Department of Plasma Proteins, Sanquin-AMC Landsteiner Laboratory, Amsterdam, The Netherlands.
J Allergy Clin Immunol. 2012 Feb;129(2):501-9, 509.e1-5. doi: 10.1016/j.jaci.2011.08.029. Epub 2011 Oct 1.
The uptake and processing of blood coagulation factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4(+) T cells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor-related protein-1 (LRP1) have been implicated in FVIII uptake.
This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis.
FVIII uptake was performed with human monocyte-derived and murine bone marrow-derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA-induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm in vitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined.
Upon treatment of DCs with mannan or LRP ligand α2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA-mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 in vivo.
Together, our observations emphasize the physiological significance of KM33-targeted residues within the C1 domain in the uptake of FVIII by DCs in vitro and in vivo.
抗原呈递细胞对凝血因子 VIII（FVIII）的摄取和加工，以及随后将 FVIII 衍生肽呈递给 CD4+T 细胞，指导了血友病 A 患者对 FVIII 的免疫反应。多种受体，包括甘露糖受体和低密度脂蛋白受体相关蛋白-1（LRP1），已被牵连到 FVIII 的摄取中。
本研究旨在研究候选受体在树突状细胞（DC）摄取 FVIII 中的作用。此外，我们还探索了介导内吞作用的 FVIII 残基。
用人单核细胞衍生和鼠骨髓衍生的 DC 进行 FVIII 摄取实验。为了研究 FVIII 的内吞作用，进行了与可溶性受体配体的竞争实验、与重组受体片段的结合研究以及小干扰 RNA 诱导的基因沉默实验。此外，还使用了 FVIII 靶向单克隆抗体 KM33 和 VK34。为了验证体外实验结果，用抗 FVIII 抗体预处理血友病 E17 敲除小鼠，然后测定抗 FVIII 滴度。
在用甘露聚糖或 LRP 配体α2-巨球蛋白处理 DC 后，我们仅观察到 FVIII 内化的轻微减少。此外，在单核细胞衍生的树突状细胞中，用小干扰 RNA 敲低 LRP、甘露糖受体或 DC-SIGN 的表达并不能阻止 FVIII 的摄取。使用 Fc 嵌合体的结合研究表明，LRP、DC-SIGN 和甘露糖受体可以与 FVIII 结合；然而，我们没有观察到这些受体在 FVIII 摄取中的关键作用。先前的研究表明，靶向 FVIII 的 C1（KM33）和 A2（VK34）结构域的人抗体干扰与内吞受体的结合。在 VK34 预孵育 FVIII 后并不影响 FVIII 的摄取；然而，KM33 完全抑制了单核细胞衍生的树突状细胞和骨髓衍生的树突状细胞对 FVIII 的内吞作用。因此，在体内预先给予 KM33 后，抗 FVIII 抗体滴度大大降低。
总的来说，我们的观察结果强调了 C1 结构域中 KM33 靶向残基在 DC 摄取 FVIII 中的生理意义，无论是在体外还是体内。
