Sarkhosh Kourosh, Tredget Edward E, Karami Ali, Uludag Hasan, Iwashina Takashi, Kilani Ruhangiz T, Ghahary Aziz
Department of Surgery, University of Alberta, Edmonton, Alberta T6G 2E1, Canada.
J Cell Biochem. 2003 Sep 1;90(1):206-17. doi: 10.1002/jcb.10593.
Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment.
吲哚胺2,3-双加氧酶（IDO）是一种色氨酸分解代谢酶，是一种具有多种免疫抑制特性的细胞内酶。在此，我们报告了该酶可能用于抑制与表达IDO的同种异体皮肤替代物成纤维细胞共培养的免疫细胞增殖。将包埋在牛胶原蛋白中的胎儿皮肤成纤维细胞用细胞因子γ干扰素（IFN-γ）处理，以诱导IDO mRNA和蛋白的表达。通过Northern分析评估IDO mRNA的表达。通过测量未处理和处理过的成纤维细胞中犬尿氨酸和色氨酸水平来评估IDO酶活性。Northern分析结果显示，响应于所使用的各种浓度的IFN-γ，IDO mRNA的表达呈剂量依赖性增加。与对照相比，作为IDO生物活性所测量的犬尿氨酸和色氨酸水平在经IFN-γ处理的成纤维细胞中存在显著差异（P < 0.001）。在持续效应实验中，在去除IFN-γ后32小时内，IDO mRNA的表达逐渐降低至无法检测的水平。然而，蛋白质印迹分析结果显示，相对于mRNA表达，IFN-γ对IDO蛋白水平的持续效应显著更长（192小时）。为了证明IDO对免疫细胞增殖的免疫抑制作用，将表达IDO的成纤维细胞与外周血单个核细胞（PBMC）共培养5天。（3）H-胸腺嘧啶核苷掺入结果显示，与与不表达IDO的成纤维细胞共培养的PBMC相比，与表达IDO的成纤维细胞共培养时PBMC的增殖显著降低（P < 0.001）。此外，添加IDO抑制剂（1-甲基-d-色氨酸）以剂量依赖性方式逆转了IDO对PBMC增殖的抑制作用。为了测试与表达IDO的成纤维细胞共培养的免疫细胞的活力，对PI染色的PBMC进行了流式细胞术分析，并且在这些细胞与对照之间未发现显著差异。在另一组实验中，我们表明表达IDO的成纤维细胞的迁移率和随后的增殖也与对照细胞相同。总之，包埋在胶原凝胶中的表达IDO的同种异体成纤维细胞抑制同种异体免疫细胞的增殖，同时它们在这种由IDO诱导的色氨酸缺乏的培养环境中仍然保持活力。
