Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.

唾液酸黏附素（Sialoadhesin）仅特异性表达于巨噬细胞的特定亚群。鉴于唾液酸黏附素阳性巨噬细胞参与多发性硬化症等炎症性自身免疫疾病，且可能参与免疫应答的生成，通过唾液酸黏附素特异性免疫偶联物靶向递送药物、毒素或抗原，或可成为一种极具应用价值的治疗策略。最初，唾液酸黏附素被鉴定为一种淋巴细胞黏附分子，而近期研究证实其可介导携带唾液酸的病原体的内化过程，这提示唾液酸黏附素是一种内吞受体。本研究显示，猪源唾液酸黏附素特异性抗体及F(ab')2片段，可在原代猪巨噬细胞与表达重组猪唾液酸黏附素的细胞中触发唾液酸黏附素的内化过程。通过化学抑制剂、双重免疫荧光染色及显性负突变体构建体实验，我们证实猪源唾液酸黏附素的内化依赖网格蛋白（clathrin）与Eps15，且其靶向早期内体而非溶酶体。除表征唾液酸黏附素的内吞机制外，本研究还评估了两种唾液酸黏附素特异性免疫偶联物：我们发现猪源唾液酸黏附素特异性免疫毒素可高效杀伤表达唾液酸黏附素的巨噬细胞；此外，猪源唾液酸黏附素特异性白蛋白免疫偶联物可在巨噬细胞中被内化，且与仅使用白蛋白免疫接种相比，使用此类免疫偶联物进行免疫接种，可快速且强效地诱导产生白蛋白特异性抗体。综上，本研究拓展了唾液酸黏附素的功能认知，证实其可作为内吞受体发挥功能；这一特性不仅可被携带唾液酸的病原体所利用，同时也可用于将毒素或抗原靶向递送至表达唾液酸黏附素的巨噬细胞。