Ohnishi H, Lin K M, Chu T M
Department of Diagnostic Immunology Research and Biochemistry, Roswell Park Memorial Institute, Buffalo, New York 14263.
Cancer Res. 1990 Feb 15;50(4):1107-12.
We reported previously using a murine model that the kidney is the organ involved in catabolism of exogenous human recombinant interleukin 2 (IL-2) and that cathepsin D, a major renal acid protease, is responsible for the degradation of IL-2. In the present report also using BALB/c mice we have investigated the effect of in vivo pepstatin, an acid protease inhibitor, treatment on serum half-life of IL-2, and generation of lymphokine-activated killer (LAK) cell activity. The in vivo pepstatin treatment by i.p. injection resulted in a significant reduction in the accumulation of 125I-IL-2 by the kidney in a reverse dose-response manner. Pepstatin treatment prolonged the serum half-life of 125I-IL-2, and the increase in serum half-life of 125I-IL-2 was pepstatin dose dependent. A significant reduction in renal cathepsin D activity, as monitored by the degradation of 125I-IL-2, was detected. In vivo pepstatin (0.6 mg/kg) treatment along with IL-2 (300,000 IU/mouse) daily for 3 or 6 days resulted in an augmentation of natural killer activity exhibited by freshly prepared and uncultured splenocytes against YAC-1 cells. An additional culturing of the splenocytes with IL-2 (3,000 IU/ml) in vitro for 1 day significantly enhanced the effect of in vivo pepstatin treatment; i.e., LAK cell activity generated from the splenocytes of animals treated with IL-2 plus pepstatin was greatly augmented in comparison with that treated with IL-2 alone. Phenotypic assessment by cell surface markers (Thy-1.2, Lyt-2, L3T4, and asialo-GM1) on the fresh splenocytes prepared from animals treated in vivo with pepstatin plus IL-2 revealed a decrease in the percentage of cells expressing Thy-1.2 and Lyt-2 and an increase in those carrying asialo-GM1. These results demonstrated that, as a result of in vivo pepstatin treatment, renal cathepsin D activity was greatly inhibited, which in turn reduced the degradation of circulating IL-2, then prolonged serum half-life of IL-2, and subsequently augmented natural killer and LAK cell activity. The in vivo pepstatin and IL-2 treatment decreased the T-cells and increased the natural killer-like LAK precursor cells, possibly also with an increase in its activity, which were further induced by in vitro IL-2 culture to generate an augmented LAK cell activity. This study also suggests the clinical potential of pepstatin in IL-2-related immunotherapy.
我们之前曾报道，利用小鼠模型发现肾脏是参与外源性人重组白细胞介素2（IL-2）分解代谢的器官，且组织蛋白酶D（一种主要的肾脏酸性蛋白酶）负责IL-2的降解。在本报告中，同样使用BALB/c小鼠，我们研究了体内使用酸性蛋白酶抑制剂胃酶抑素治疗对IL-2血清半衰期以及淋巴因子激活的杀伤（LAK）细胞活性产生的影响。通过腹腔注射进行体内胃酶抑素治疗，导致肾脏对125I-IL-2的摄取量显著降低，呈反向剂量反应关系。胃酶抑素治疗延长了125I-IL-2的血清半衰期，且125I-IL-2血清半衰期的延长呈胃酶抑素剂量依赖性。通过监测125I-IL-2的降解发现肾脏组织蛋白酶D活性显著降低。体内胃酶抑素（0.6毫克/千克）与IL-2（300,000国际单位/只小鼠）联合每日治疗3天或6天，导致新鲜制备且未经培养的脾细胞对YAC-1细胞表现出的自然杀伤活性增强。脾细胞在体外与IL-2（3,000国际单位/毫升）再培养1天，显著增强了体内胃酶抑素治疗的效果；即与单独用IL-2治疗的动物相比，用IL-2加胃酶抑素治疗的动物脾细胞产生的LAK细胞活性大大增强。对体内用胃酶抑素加IL-2治疗的动物制备的新鲜脾细胞进行细胞表面标志物（Thy-1.2、Lyt-2、L3T4和去唾液酸GM1）的表型评估，结果显示表达Thy-1.2和Lyt-2的细胞百分比降低，而携带去唾液酸GM1的细胞百分比增加。这些结果表明，体内胃酶抑素治疗的结果是肾脏组织蛋白酶D活性受到极大抑制，这反过来减少了循环中IL-2的降解，进而延长了IL-2的血清半衰期，随后增强了自然杀伤和LAK细胞活性。体内胃酶抑素和IL-2治疗减少了T细胞数量，增加了自然杀伤样LAK前体细胞数量，其活性可能也有所增加，体外IL-2培养进一步诱导这些细胞产生增强的LAK细胞活性。本研究还提示胃酶抑素在与IL-2相关的免疫治疗中的临床应用潜力。
