SNAP-23 and syntaxin-3 are required for chemokine release by mature human mast cells.

Mast cells play a key role in allergic and non-allergic disease by releasing a broad array of mediators. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) are necessary for membrane fusion events during mast cell exocytosis. We have shown recently that the SNAREs SNAP-23, syntaxin (STX)-4, vesicle associated membrane protein (VAMP)-7, and VAMP-8 are required for release of pre-stored histamine by mast cells. Here we analyze the involvement of different SNARE isoforms in exocytosis of de novo synthesized chemokines in mast cells isolated from human intestine. Following IgE receptor cross-linking, mast cells released substantial amounts of the chemokines CXCL8, CCL2, CCL3, and CCL4. Measurement of SNARE mRNA expression revealed only a moderate up-regulation of mRNA for STX-4 after stimulation for 1.5h. Inhibition of SNAP-23 or STX-3 abolished IgE mediated release of the chemokines CXCL8, CCL2, CCL3, and CCL4. In contrast, blocking of STX-2, or VAMP-3 did not affect the chemokine release. Inhibition of STX-4 or VAMP-8 resulted in a reduced release of CXCL8, but not of CCL2, CCL3, or CCL4. Inhibition of STX-6 attenuated the release of CXCL8 and CCL2, inhibition of VAMP-7 that of CCL3. In summary, STX-3 and SNAP-23 are crucial for the release of all chemokines in mature human mast cells whereas other SNAREs affect only release of selected chemokines.

Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells.

Mediator release from mast cells (MC) is a crucial step in allergic and non-allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N-ethylmaleide sensitive factor attachment protein (SNAP)-23, Syntaxin (STX)-1B, STX-2, STX-3, STX-4, and STX-6 but not SNAP-25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)-3, VAMP-7 and VAMP-8 and, in contrast to previous reports about rodent MC, only low levels of VAMP-2. Furthermore, VAMP-7 and VAMP-8 were found to translocate to the plasma membrane and interact with SNAP-23 and STX-4 upon activation. Inhibition of SNAP-23, STX-4, VAMP-7 or VAMP-8, but not VAMP-2 or VAMP-3, resulted in a markedly reduced high-affinity IgE receptor-mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP-7 and VAMP-8, but not VAMP-2, are required for rapid degranulation.