Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.
Blood. 2012 Apr 19;119(16):3828-35. doi: 10.1182/blood-2011-09-377754. Epub 2012 Jan 30.
ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura. The formation of autoantibodies depends on the activation of CD4(+) T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4(+) T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.
ADAMTS13 是一种血浆金属蛋白酶，通过在血管内皮细胞表面切割超大 VWF 多聚体来调节血小板黏附和聚集。针对 ADAMTS13 的自身抗体阻止了 VWF 多聚体的加工，从而引发一种罕见且危及生命的疾病，称为获得性血栓性血小板减少性紫癜。自身抗体的形成取决于 CD4(+) T 细胞的激活。这一过程需要免疫识别、内吞作用，以及随后 ADAMTS13 被加工成肽，由树突状细胞 (DC) 将这些肽呈递到 MHC Ⅱ类分子上，以供 CD4(+) T 细胞识别。在本研究中，我们使用流式细胞术和共聚焦显微镜研究了未成熟单核细胞来源的 DC 对重组 ADAMTS13 的内吞作用。在用荧光标记的 ADAMTS13 孵育 DC 后，观察到 ADAMTS13 的明显摄取。添加 EGTA 和甘露聚糖完全阻断了 ADAMTS13 的内吞作用。在存在针对巨噬细胞甘露糖受体 (MR) 的阻断 mAb 的情况下，ADAMTS13 的内吞作用减少。此外，MR 的 siRNA 沉默降低了 DC 对 ADAMTS13 的摄取。此外，体外结合研究证实了 ADAMTS13 与 MR 的碳水化合物识别结构域的相互作用。本研究的结果表明，ADAMTS13 上的糖基与 MR 相互作用，从而促进 APC 对其的内吞作用。
