Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events ( granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.

Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. 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 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted 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 but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, 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.

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