Control of Mast Cell Exocytosis by Munc18 Proteins

RATIONALEUpon stimuli, mast cells (MCs) release inflammatory mediators stored in secretory granules (degranulation) which contribute to thepathophysiology of anaphylaxis and asthma. This process involves granule to plasma membrane fusion (regulated exocytosis) and granuleto granule fusion (compound exocytosis). The SM (Sec/Munc18) protein family includes essential regulators of vesicular trafficking andthree isoforms of Munc18 (-1, -2, and -3) are involved in exocytosis in many mammalian cells. Since MCs express all three isoforms, weattempted to identify which functions were unique, redundant and complementary to each Munc18 and how they participate inregulated and compound exocytosis using mature MCs and models.in vivoMETHODSThe global deletions (-/-) of Munc18-1, -2 and -3 are lethal, therefore we studied heterozygote (+/-), conditional KO (F/F), and exclusiveMC-deletant (Δ/Δ) mice. Expression in peritoneal MCs was assessed by qPCR and immunoblot. Degranulation was measured as an increaseof membrane capacitance in patch-clamped MCs stimulated with a pipette solution containing GTPγS and calcium. With electronmicroscopy (EM), we studied ultrastructural changes of stimulated peritoneal MCs. We evaluated regulated, constitutive and non-exocyticeffector responses using populations of MCs. We determined the number, distribution, migration and differentiation of MCs. We testedthe pathological consequences of our mutants with a passive systemic anaphylaxis (PSA) model.in vivoRESULTSMunc18-2 was the main isoform expressed in MCs followed by Munc18-3 and -1. Electrophysiological recordings showed impairedexocytosis only in the MCs from Munc18-2Δ/Δ, and an intermediate defect in the Munc18-2+/- MCs, indicating that Munc18-2 is a limitingfactor in this process and ruling out a redundant function of the three proteins in regulated exocytosis. A severe defect in granule toplasma membrane and granule to granule fusion was observed in EM studies of Munc18-2Δ/Δ MCs, meaning that Munc18-2 plays asignificant role in compound exocytosis also. The secretion assays showed a selective defect in regulated exocytosis on the Munc18-2Δ/Δ.Although mice lacking Munc18-2 in their MCs showed a normal development and distribution of their MCs, they had a blunted responseto the PSA model, corroborating that this MC-exclusive pure functional defect has an important impact on the anaphylactic reaction.CONCLUSIONSAlthough other isoforms are expressed in MCs, Munc18-2 is the only one involved in MC degranulation probably by its interaction withSyntaxin-3. Munc18-2 is critical for regulated exocytosis, including compound exocytosis, but not constitutive exocytosis in MCs. MCsregulated exocytosis is required for a full anaphylactic response.