Department of Surgery, Osaka University Graduate School of Medicine, Suita, Japan.
Xenotransplantation. 2018 Sep;25(5):e12396. doi: 10.1111/xen.12396. Epub 2018 Apr 8.
Xenotransplantation is one of the promising strategies for overcoming the shortage of organs available for transplant. However, many immunological obstructions need to be overcome for practical use. Increasing evidence suggests that neutrophils contribute to xenogeneic cellular rejection. Neutrophils are regulated by activation and inhibitory signals to induce appropriate immune reactions and to avoid unnecessary immune reactivity. Therefore, we hypothesized that the development of neutrophil-targeted therapies may have the potential for increased graft survival in xenotransplantation.
A plasmid containing a cDNA insert encoding the human CD31 gene was transfected into swine endothelial cells (SEC). HL-60 cells were differentiated into neutrophil-like cells by culturing them in the presence of 1.3% dimethyl sulfoxide for 48 hours. The cytotoxicity of the differentiated HL-60 cells (dHL-60) and peripheral blood-derived neutrophils was evaluated by WST-8 assays. To investigate the mechanism responsible for hCD31-induced immunosuppression, citrullinated histone 3 (cit-H3) and phosphorylation of SHP-1 were detected by a cit-H3 enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively.
A significant decrease in dHL-60 and neutrophil-mediated cytotoxicity in SEC/hCD31 compared with SEC was seen, as evidenced by a cytotoxicity assay. Furthermore, the suppression of NETosis and the induction of SHP-1 phosphorylation in neutrophils that had been co-cultured with SEC/CD31 were confirmed by cit-H3 ELISA and Western blotting with an anti-phosphorylated SHP-1.
These data suggest that human CD31 suppresses neutrophil-mediated xenogenic cytotoxicity via the inhibition of NETosis. As CD31 is widely expressed in a variety of inflammatory cells, human CD31-induced suppression may cover the entire xenogeneic cellular rejection, thus making the generation of human CD31 transgenic pigs very attractive for use in xenografts.
异种移植是克服可供移植器官短缺的有前途的策略之一。然而，为了实际应用，许多免疫障碍需要克服。越来越多的证据表明，中性粒细胞有助于异种细胞排斥。中性粒细胞通过激活和抑制信号来调节，以诱导适当的免疫反应并避免不必要的免疫反应。因此，我们假设开发针对中性粒细胞的治疗方法可能有潜力增加异种移植中的移植物存活率。
将包含编码人 CD31 基因 cDNA 插入物的质粒转染到猪内皮细胞（SEC）中。通过在存在 1.3%二甲基亚砜的情况下培养 HL-60 细胞 48 小时将 HL-60 细胞分化为类似于中性粒细胞的细胞。通过 WST-8 测定评估分化的 HL-60 细胞（dHL-60）和外周血衍生的中性粒细胞的细胞毒性。为了研究导致 hCD31 诱导免疫抑制的机制，通过 cit-H3 酶联免疫吸附测定（ELISA）和 Western 印迹分别检测了瓜氨酸化组蛋白 3（cit-H3）和 SHP-1 的磷酸化。
通过细胞毒性测定证实，与 SEC 相比，SEC/hCD31 中的 dHL-60 和中性粒细胞介导的细胞毒性显着降低。此外，通过 cit-H3 ELISA 和抗磷酸化 SHP-1 的 Western 印迹证实，与 SEC/CD31 共培养的中性粒细胞中 NETosis 的抑制和 SHP-1 磷酸化的诱导。
这些数据表明，人 CD31 通过抑制 NETosis 抑制中性粒细胞介导的异种细胞毒性。由于 CD31 在各种炎症细胞中广泛表达，因此人 CD31 诱导的抑制可能涵盖整个异种细胞排斥反应，从而使生成人 CD31 转基因猪非常适合用于异种移植物。
