SNARE isoforms in granule-derived mediator secretion from human eosinophils and neutrophils*1A conserved mechanism for granule docking?

Rationale Exocytosis is dependent on interaction of vesicle (v-) and plasma membrane (t-) SNAREs to form SNARE complexes. SNARE assembly is a prerequisite for secretion, which is specifically blocked by clostridial neurotoxins. We showed that the v-SNARE, VAMP-2, was localized to cytoplasmic vesicles in eosinophils, as shown in neutrophils. The t-SNAREs, SNAP-23 and syntaxin-4 are conserved in hematopoeitic cells and localized to eosinophil plasma membranes. We hypothesized that SNAREs are essential in granule-derived mediator release in eosinophils and neutrophils. Methods Purified human peripheral blood eosinophils and neutrophils were preincubated with or without inhibitory antibodies in the presence of the permeabilization agent, streptolysin-O, then stimulated with Ca2+ and GTPγS and supernatants examined for mediator release. Syntaxin-4 immunoprecipitates were examined for binding with SNARE isoforms. Expression of SNARE isoforms was determined using RT-PCR analysis. Protein expression was determined by Western blot and confirmed by FACS analysis of whole cells and granule fractions. Results Anti-syntaxin-4 impaired peroxidase secretion from both granulocytes. In contrast, anti-VAMP-2 blocked only eosinophil peroxidase release. Release of lactoferrin and MMP-9 from secondary and tertiary neutrophil granules, respectively, was modestly reduced by anti-VAMP-2. VAMP-7, -8 and syntaxin-6 mRNA was detected in both granulocyte types. Negligible expression of syntaxin-6 protein was found in both cell types. Analysis of purified CD63+ granules indicated that VAMP-7 is weakly expressed, while significant levels of VAMP-8 expression were detected in both cell types. Conclusions Our observations suggest that mediator release from granulocytes is SNARE-dependent and that subsets of v-SNAREs may be implicated in exocytosis of specific granules.