Zhao Z, Termignon J L, Cardoso J, Chéreau C, Gautreau C, Calmus Y, Houssin D, Weill B
Laboratoire de Recherche Chirurgicale, Faculté Cochin-Port Royal, Paris, France.
Transplantation. 1994 Jan;57(2):245-9. doi: 10.1097/00007890-199401001-00016.
Complement activation is central to the rejection of discordant xenografts. In order to assess the respective roles of direct and alternative pathways, an in vitro model of hyperacute rejection in the swine-to-human donor-recipient combination was designed, using a complement-dependent cytotoxicity test with swine endothelial cells in culture as targets, and fresh human serum as the source of xenogeneic antibodies and complement. The cytotoxic activity of the sera was evaluated by a colorimetric assay using (3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Pure human serum lysed 58 +/- 5% of swine endothelial cells. Selective inhibition of the direct pathway by adding EGTA to the serum reduced cytolysis to 51 +/- 2% (P < 0.01 versus normal serum). Similarly, when using C1q-deficient human sera, only 37 +/- 7% of swine endothelial cells were killed (P < 0.001 versus normal serum). When the alternative pathway was selectively inhibited by heating for 20 min at 50 degrees C, the lytic activity of human serum dropped to 42 +/- 5% (P < 0.001 versus normal serum). Factor B-deficient human serum could only lyse 42 +/- 10% of porcine endothelial cells (P < 0.001 versus normal serum). Syngeneic normal swine serum and heat-inactivated serum were not cytotoxic. Mixing serum with deficient direct pathway and serum with deficient alternative pathway restored the cytotoxicity to normal levels. Similarly, the cytotoxic activity of deficient serum supplemented with purified C1q or factor B at physiological concentrations reached that of normal human serum. In this model of in vitro hyperacute rejection, both pathways of complement activation are involved, suggesting that regimens designed to inhibit hyperacute rejection of swine xenografts into humans should take into account the dual activation of complement in this donor-recipient combination.
补体激活在异种移植排斥反应中起着核心作用。为了评估经典途径和替代途径各自的作用，设计了猪到人的供受体组合的超急性排斥反应体外模型，采用以培养的猪内皮细胞为靶细胞的补体依赖细胞毒性试验，并以新鲜人血清作为异种抗体和补体的来源。使用（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四氮唑）（MTT）比色法评估血清的细胞毒性活性。纯人血清可裂解58±5%的猪内皮细胞。通过向血清中添加乙二醇双四乙酸（EGTA）选择性抑制经典途径，可将细胞溶解率降低至51±2%（与正常血清相比，P<0.01）。同样，当使用缺乏C1q的人血清时，只有37±7%的猪内皮细胞被杀死（与正常血清相比，P<0.001）。当通过在50℃加热20分钟选择性抑制替代途径时，人血清的溶解活性降至42±5%（与正常血清相比，P<0.001）。缺乏B因子的人血清只能裂解42±10%的猪内皮细胞（与正常血清相比，P<0.001）。同基因正常猪血清和热灭活血清无细胞毒性。将缺乏经典途径的血清与缺乏替代途径的血清混合可使细胞毒性恢复到正常水平。同样，补充生理浓度纯化C1q或B因子的缺陷血清的细胞毒性活性达到正常人血清水平。在这个体外超急性排斥反应模型中，补体激活的两条途径均参与其中，这表明旨在抑制猪异种移植到人体的超急性排斥反应的方案应考虑到该供受体组合中补体的双重激活。
