Expression of human ecto-5'-nucleotidase in pig endothelium increases adenosine production and protects from NK cell-mediated lysis
Expression of human ecto-5'-nucleotidase in pig endothelium increases adenosine production and protects from NK cell-mediated lysis
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DOI：
10.1111/j.1600-6143.2005.00868.x
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发表时间：
2005-06-01
影响因子：
8.8
通讯作者：
Smolenski, RT
中科院分区：
文献类型：
--
作者：
Osborne, FN;Kalsi, KK;Smolenski, RT
ECTO-5‘-核苷酸酶(E5’N)是一种内皮细胞表面酶，控制细胞外核苷酸转化为免疫抑制的腺苷。我们评估了人E5‘N在猪内皮细胞(EC)上的表达是否减弱了人NK细胞介导的细胞毒作用。将人E5‘N稳定导入猪内皮细胞系，用流式细胞仪和细胞内酶释放法检测人NK细胞对培养的猪内皮细胞的黏附和细胞毒作用。未转染组和转染组的E5‘N活性分别从0.68+/-0.07增加到1013+/-293nmol/min/mg蛋白，而完整细胞的AMP/腺苷代谢率从0.37+/-0.05增加到>；300nmol/min/mg蛋白。以三磷酸腺苷为细胞外底物时，转染腺苷的速率也增加。人NK细胞的杀伤活性由未转染猪EC的10.7+/-0.4%和11.1+/-1.1%降至转染组的5.2+/-0.2%和5.0+/-0.2%。E5‘N抑制剂能消除E5’N转染组EC的细胞毒作用，而未转染组的细胞毒作用与腺苷类似，说明E5‘N产生的腺苷在抑制NK细胞杀伤作用中起关键作用。我们认为在转基因猪的EC中过表达E5‘N可能是减轻异种移植后排斥反应的一种策略。
Ecto-5'-nucleotidase (E5'N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. We evaluated whether expression of human E5'N on pig endothelial cells (EC) attenuates human NK cell-mediated cytotoxicity. A pig EC line was stably transfected with human E5'N and human NK cell adhesion and cytotoxicity toward pig EC cultures was measured by flow cytometry and intracellular enzyme release. E5'N activity in pig EC lysates increased from 0.68 +/- 0.07 to 1013 +/- 293 nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37 +/- 0.05 to > 300 nmol/min/mg protein in non-transfected and transfected cells, respectively. The rate of adenosine production in transfected cells increased also with ATP as the extracellular substrate. Cytotoxicity of human NK cells was reduced from 10.7 +/- 0.4% and 11.1 +/- 1.1% with non-transfected pig EC to 5.2 +/- 0.2% and 5.0 +/- 0.2% in transfected cells with 50 mu M and 250 mu M AMP, respectively. Reduction of cytotoxicity in E5'N-transfected EC was abolished by the E5'N inhibitor and was mimicked in non-transfected EC by the addition of adenosine, demonstrating the key role of adenosine produced by E5'N in inhibiting NK cell cytotoxicity. We suggest that overexpression of E5'N in EC of transgenic pigs is a possible strategy to ameliorate rejection after xeno-transplantation.