Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Abstract

Mast cell degranulation is a highly regulated, calciumdependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis. Mast cells (MCs)2 participate in adaptive and innate immune responses. Their secreted products play important roles in immunoglobulin E (IgE)-dependent inflammatory reactions such as allergic asthma and anaphylaxis (1) and are also involved in other forms of inflammation such as immune arthritis (2, 3) and innate immune responses to bacterial infections (4, 5). Upon activation, MCs exhibit three main secretory responses: release of granule contents (i.e. degranulation), secretion of prostaglandins and leukotrienes, and secretion of cytokines and growth factors (6). The exocytic release of preformed mediators (e.g. histamine and proteases) stored in secretory granules is immediate and regulated at the step of fusion between the membrane of the granule and the plasma membrane. Thus, it is an example of regulated exocytosis, like neuronal synaptic neurotransmitter release and insulin secretion (7). Another early event is the release of metabolites of arachidonic acid (e.g. prostaglandin D 2 (PGD 2 ) and leukotriene C 4 (LTC 4 )). These eicosanoids cross the plasma membrane using transmembrane transporters (8), and their production is regulated by the activation of their synthetic enzymes (9). A late response after MC activation is the secretion of cytokines and growth factors (e.g. tumor necrosis factor-␣ (TNF-␣) and interleukin-4 (IL-4)). The gap in time of minutes to hours between stimulation and the secretion of these mediators is explained by the fact that regulation is at the transcriptional and post-transcriptional levels, with secretion occurring via constitutive exocytosis (10).A common intracellular mediator linking the stimulation event to these three MC responses is calcium (Ca 2ϩ ) that is released into the cytoplasm from intracellular stores and introduced from the extracellular environment via specialized channels. Increase in the cytoplasmic concentration of Ca 2ϩ ([Ca 2ϩ ] i ) is required for the activation of phospholi...

Role of MARCKS in regulated secretion from mast cells and airway goblet cells

“…Additionally, sensitized BMMCs and eHMCs exhibited robust, concentration-dependent responses to ionomycin and concentration-dependent enhancement of ionomycin-induced secretory responses by PMA (data not shown). The magnitude of secretory responses observed, the concentration dependencies, including the domed shape of the DNP-BSA concentration-response curves, and the enhanced secretory responses of adherent cells are all consistent, qualitatively and quantitatively, with reports in the literature from other laboratories (19,25,34,46,54).…”

“…Consequently, both cell types depend on the SNARE and SM proteins of the core vesicle trafficking machinery for exocytosis (11,23,30,53), and exocytosis for both is Ca 2ϩ dependent (33,34). From the data in hand, it is likely that the exocytic machinery in these two cells is more similar than different and that any differences are most likely to be due to differences in the specific isoforms involved in the SNARE/SM complex and its regulation, and in second messenger pathways, e.g., how intracellular Ca 2ϩ is handled (11,30).…”

Inhibitory effect of a new orally active cedrol-loaded nanostructured lipid carrier on compound 48/80-induced mast cell degranulation and anaphylactic shock in mice

“…61,62 To investigate the mechanisms of cedrol and CR-NLC in histamine release inhibition, intracellular calcium levels were examined. Upon treatment with compound 48/80 (4 µg/mL), the intracellular calcium of mast cells gets stimulated which are presented in Figure 8A-C. Preincubation of cromolyn sodium ( Figure 8A), cedrol ( Figure 8B), and CR-NLC ( Figure 8C) decreased intracellular calcium Systemic anaphylaxis was induced in mice by IP injection of 10 mg/kg of C-48/80.…”

The impact of bacterial infection on mast cell degranulation

“…While BMMCs contain three granule populations, RBL-2H3 mast cells only possess two distinct granule populations [26]. Additionally, RBL-2H3 mast cells are mucosal-like while the BMMCs from the Syt2 knock-out mice are connective tissue-type mast cells [50,51]. Interestingly, Syt2 in RBL-2H3 mast cells appears to be localized on the lysosomes rather than on the secretory granules.…”

“…Overexpression of Syt1 and Syt2 in RBL-2H3 mast cells results in increased b-hexosaminidase release when compared with the control [47,49], suggesting that Syt1 and Syt2 are negative regulators of degranulation. However, bone marrow-derived mast cells (BMMC) from Syt2 knockout mice showed 70% decrease in the secretion of histamine and b-hexosaminidase [50], suggesting that Syt2 functions as a positive regulator of degranulation. The difference of granule composition between RBL-2H3 and BMMCs might explain such inconsistencies.…”