Logan M R, Lacy P, Odemuyiwa S O, Steward M, Davoine F, Kita H, Moqbel R
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, AB, Canada, and Department of Medicine and Immunology, Mayo Clinic, Rochester, MN, USA.
Allergy. 2006 Jun;61(6):777-84. doi: 10.1111/j.1398-9995.2006.01089.x.
Granulocyte exocytosis is proposed to be critically dependent on the interaction of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) located on granules/vesicles (v-SNAREs) and plasma membrane (t-SNAREs). Previous studies indicated that the v-SNARE, vesicle-associated membrane protein (VAMP)-2, as well as t-SNAREs (SNAP-23, syntaxin-4 and -6) are implicated in exocytosis from human granulocytes. Vesicle-associated membrane proteins-7 and -8 have been implicated in endosome/lysosome trafficking, however, their role in granulocyte exocytosis remains obscure.
We sought to investigate the expression and functional role of SNARE isoforms in the secretion of different granule-derived mediators in human eosinophils and neutrophils.
The expression of SNAREs was determined by subcellular fractionation and flow cytometry. SNARE-specific antibodies were examined for their ability to impair mediator release from permeabilized eosinophils and neutrophils.
Vesicle-associated membrane proteins-7 and -8 were localized to granule and membrane-enriched fractions in eosinophils and neutrophils, whereas syntaxin-6 was not detectable. In permeabilized cells, anti-VAMP-7, but not anti-VAMP-8, antibody impaired the secretion of all mediators examined (in eosinophils, eosinophil peroxidase and eosinophil-derived neurotoxin; in neutrophils, myeloperoxidase, lactoferrin and matrix metalloprotease-9) in a dose-dependent manner. In contrast, anti-VAMP-2 modestly and selectively impaired secretion from small granules and vesicles. Syntaxin-4, but not syntaxin-6, was found to interact with SNAP-23 and was partially involved in mediator secretion from multiple compartments.
Our observations indicate for the first time a critical role for VAMP-7 in both eosinophil and neutrophil mediator release.
粒细胞胞吐作用被认为严重依赖于位于颗粒/囊泡（v-SNAREs）和质膜（t-SNAREs）上的可溶性N-乙基马来酰亚胺敏感因子附着蛋白（SNAP）受体（SNAREs）之间的相互作用。先前的研究表明，v-SNARE、囊泡相关膜蛋白（VAMP）-2以及t-SNAREs（SNAP-23、 syntaxin-4和-6）参与人粒细胞的胞吐作用。囊泡相关膜蛋白-7和-8参与内体/溶酶体运输，然而，它们在粒细胞胞吐作用中的作用仍不清楚。
我们试图研究SNARE亚型在人嗜酸性粒细胞和中性粒细胞不同颗粒来源介质分泌中的表达及功能作用。
通过亚细胞分级分离和流式细胞术测定SNAREs的表达。检测SNARE特异性抗体损害通透化嗜酸性粒细胞和中性粒细胞介质释放的能力。
囊泡相关膜蛋白-7和-8定位于嗜酸性粒细胞和中性粒细胞的颗粒和富含膜的部分，而未检测到syntaxin-6。在通透化细胞中，抗VAMP-7抗体而非抗VAMP-8抗体以剂量依赖性方式损害所有检测介质的分泌（在嗜酸性粒细胞中为嗜酸性粒细胞过氧化物酶和嗜酸性粒细胞衍生神经毒素；在中性粒细胞中为髓过氧化物酶、乳铁蛋白和基质金属蛋白酶-9）。相比之下，抗VAMP-2适度且选择性地损害小颗粒和囊泡的分泌。发现syntaxin-4而非syntaxin-6与SNAP-23相互作用，并部分参与多个区室的介质分泌。
我们的观察首次表明VAMP-7在嗜酸性粒细胞和中性粒细胞介质释放中起关键作用。
