Department of Medicine, University of Louisville, Louisville, KY 40202, USA.
J Immunol. 2011 Jul 1;187(1):391-400. doi: 10.4049/jimmunol.1003112. Epub 2011 Jun 3.
The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.
采用含有 HIV 转录激活因子（TAT）细胞穿透序列和突触相关蛋白-23（SNAP-23）N 端 SNARE 结构域的融合蛋白（TAT-SNAP-23）来确定胞吐作用在人类中性粒细胞呼吸爆发中的作用。该试剂抑制了分泌囊泡和明胶酶以及特异性颗粒的刺激胞吐作用，但不抑制嗜中性颗粒的胞吐作用。GST 下拉显示 TAT-SNAP-23 与囊泡相关膜蛋白-2 和突触融合蛋白-4 的复合物结合，但不与任一个单独结合。TAT-SNAP-23 使吞噬作用刺激的过氧化氢产生减少了 60％，而不影响吞噬作用或吞噬体中 HOCl 的生成。TAT-SNAP-23 对 fMLF 刺激的超氧化物释放没有影响，但显著抑制了 TNF-α 和血小板激活因子对该反应的引发作用。TAT-SNAP-23 的预处理抑制了 TNF-α 引发的中性粒细胞中 gp91（phox）质膜表达的增加，而 TNF-α 对 ERK1/2 和 p38 MAPK 的激活没有影响。数据表明，中性粒细胞颗粒胞吐作用有助于吞噬作用诱导的呼吸爆发活性，并通过增加 NADPH 氧化酶的膜成分表达在呼吸爆发的引发中起关键作用。
