Luo Lei, Lu Jun, Wei Liang, Long Dan, Guo Jia Y, Shan Juan, Li Fu S, Lu Ping Y, Li Ping Y, Feng Li
Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China.
BMC Cell Biol. 2010 Nov 23;11:91. doi: 10.1186/1471-2121-11-91.
Human major histocompatibility complex class I-related chain A (MICA) plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI) could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity.
We find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2) cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased.
These results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is through a HIF-1 pathway. The increased IFNgamma secretion and enhanced NK cell cytotoxicity was mainly due to the surface expression of MICA induced by over-expression of HIF-1alpha. This study enhances our understanding of MICA activation mechanisms during kidney transplantation and provides insights into how IRI can influence transplant outcome. Moreover, these findings might be also important for developing strategies to reduce the effect of MICA in kidney transplant outcomes in the future.
人类主要组织相容性复合体 I 类相关链 A（MICA）在适应性免疫和先天性免疫反应中发挥双重作用。越来越多的证据表明，MICA 与急性和慢性肾移植排斥反应密切相关。因此，了解 MICA 的激活机制对肾移植很重要。我们之前证明，缺血/再灌注损伤（IRI）可上调小鼠肾移植组织上的 MICA 表达。由于缺氧诱导因子-1（HIF-1）是 IRI 期间细胞对缺氧适应性反应的主要调节因子，在此我们研究 HIF-1 是否可上调 MICA 表达及其对自然杀伤（NK）细胞细胞毒性的影响。
我们发现 HIF-1α在缺氧/复氧过程中上调 MICA 表达、诱导γ干扰素（IFNγ）分泌及 NK 细胞细胞毒性方面发挥重要作用。首先，我们构建了表达 HIF-1αΔODD 的腺病毒，以在常氧条件下在人肾近端小管上皮（HK-2）细胞中稳定且功能性地表达 HIF-1α。HK-2 细胞中 HIF-1α的过表达诱导 MICA 表达，并增强 NK 细胞对表达 HIF-1α细胞的细胞毒性活性。其次，我们使用缺氧/复氧细胞模型在体外模拟 IRI，发现 RNA 干扰（RNAi）抑制 HIF-1α可诱导 MICA 表达下调并抑制 NK 细胞毒性。在抗体阻断实验中，抗 MICA 单克隆抗体（mAb）能够下调 NK 细胞对过表达 HIF-1α的 HK-2 细胞的细胞毒性活性。此外，当 NK 细胞与表达由 HIF-1α过表达上调 MICA 的 HK-2 细胞共培养时，IFNγ分泌显著增加。在存在阻断性 MICA mAb 的情况下，IFNγ分泌显著减少。
这些结果表明，缺氧/复氧促进 HK-2 细胞上 MICA 的表达是通过 HIF-1 途径。IFNγ分泌增加和 NK 细胞细胞毒性增强主要归因于 HIF-1α过表达诱导的 MICA 表面表达。本研究增进了我们对肾移植过程中 MICA 激活机制的理解，并为 IRI 如何影响移植结果提供了见解。此外，这些发现对于未来制定降低 MICA 对肾移植结果影响的策略可能也很重要。
