Verbij Fabian C, Sorvillo Nicoletta, Kaijen Paul H P, Hrdinova Johana, Peyron Ivan, Fijnheer Rob, Ten Brinke Anja, Meijer Alexander B, van Alphen Floris P J, van den Berg Timo K, Graversen Jonas J H, Moestrup Soren K, Voorberg Jan
Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.
Department of Hematology, University Medical Center Utrecht, Utrecht, The Netherlands.
Blood Adv. 2017 Jan 16;1(5):293-305. doi: 10.1182/bloodadvances.2016001321. eCollection 2017 Jan 24.
Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.
巨噬细胞对ADAMTS13的内化作用可能有助于其从循环中清除。在此，我们研究了促成巨噬细胞摄取ADAMTS13的内吞机制。使用人单核细胞衍生的巨噬细胞，通过流式细胞术监测荧光标记的重组ADAMTS13的摄取情况。添加细胞可渗透的发动蛋白抑制剂dynasore后，ADAMTS13的内化作用被阻断。使用甘露聚糖可观察到ADAMTS13摄取的部分阻断；然而，摄取不受阻断与巨噬细胞甘露糖受体CD206结合的抗体的影响，这表明其他内吞受体有助于巨噬细胞对ADAMTS13的内化。用ADAMTS13进行下拉实验并随后进行质谱分析，确定I类清道夫受体CD163为ADAMTS13的候选受体。用单克隆抗CD163抗体EDHu-1进行阻断实验，导致巨噬细胞对ADAMTS13的内化作用降低。在高表达CD163的巨噬细胞中观察到EDHu-1对ADAMTS13摄取有明显抑制作用。与这些发现一致，表达CD163的中国仓鼠卵巢细胞能够快速内化ADAMTS13。表面等离子体共振显示ADAMTS13与CD163的富含半胱氨酸的清道夫受体结构域1-9和1-5结合。综上所述，我们的数据确定CD163是巨噬细胞上ADAMTS13的主要内吞受体。
