Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production

Mast cellscontribute to the pathology of allergic and other disorders. Strategies to interfere with harmful mast cell-related activities are therefore warranted. Previously we established a principle for inducing selective apoptosis of mast cells, by the use of lysosomotropic agents that cause secretory granulepermeabilization, leading to production of reactive oxygen species (ROS). However, the mechanism of ROS production has not been known. Here we addressed this issue. Live microscopy analysis showed that the secretory granulescomprise major subcellular compartments for ROS production in response to mefloquine. As further signs for the primary involvement of secretory granules, both ROS production and cell death was blunted in mast cellslacking serglycin, a secretory granule-restricted proteoglycan. Inhibition of granuleacidification caused an essentially complete blockade of granulepermeabilization, ROS production and cell death in response to mefloquine. ROS production was also attenuated in the presence of an iron chelator, and after inhibition of either granzyme Bor the ERK1/2 MAP kinase signaling pathway. Together, our findings reveal that the mast cellsecretory granulesconstitute major sites for ROS production in mast cellssubjected to lysosomotropic challenge. Moreover, this study reveals a central role for granuleacidification in ROS generation and the pro-apoptotic response triggered downstream of secretory granulepermeabilization.