Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules

Background—Cysteinyl leukotrienes are recognized to act via receptors (CysLTRs) expressed on cell surface plasma membranes. Agents that block CysLT1R, are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis and urticaria, but have unknown functional capabilities. Objective—We evaluated the expression of CysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules. Methods—We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysteinyl leukotrienes and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and western blot. Results—We report that receptors for cysteinyl leukotrienes, CysLT1R, CysLT2R and the purinergic P2Y12 receptor (P2Y12R), are expressed on eosinophil granule membranes. Leukotriene (LT) C4 and extracellularly generated LTD4 and LTE4 stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12R antagonist, inhibited cysteinyl leukotrienes-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of CysLT1R, inhibited eosinophil cationic protein release elicited by LTC4 and LTD4 as well as by LTE4. Conclusion—These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysteinyl leukotriene-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly