Watier H, Guillaumin J M, Piller F, Lacord M, Thibault G, Lebranchu Y, Monsigny M, Bardos P
Equipe Interactions Hôte-Greffon, Faculté de Médecine, Université François Rabelais, Tours, France.
Transplantation. 1996 Jul 15;62(1):105-13. doi: 10.1097/00007890-199607150-00020.
To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the alpha-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts.
为了确定末端α-半乳糖基残基在人异种反应性天然抗体（IgM和IgG）介导的内皮损伤中的作用，我们用绿咖啡豆α-半乳糖苷酶处理培养的猪内皮细胞。通过使用简单叶豆凝集素B4进行流式细胞术评估，几乎完全去除了末端α-Gal残基，并同时暴露了N-乙酰乳糖胺，且未改变细胞活力。观察到IgM和IgG结合（来自人血清池）显著减少，证实了α-半乳糖基残基起关键作用。酶处理未诱导任何非特异性免疫球蛋白结合位点，但导致一小部分IgM的新表位暴露。主要的残留IgM和IgG结合可能归因于除α-半乳糖基残基之外的异种抗原。当用α-半乳糖苷酶处理的内皮细胞作为细胞毒性实验的靶标时，它们比未处理的细胞对新鲜人血清诱导的补体介导的细胞毒性更不敏感。相反，尽管IgG结合减少，但它们并未获得对人IgG依赖性细胞毒性的抗性。因为已知由CD16 + NK细胞介导的抗体依赖性细胞毒性依赖于IgG1和IgG3，而不依赖于IgG2或IgG4，这通过阻断实验得到证实，所以我们研究了所有四个亚类与完整的和α-半乳糖苷酶处理的内皮细胞的结合。两个主要亚类IgG1和IgG2与未处理的内皮细胞结合，而IgG3结合较低，IgG4结合可忽略不计。α-半乳糖苷酶处理后观察到IgG1、IgG2和IgG3结合减少，表明属于这三个亚类的抗体识别α-半乳糖基残基。IgG2结合的减少比IgG1结合的减少更明显。总体而言，这些数据表明，IgG1异种反应性天然抗体，包括那些不针对α-半乳糖基残基的抗体，可能在猪异种移植物早期延迟性血管排斥中起主要作用。
