RESUMO
BACKGROUND: Exocytosis of eosinophil granule-derived mediators is thought to be an important effector response contributing to allergic inflammation. Secretion from many cell types has been shown to be dependent on the formation of a docking complex composed of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on the vesicle (v-SNAREs) and the target membrane (t-SNAREs). The SNARE isoforms VAMP-2, SNAP-23, and syntaxin-4 have been described in secretory processes in myeloid cells. Previously, we have demonstrated that the v-SNARE VAMP-2 is a candidate v-SNARE involved in eosinophil exocytosis and is localized to a pool of RANTES-positive vesicles that translocate to the cell periphery after IFN-gamma-induced degranulation. OBJECTIVE: We sought to determine whether eosinophils express the t-SNARE isoforms SNAP-23 and syntaxin-4 as potential binding targets for VAMP-2 during exocytosis. METHODS: Human peripheral blood eosinophils (>97%) from atopic subjects were subjected to RT-PCR and sequence analysis by using specific primers for SNAP-23 and syntaxin-4. Protein expression and localization was determined by means of Western blot analysis of eosinophil subcellular fractions and confirmed with confocal laser scanning microscopy. RESULTS: Nucleotide sequences obtained from PCR products exhibited nearly identical (>95%) homology with reported sequences for human SNAP-23 and syntaxin-4. Both SNAP-23 and syntaxin-4 were present in plasma membranes, with some staining in endoplasmic reticulum and Golgi membranes. Negligible expression was detected in crystalloid and small secretory granules. CONCLUSIONS: The plasma membrane-associated t-SNAREs SNAP-23 and syntaxin-4 are expressed in human eosinophils and are likely candidates for association with VAMP-2 during docking, which is followed by exocytosis. These findings support a role for SNARE molecules in eosinophil mediator release.
