Shimizu Y, Hata K, Herberman R B, Whiteside T L
Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Japan.
J Immunol Methods. 1993 Aug 26;164(1):69-77. doi: 10.1016/0022-1759(93)90277-e.
Previous studies on lymphocyte cytotoxicity against autologous human hepatocytes had been studied using Terasaki plates, in which dead hepatocytes after incubation with lymphocytes were counted visually. No studies with 51Cr-labeled human hepatocytes as targets have been reported, although it can give us more objective results. In the present study, we established procedures for labeling human hepatocytes with 51Cr and for measuring cytotoxicity of freshly isolated liver-infiltrating lymphocytes (LIL) against 51Cr-labeled human autologous hepatocytes. Hepatocytes were isolated from diseased and 'normal' liver tissues, cultured overnight, and labeled with 51Cr 'in situ' in the wells of 96-well round bottom plates. Human hepatocytes isolated from either 'normal' or diseased liver tissues labeled well with 51Cr, and mean spontaneous 51Cr release was less than 15% in the presence of 5% fetal bovine serum or human AB serum. Freshly isolated LIL obtained from chronic viral hepatitis but not from other liver diseases showed cytotoxicity against 51Cr-labeled autologous hepatocytes in 4 h 51Cr release assays, and the percent specific lysis was linearly related to the E:T cell ratio. LIL from viral hepatitis were able to mediate natural killer (NK) activity against K562 targets, lymphokine-activated killer-like activity against Daudi cells, and lectin-dependent cellular cytotoxicity. Two-color flow cytometry analysis showed that these LIL contained both CD3+ T cells and CD3-CD56+ NK cells. This is the first report which examined the cytotoxicity of liver-infiltrating lymphocytes against 51Cr-labeled human hepatocytes, and it will be useful in assessing local immune response against autologous hepatocytes in chronic liver diseases.
以往关于淋巴细胞对自体人肝细胞细胞毒性的研究是使用寺崎板进行的，在该实验中，与淋巴细胞孵育后的死亡肝细胞通过肉眼计数。虽然使用51Cr标记的人肝细胞作为靶标能给出更客观的结果，但尚未有相关研究报道。在本研究中，我们建立了用51Cr标记人肝细胞以及测量新鲜分离的肝浸润淋巴细胞（LIL）对51Cr标记的人自体肝细胞细胞毒性的方法。从患病和“正常”肝组织中分离肝细胞，培养过夜，并在96孔圆底板的孔中“原位”用51Cr标记。从“正常”或患病肝组织中分离的人肝细胞用51Cr标记良好，在5%胎牛血清或人AB血清存在的情况下，平均自发51Cr释放率小于15%。在4小时的51Cr释放试验中，从慢性病毒性肝炎而非其他肝病中获得的新鲜分离的LIL对51Cr标记的自体肝细胞表现出细胞毒性，特异性裂解百分比与E:T细胞比值呈线性相关。来自病毒性肝炎的LIL能够介导对K562靶标的自然杀伤（NK）活性、对Daudi细胞的淋巴因子激活的杀伤样活性以及凝集素依赖性细胞毒性。双色流式细胞术分析表明，这些LIL同时含有CD3+ T细胞和CD3-CD56+ NK细胞。这是首篇研究肝浸润淋巴细胞对51Cr标记的人肝细胞细胞毒性的报告，它将有助于评估慢性肝病中针对自体肝细胞的局部免疫反应。
