Control of mast cell regulated exocytosis by Munc18 proteins

Abstract

Mast cells (MCs) play pivotal roles in many inflammatory conditions including allergic diseases. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Here, we tested whether mature MCs express some or all three mammalian isoforms: Munc18-1, -2 and -3. Then, in order to study the functions of Munc18 proteins expressed in MCs in vitro and in vivo, we used conditional knockout (cKO) mice for each Munc18 protein to delete each of them exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2 deficient MCs, while exocytosis in MCs lacking Munc18-1 or Munc18-3 was normal. Stereological analysis of EM images of stimulated MCs confirmed that MCs were unable to release their granules in the absence of Munc18-2. Furthermore, deletion of Munc18-2 also abolished the homotypic granule-to-granule membrane fusion required for compound exocytosis. The severe defect in MC exocytosis in the absence of Munc18-2 impaired the release of mediators stored in MC granules in our secretion assays. Munc18-2 cKO mice were viable. Their MCs had normal morphology, development and tissue distribution, indicating that Munc18-2 is not essential for the migration, retention and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.