Clayton Aled, Turkes Attilla, Navabi Hossein, Mason Malcolm D, Tabi Zsuzsanna
Department of Clinical Oncology and Palliative Medicine, Wales College of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff, CF14 2TL, UK.
J Cell Sci. 2005 Aug 15;118(Pt 16):3631-8. doi: 10.1242/jcs.02494. Epub 2005 Jul 26.
Exosomes are nanometer-sized vesicles secreted by a diverse range of live cells that probably have physiological roles in modulating cellular immunity. The extracellular factors that regulate the quantity and phenotype of exosomes produced are poorly understood, and the properties of exosomes that dictate their immune functions are not yet clear. We investigated the effect of cellular stress on the exosomes produced by B-lymphoblastoid cell lines. Under steady-state conditions, the exosomes were positive for hsp27, hsc70, hsp70 and hsp90, and other recognised exosome markers such as MHC class I, CD81, and LAMP-2. Exposing cells to heat stress (42 degrees C for up to 3 hours), resulted in a marked increase in these heat shock proteins (hsps), while the expression of other stress proteins such as hsp60 and gp96 remained negative, and other exosome markers remained unchanged. Stress also triggered a small increase in the quantity of exosomes produced [with a ratio of 1.245+/-0.07 to 1 (mean+/-s.e.m., n=20) of 3-hour-stress-exosomes to control-exosomes]. Flow-cytometric analysis of exosome-coated beads and immuno-precipitation of intact exosomes demonstrated that hsps were located within the exosome lumen, and not present at the exosome-surface, suggesting that such exosomes may not interact with target cells through cell-surface hsp-receptors. Functional studies further supported this finding, in that exosomes from control or heat-stressed B cells did not trigger dendritic cell maturation, assessed by analysis of dendritic-cell-surface phenotype, and cytokine secretion profile. Our findings demonstrate that specific alterations in exosome phenotype are a hitherto unknown component of the cellular response to environmental stress and their extracellular function does not involve the direct activation of dendritic cells.
外泌体是由多种活细胞分泌的纳米级囊泡，可能在调节细胞免疫中发挥生理作用。目前对调节外泌体产生的数量和表型的细胞外因素了解甚少，决定其免疫功能的外泌体特性也尚不清楚。我们研究了细胞应激对B淋巴母细胞系产生的外泌体的影响。在稳态条件下，外泌体对热休克蛋白27（hsp27）、热休克蛋白70前体（hsc70）、热休克蛋白70（hsp70）和热休克蛋白90（hsp90）呈阳性，对其他公认的外泌体标志物如MHC I类分子、CD81和溶酶体相关膜蛋白2（LAMP-2）也呈阳性。将细胞暴露于热应激（42℃，长达3小时）会导致这些热休克蛋白（hsps）显著增加，而其他应激蛋白如热休克蛋白60（hsp60）和糖蛋白96（gp96）的表达仍为阴性，其他外泌体标志物保持不变。应激还引发了所产生外泌体数量的小幅增加[3小时应激外泌体与对照外泌体的比例为1.245±0.07比1（平均值±标准误，n = 20）]。对外泌体包被微珠的流式细胞术分析和完整外泌体的免疫沉淀表明，hsps位于外泌体腔内，而非外泌体表面，这表明此类外泌体可能不会通过细胞表面hsp受体与靶细胞相互作用。功能研究进一步支持了这一发现，即通过分析树突状细胞表面表型和细胞因子分泌谱评估，来自对照或热应激B细胞的外泌体均未触发树突状细胞成熟。我们的研究结果表明，外泌体表型的特定改变是细胞对环境应激反应中一个迄今未知的组成部分，其细胞外功能不涉及树突状细胞的直接激活。
