ABSTRACT
Mast cells (MCs) participate in allergy, inflammation, and defense against pathogens. They release multiple immune mediators via exocytosis, a process that requires SNARE proteins, including syntaxins (Stxs). The identity of the Stxs involved in MC exocytosis remains controversial. Here, we studied the roles of Stx3 and -4 in fully developed MCs from conditional knockout mice by electrophysiology and EM, and found that Stx3, and not Stx4, is crucial for MC exocytosis. The main defect seen in Stx3-deficient MCs was their inability to engage multigranular compound exocytosis, while leaving most single-vesicle fusion events intact. We used this defect to show that this form of exocytosis is not only required to accelerate MC degranulation but also essential to achieve full degranulation. The exocytic defect was severe but not absolute, indicating that an Stx other than Stx3 and -4 is also required for exocytosis in MCs. The removal of Stx3 affected only regulated exocytosis, leaving other MC effector responses intact, including the secretion of cytokines via constitutive exocytosis. Our in vivo model of passive systemic anaphylaxis showed that the residual exocytic function of Stx3-deficient MCs was sufficient to drive a full anaphylactic response in mice.
Subject(s)
Exocytosis , Mast Cells/cytology , Qa-SNARE Proteins/metabolism , Animals , Cell Count , Cell Degranulation , Cell Differentiation , Gene Knockout Techniques , Kinetics , Mice , Qa-SNARE Proteins/deficiency , Qa-SNARE Proteins/geneticsABSTRACT
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.
Subject(s)
Exocytosis/physiology , Mast Cells/metabolism , Munc18 Proteins/physiology , Anaphylaxis/physiopathology , Animals , Cell Degranulation , Gene Deletion , Mast Cells/ultrastructure , Membrane Fusion/physiology , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Munc18 Proteins/genetics , Patch-Clamp TechniquesABSTRACT
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 Caenorhabditis elegans 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. In vitro 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 (i.e. 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.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Anaphylaxis , Animals , Disease Models, Animal , Exocytosis/physiology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Mast Cells/metabolism , Mast Cells/physiology , Membrane Proteins/genetics , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Protein Isoforms , Protein TransportABSTRACT
BACKGROUND: We developed a study using low dose radioactive iodine creating an animal model of transient elevation of thyroid stimulating hormone (TSH). Male derived bone marrow cells were transplanted to asses their effect on thyroid function and their capability to repair the thyroid parenchyma. RESULTS: At 40 an 80 days after I131 treatment, the study groups TSH and T4 serum values both increased and decreased significantly respectively compared to the negative control group. Eight weeks after cell transplantation, neither TSH nor T4 showed a significant difference in any group. The mean number of SRY gene copies found in group I (Left Intracardiac Transplant) was 523.3 and those in group II (Intrathyroid Transplant) were only 73. Group III (No Transplant) and IV had no copies. Group I presented a partial restore of the histological pattern of rat thyroid with approximately 20%-30% of normal-sized follicles. Group II did not show any histological differences compared to group III (Positive control). CONCLUSION: Both a significant increase of TSH and decrease of T4 can be induced as early as day 40 after a low dose of I131 in rats. Restore of normal thyroid function can be spontaneously achieved after using a low dose RAI in a rat model. The use of BM derived cells did not affect the re-establishment of thyroid function and might help restore the normal architecture after treatment with RAI.
