Scherrer L, Leventhal J R, Matas A J, Dalmasso A P
Department of Surgery, University of Minnesota, Minneapolis 55455.
Transplantation. 1994 Aug 27;58(4):458-66. doi: 10.1097/00007890-199408270-00012.
Xenoreactive antibodies are an integral part of the natural immune barrier to successful xenotransplantation between phylogenetically disparate species. Studies in primates suggest that the critical targets involved in hyperacute rejection of pig organs are glycoproteins expressed on endothelial cells and platelets. However, there is little information regarding the targets of xenoreactive antibodies and their cellular distribution in other experimental models of hyperacute xenograft rejection, including the commonly used guinea pig-to-rat model. The aim of this study was to characterize the target antigens on guinea pig platelets and endothelial cells that are recognized by rat anti-guinea pig antibodies. Using guinea pig platelet membrane proteins or intact guinea pig endothelial cells as antigens in an ELISA, we demonstrated that rat serum contains IgG* and IgM anti-guinea pig antibodies; levels of antiplatelet antibodies correlated with those against endothelial cells. Serum from naive or complement-depleted rats transplanted with a guinea pig heart was investigated by immunoblotting against membrane proteins extracted from guinea pig platelets and endothelial cells. Normal rat serum revealed numerous bands on either platelet or endothelial cell immunoblots, without obvious similarities in banding pattern under reduced or nonreduced conditions. Absorption of rat serum with intact guinea pig endothelial cells reduced reactivity against numerous bands on endothelial cell membrane blots, but only partially reduced reactivity with three platelet bands (141, 155, and 210 kDa). Absorption of rat serum with intact guinea pig platelets resulted in reduction in reactivity against both platelet and endothelial cell immunoblots. Antibodies eluted from those intact guinea pig endothelial cells and platelets used to absorb rat sera were found to yield patterns of membrane blot reactivity similar to those with unabsorbed sera, suggesting that the proteins recognized are expressed on the cell surface. Lectin affinity blotting demonstrated many of the guinea pig endothelial cell and platelet membrane proteins to be glycosylated. However, digestion of endothelial cell and platelet immunoblots with a mixture of glycosidases failed to change the xenoreactivity with naive rat serum. Immunoblot analysis of serum samples taken daily from complement-depleted rats carrying a functioning guinea pig heart for 3-4 days showed an increase in intensity for specific protein bands on platelets (38, 48, 56, 141, and 155 kDa) and endothelial cells (30, 210, and 270 kDa); no new protein bands were observed. Increased reactivity at several bands correlated with highly elevated rat serum anti-guinea pig antibody titers, as determined by ELISA.(ABSTRACT TRUNCATED AT 400 WORDS)
异种反应性抗体是在系统发育上不同物种之间成功进行异种移植的天然免疫屏障的一个组成部分。对灵长类动物的研究表明，猪器官超急性排斥反应中涉及的关键靶点是内皮细胞和血小板上表达的糖蛋白。然而，关于异种反应性抗体的靶点及其在其他超急性异种移植排斥实验模型中的细胞分布的信息很少，包括常用的豚鼠到大鼠模型。本研究的目的是鉴定豚鼠血小板和内皮细胞上被大鼠抗豚鼠抗体识别的靶抗原。在酶联免疫吸附测定（ELISA）中使用豚鼠血小板膜蛋白或完整的豚鼠内皮细胞作为抗原，我们证明大鼠血清中含有IgG和IgM抗豚鼠抗体；抗血小板抗体的水平与抗内皮细胞抗体的水平相关。对移植了豚鼠心脏的未免疫或补体缺失大鼠的血清进行免疫印迹分析，以检测从豚鼠血小板和内皮细胞中提取的膜蛋白。正常大鼠血清在血小板或内皮细胞免疫印迹上显示出许多条带，在还原或非还原条件下条带模式没有明显相似性。用完整的豚鼠内皮细胞吸收大鼠血清可降低对内皮细胞膜印迹上许多条带的反应性，但仅部分降低对三条血小板条带（141、155和210 kDa）的反应性。用完整的豚鼠血小板吸收大鼠血清导致对血小板和内皮细胞免疫印迹的反应性降低。从用于吸收大鼠血清的那些完整豚鼠内皮细胞和血小板上洗脱的抗体，其膜印迹反应模式与未吸收血清相似，表明所识别的蛋白质在细胞表面表达。凝集素亲和印迹显示许多豚鼠内皮细胞和血小板膜蛋白是糖基化的。然而，用糖苷酶混合物消化内皮细胞和血小板免疫印迹未能改变与未免疫大鼠血清的异种反应性。对携带功能正常的豚鼠心脏3 - 4天的补体缺失大鼠每天采集的血清样本进行免疫印迹分析，结果显示血小板（38、48、56、141和155 kDa）和内皮细胞（30、210和270 kDa）上特定蛋白条带的强度增加；未观察到新的蛋白条带。如通过ELISA所测定的，几个条带反应性的增加与大鼠血清抗豚鼠抗体滴度的高度升高相关。（摘要截断于400字）
