Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5.
Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5.
复制标题
DOI：
10.1097/shk.0000000000000995
复制
发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Pritts TA
中科院分区：
文献类型：
--
作者：
Kim Y;Abplanalp WA;Jung AD;Schuster RM;Lentsch AB;Gulbins E;Caldwell CC;Pritts TA
微粒是从老化的红细胞中脱落的亚微米小泡，是红细胞(RBC)储存损伤的特征。肺内皮细胞暴露于RBC衍生的微粒子可促进炎症反应，但微粒子诱导内皮细胞激活的机制尚不清楚。本研究用从老化的小鼠红细胞或载体中分离出的微粒处理培养的小鼠肺内皮细胞(MLEC)。微粒子处理的细胞显示黏附分子ICAM和E-选择素以及细胞因子IL-6的表达增加。为了确定微粒子对MLECs的这些影响的机制，用与羧基荧光素琥珀酰亚胺酯(CFSE)共价结合的微粒子处理细胞，并通过流式细胞仪定量细胞对微粒子的摄取。与对照组相比，从15分钟到24小时，CFSE阳性的MLECs比例更高，这表明内皮细胞吞噬了微粒。通过免疫荧光观察到微粒与溶酶体共定位，表明内吞作用和内溶酶体转运。这一过程被内吞作用抑制剂抑制。与无意义的siRNA处理细胞相比，内皮细胞中Rab5信号蛋白的siRNA敲除导致微粒子摄取受损，以及对微粒子处理的炎症反应的减弱。综上所述，这些数据表明，肺内皮细胞内吞RBC来源的微粒导致内皮细胞激活。这一反应似乎部分是由Rab5信号蛋白介导的。
Microparticles are submicron vesicles shed from aging erythrocytes as a characteristic feature of the red blood cell (RBC) storage lesion. Exposure of pulmonary endothelial cells to RBC-derived microparticles promotes an inflammatory response, but the mechanisms underlying microparticle-induced endothelial cell activation are poorly understood. In the present study, cultured murine lung endothelial cells (MLECs) were treated with microparticles isolated from aged murine packed RBCs or vehicle. Microparticle-treated cells demonstrated increased expression of the adhesion molecules ICAM and E-selectin, as well as the cytokine, IL-6. To identify mechanisms that mediate these effects of microparticles on MLECs, cells were treated with microparticles covalently bound to carboxyfluorescein succinimidyl ester (CFSE) and cellular uptake of microparticles was quantified via flow cytometry. Compared with controls, there was a greater proportion of CFSE-positive MLECs from 15 minutes up to 24 hours, suggesting endocytosis of the microparticles by endothelial cells. Co-localization of microparticles with lysosomes was observed via immunofluorescence, indicating endocytosis and endolysosomal trafficking. This process was inhibited by endocytosis inhibitors. SiRNA knockdown of Rab5 signaling protein in endothelial cells resulted in impaired microparticle uptake as compared to nonsense siRNA-treated cells, as well as an attenuation of the inflammatory response to microparticle treatment. Taken together, these data suggest that endocytosis of RBC-derived microparticles by lung endothelial cells results in endothelial cell activation. This response appears to be mediated, in part, by the Rab5 signaling protein.