Pathogenesis of immune-mediated cholangiopathy

在肝移植物抗宿主病（GVHD）中观察到的胆管损伤被认为是一种免疫介导的损伤，尽管其确切机制尚不清楚。然而，最近的研究表明，fas介导的细胞死亡参与了这种免疫介导的胆管病。在本研究中，我们首次证实了正常BALB/c小鼠的胆管细胞原位表达Fas受体，该受体在GVHD小鼠中上调。同时，我们通过免疫细胞化学和免疫印迹法证实了正常BALB/c小鼠离体胆管细胞中Fas蛋白的表达。此外，通过dna阶梯形成和膜联蛋白V染色证实，加入激动性Fas抗体（Jo2）可诱导胆管细胞凋亡。fas缺陷小鼠的胆管细胞（MRL lpr/lpr）未显示jo2诱导的凋亡。干扰素γ分别增强Fas表达和Fas介导的细胞死亡。根据这些观察结果，通过转移B10的脾细胞诱导实验性GVHD。D2小鼠与辐照（800 rad） BALB/c小鼠。来自受体的肝浸润淋巴细胞对从BALB/c小鼠分离的^<51> cr标记的胆管细胞表现出剂量依赖性的细胞毒性。此外，阻断Fas-Fc融合蛋白的加入将细胞毒性降低到44.7%。最后，将这种Fas-Fc蛋白给予BALB/c小鼠，BALB/c小鼠已与B10脾细胞过继转移。D2小鼠体内抑制GVHD的发生。这些结果表明，fas介导的细胞死亡参与了GVHD中观察到的胆管病变，可溶性Fas-Fc蛋白可能具有治疗肝GVHD的潜力。

Bile duct injury observed in hepatic graft versus host disease (GVHD) is regarded as an immune-mediated injury, although its precise mechanism is unclear. However, recent studies have suggested the involvement of Fas-mediated cell death in this immune-mediated cholangiopathy. In this study, we first demonstrated the constitutive expression of Fas receptor by cholangiocytes in situ from normal BALB/c mice, which was upregulated in GVHD mice. Also, we confirmed the Fas protein expression in the isolated cholangiocytes from normal BALB/c mice by immunocytochemistry and immunoblotting. Furthermore, the addition of agonistic Fas antibody (Jo2) induced cholangiocyte apoptosis confirmed by DNA-ladder formation and annexin V staining. Cholangiocytes from Fas-deficient mice (MRL lpr/lpr) did not show Jo2-induced apoptosis. Interferon γ augmented Fas expression and Fas-mediated cell death, respectively. Following these observations, experimental GVHD was induced by transfer of splenocytes from B10.D2 mice to irradiated (800 rad) BALB/c mice. Liver infiltrating lymphocytes from the recipient showed dose-dependent cytotoxicity against ^<51>Cr-labeled cholangiocytes isolated from BALB/c mice. Moreover, the addition of blocking Fas-Fc fusion protein reduced this cytotoxicity to 44.7%. Finally, administration of this Fas-Fc protein to the BALB/c mice, which had been adoptively transferred with splenocytes of B10.D2 mice, prevented the development of hepatic GVHD in vivo. These results demonstrated the involvement of Fas-mediated cell death in cholangiopathy observed in GVHD, and a soluble Fas-Fc protein may have a therapeutic potential for hepatic GVHD.