VAMP 3 and 8 Define Distinct Vesicle Pools that Regulate the Secretion of Inflammatory Mediators in Human Mast Cells

Abstract

Mast cells form an integral part of both innate and adaptive immunity, playing an important role in the inflammatory response. Adverse reaction to allergens can lead to activation of mast cells, causing degranulation and release of a range of pro-inflammatory mediators contributing to the onset of allergy. Mast cells have been further implicated in many other diseases associated with aberrant mediator release, including irritable bowel disease. Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins have been identified as essential proteins for the exocytosis of vesicles containing these mediators. Inhibiting secretion though disruption of SNARE-mediated vesicle fusion could provide a novel therapeutic strategy for a variety of inflammatory diseases.The aim of this study was to identify and characterise SNARE proteins involved in the release of inflammatory mediators in human mast cells. using LAD 2 human mast cells, PCR identified expression of a variety of Syntaxins and VAMPS, as well as the ubiquitously expressed SNAP 23.To study the roles of individual VAMPS in exocytosis a novel technique utilising pH sensitive pHluorins was developed. using VAMPS tagged with pHluorins, we have studied the cellular distribution of VAMP 3 and VAMP 8 containing vesicles, and their behaviour upon IgE stimulation in live cells. In unstimulated cells, VAMPS 3 and 8 were found to have distinct cellular distributions. Upon IgE stimulation both VAMP 3 and VAMP 8 containing vesicles translocate to the membrane and undergo membrane fusion, consistent with roles in exocytosis. However, their responses show distinct time courses and calcium dependences. The data presented here supports the notion that distinct vesicle pools, defined in part by expression of VAMP 3 and VAMP 8, regulate the release of inflammatory mediators from mast cells.