Wever P C, Boonstra J G, Laterveer J C, Hack C E, van der Woude F J, Daha M R, ten Berge I J
Renal Transplant Unit, Academic Medical Center, University of Amsterdam, The Netherlands.
Transplantation. 1998 Jul 27;66(2):259-64. doi: 10.1097/00007890-199807270-00021.
Graft-infiltrating T-cell (GIC) lines cultured from biopsies obtained during acute renal allograft rejection exhibit donor-specific cytotoxicity toward proximal tubular epithelial cell (PTEC) lines cultured from corresponding biopsies. This system allows for study of the relative contributions of perforin/granzyme B (GrB)- and Fas ligand (FasL)-based cytotoxicity to killing of PTEC.
Expression of perforin, GrB and FasL was analyzed by immunocytochemical staining of cytocentrifuge preparations of GIC lines cultured from 10 renal allograft biopsies. Specific inhibitors of the perforin/GrB- and FasL-based pathways were used in 51Cr release and apoptosis assays to determine their relative contributions to cytotoxicity of GIC lines toward corresponding donor PTEC lines.
Cells with a strong granular pattern were observed upon immunocytochemical staining of GIC lines with anti-perforin or anti-GrB monoclonal antibodies. A diffuse staining pattern was observed upon staining with anti-FasL polyclonal antibodies. Six of eight GIC lines cultured from biopsies with acute rejection showed cytotoxicity toward corresponding donor PTEC lines, whereas two GIC lines cultured from biopsies without rejection did not. Preincubation of cytotoxic GIC lines with concanamycin A, an inhibitor of the perforin/GrB-based pathway, caused inhibition of both lysis and apoptosis of PTEC. Inhibition was not observed upon incubation with monoclonal antibodies that inhibit Fas.
The perforin/GrB-based pathway is mainly responsible for cytotoxicity of GIC lines toward corresponding donor PTEC lines, suggesting that this pathway predominates in tubular epithelial cell destruction by cytotoxic T lymphocytes during acute renal allograft rejection in vivo.
从急性肾移植排斥反应期间获取的活检组织中培养出的移植物浸润性T细胞（GIC）系，对从相应活检组织中培养出的近端肾小管上皮细胞（PTEC）系表现出供体特异性细胞毒性。该系统有助于研究穿孔素/颗粒酶B（GrB）和Fas配体（FasL）介导的细胞毒性对PTEC杀伤作用的相对贡献。
通过对从10例肾移植活检组织中培养出的GIC系的细胞离心涂片进行免疫细胞化学染色，分析穿孔素、GrB和FasL的表达。在51Cr释放试验和凋亡试验中使用基于穿孔素/GrB和FasL途径的特异性抑制剂，以确定它们对GIC系对相应供体PTEC系细胞毒性的相对贡献。
用抗穿孔素或抗GrB单克隆抗体对GIC系进行免疫细胞化学染色时，观察到细胞呈现强烈的颗粒状染色模式。用抗FasL多克隆抗体染色时观察到弥漫性染色模式。从急性排斥反应活检组织中培养出的8个GIC系中有6个对相应供体PTEC系表现出细胞毒性，而从无排斥反应活检组织中培养出的2个GIC系则没有。用基于穿孔素/GrB途径的抑制剂ConcA预处理具有细胞毒性的GIC系，可导致PTEC的裂解和凋亡均受到抑制。用抑制Fas的单克隆抗体孵育时未观察到抑制作用。
基于穿孔素/GrB的途径主要负责GIC系对相应供体PTEC系的细胞毒性，这表明该途径在体内急性肾移植排斥反应期间细胞毒性T淋巴细胞对肾小管上皮细胞的破坏中起主导作用。
