Abstract
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.
Highlights
Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma
Exocytosis in many cells requires the participation of Munc18 proteins, and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3
We have shown that Synaptotagmin-2 and Munc13-4 control regulated exocytosis in MCs [20, 21], but the role of Munc18 proteins in MC regulated exocytosis has not been studied in mature MCs
Summary
Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. Exocytosis in many cells requires the participation of Munc proteins ( 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 Munc 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 Munc or Munc. We conditionally deleted all three isoforms in MCs and observed a failure in regulated exocytosis only in Munc18-2– deficient MCs. The defect was severe—no exocytosis, single or compound, could be detected in mutant MCs— but it did not affect MC differentiation, number, distribution, or granule biogenesis. These abnormal MC responses had prominent effects on the reactions of mutant mice to systemic anaphylaxis
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