SUPPRESSION OF TNF-ALPHA SECRETION BY AZELASTINE IN A RAT MAST (RBL-2H3) CELL-LINE - EVIDENCE FOR DIFFERENTIAL REGULATION OF TNF-ALPHA RELEASE, TRANSCRIPTION, AND DEGRANULATION

The mast cell plays a pivotal role in initiating allergic inflammation by secreting several cytokines including TNF-alpha, in addition to gr anule mediators such as histamine. Anti-allergic drugs including azela stine prevent immediate-type hypersensitivity by inhibiting mast cell degranulation, as well as blocking histamine H-1 receptors. However, t heir effects on cytokine release from mast cells remain unknown. In a rat mast RBL-2H3 cell line, azelastine inhibited Ag- and ionomycin-ind uced TNF-alpha release with IC50 values of 25.7 +/- 3.4 mu M and 1.66 +/- 0.45 mu M respectively. These effects were observed at lower conce ntrations than needed for the inhibition of degranulation. In Ag-stimu lated cells, azelastine also inhibited TNF-alpha mRNA expression, TNF- alpha protein synthesis and release, and, possibly related to these ef fects, Ca2+ influx. In ionomycin-stimulated cells, however, azelastine inhibited TNF-alpha release to a greater extent than mRNA expression/ protein synthesis and Ca2+ influx, suggesting that azelastine inhibits the release process more potently than transcription or production of TNF-alpha by interfering with a signal other than Ca2+. Azelastine ad ded 1 h after ionomycin stimulation also immediately blocked subsequen t release of TNF-alpha which had been produced in the cells, without a ffecting Ca2+ influx. Pretreatment with 1 mu M azelastine inhibited io nomycin-induced, but not Ag-induced, protein kinase C translocation to the membranes. These results suggest that the release process of TNF- alpha in mast cells is regulated by a mechanism distinct from that of degranulation, and that in Ca2+-ionophore-stimulated cells, it is also different from that of transcription/production, and possibly involve s protein kinase C activation.