidly altered by GCs. Thus, we conclude that GCs' nongenomic effect was not through direct action on secretory machinery, but was mediated by a reduction in the [Ca2+]j elevation of mast cells.GCs could rapid inhibited the phosphorylation of PKC in activation of mast cells, neither glucocorticoid nuclear receptor antagonist nor protein synthesis inhibitor could block GCs' rapid action;The study expanded the research of GCs' nongenomic effect from neuro-endocrine system to immune system. And we proposed for the first time that GCs could rapidly influence the signal pathway in the activation of mast cells with intracellular Ca2+ as the core, including the phosphorylation of PKC, and then decrease exocytosis and release of inflammation mediator, finallyresulting in the inhibition of type I allergic reaction independent of classicgenomic mechanism. Further study might raise the possibility of new therapeutic strategies for allergic diseases. It also indicates that GCs' nongenomic effects might play an important role in modulating stress and immune response.