Carmo Lívia A S, Bonjour Kennedy, Ueki Shigeharu, Neves Josiane S, Liu Linying, Spencer Lisa A, Dvorak Ann M, Weller Peter F, Melo Rossana C N
Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil;
Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan;
J Leukoc Biol. 2016 Aug;100(2):391-401. doi: 10.1189/jlb.3A1015-480R. Epub 2016 Mar 10.
Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.
嗜酸性粒细胞活化导致预先合成的、储存于颗粒中的介质分泌，这些介质决定了过敏、炎症和免疫调节反应的进程。CD63是跨膜4糖蛋白超家族（四跨膜蛋白）的成员，存在于嗜酸性粒细胞特异性（分泌性）颗粒的界膜上，被认为是嗜酸性粒细胞脱颗粒的潜在表面标志物。然而，CD63在嗜酸性粒细胞和其他白细胞中的细胞内分泌运输情况尚不清楚。在这里，我们对炎症刺激物CCL11和肿瘤坏死因子α刺激的人类嗜酸性粒细胞内的CD63运输进行了高分辨率的全面研究，这两种刺激物分别在嗜酸性粒细胞中诱导截然不同的分泌过程：逐个脱颗粒和复合胞吐作用。通过使用不同的透射电子显微镜方法，包括免疫纳米金技术，以增强亚细胞区室中CD63的检测，我们确定了一个主要的细胞内CD63池，它与嗜酸性粒细胞脱颗粒事件直接相关。透射电子显微镜定量分析表明，在刺激下，CD63集中在通过逐个脱颗粒或复合胞吐作用进行分泌的颗粒内，并且CD63随着细胞质中囊泡和颗粒的移动而移动。尽管刺激后在细胞表面观察到了CD63，但免疫纳米金电子显微镜显示，细胞质中仍有大量的CD63池。值得注意的是，CCL11和肿瘤坏死因子α触发了CD63(+)大囊泡管状载体（嗜酸性粒细胞墨西哥帽小泡）形成增加，这些载体在分泌过程中与颗粒融合，可能在CD63的细胞内转运中起作用。总之，我们确定了与嗜酸性粒细胞颗粒衍生分泌途径相关的活跃的细胞内CD63运输。这对于理解免疫反应中嗜酸性粒细胞复杂的分泌活动很重要。
