Role of SNARE isoforms in exocytosis of stored mediators from human granulocytes

It is postulated that the release of stored mediators from eosinophils and neutrophils is a critical element contributing to the pathogenesis of pulmonary diseases such as asthma. Pre-formed mediators in these cells are stored within cytoplasmic storage granules and small secretory vesicles and released by the process of exocytosis. Several stored mediators have been shown to damage airway epithelium, promote airway constriction and activate other inflammatory cells. The intracellular molecules that are essential for regulating mediator secretion from these cells remain poorly understood.; The formation of a SNARE (SNAP receptor) complex during secretory/granule docking at the plasma membrane has been identified as an essential step for exocytosis. The synaptic SNARE complex is comprised of one vesicular (v-) SNARE, VAMP-2 (vesicle associated membrane protein) and two target (t-) SNAREs: syntaxin-1 and SNAP-25 (synaptosome-associated protein of 25 kDa). Botulinum and tetanus toxins impair exocytosis by specifically cleaving SNAREs, and preventing a functional SNARE complex. It is postulated that distinct SNARE isoforms may, in part, regulate the specificity of vesicle/granule trafficking. The research objective of this project was to investigate the expression and functional role of candidate SNARE isoforms in exocytosis of pre-formed mediators from eosinophils and neutrophils.; Our results demonstrated that the v-SNARE, VAMP-2, was expressed on small cytoplasmic vesicles but not large granules in eosinophils or neutrophils. In contrast, tetanus-insensitive VAMP (TI-VAMP) and endobrevin (VAMP-8) were expressed on large granules, in addition to membrane fractions containing secretory vesicles. Antibody-mediated inhibition of VAMP-2 in permeabilized cells supported the notion that this v-SNARE is primarily involved in vesicle-mediated trafficking. TI-VAMP, but not VAMP-8, antibodies impaired the release of mediators from more than one granule compartment, suggesting that TI-VAMP is a common element in exocytosis. The t-SNAREs, SNAP-23 and syntaxin-4, were expressed on plasma membranes and shown to interact with each other. Antibody-mediated inhibition of syntaxin-4 suppressed the release of all stored mediators examined, indicating a central role for this isoforms in exocytosis from human granulocytes. In contrast to a previous study, our data did not indicate a significant role for the t-SNARE, syntaxin-6 in exocytosis from either eosinophils or neutrophils. These observations suggest that specific SNARE isoforms play distinct roles in mediator release from human granulocytes. SNAREs and/or regulators of their assembly are, therefore, candidates for targeted inhibition which may prove to be a novel and effective anti-inflammatory treatment strategy.