Morigi M, Zoja C, Colleoni S, Angioletti S, Imberti B, Remuzzi A, Remuzzi G
Mario Negri Institute for Pharmacological Research, Bergamo, Italy.
Xenotransplantation. 1998 Feb;5(1):57-60. doi: 10.1111/j.1399-3089.1998.tb00009.x.
Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.
内皮细胞活化和白细胞浸润是异种移植排斥反应不一致的一个持续特征。在此，我们评估了作为异种反应性天然抗体和补体来源的异种血清，是否会在流动条件下诱导内皮细胞活化并导致白细胞黏附。将猪主动脉内皮细胞（PAEC）与10%同源猪血清（对照）或10%异种人血清孵育1小时30分钟或5小时，然后在层流（1.5达因/平方厘米）下在平行板流动腔中用全人类白细胞灌注。通过数字图像分析对黏附细胞进行计数。与猪血清相比，异种人血清显著（P < 0.01）增加了黏附白细胞的数量。内皮细胞黏附特性最有效的诱导剂之一肿瘤坏死因子α（100 U/ml）引发了类似的黏附反应（在此用作阳性对照）。为了阐明异种血清激活内皮细胞的可能机制，我们聚焦于转录因子核因子κB，它是诱导包括黏附分子和趋化因子在内的不同基因的核心调节因子。通过共聚焦荧光显微镜观察，我们发现暴露于人类血清1小时30分钟的PAEC细胞核中核因子κB（p65亚基）呈阳性染色，这表明在此情况下核因子κB被激活。相比之下，在与同源血清孵育的PAEC中，核因子κB严格定位于细胞质中。用核因子κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC，25 microM）和甲苯磺酰苯丙氨酰氯甲基酮（TPCK，25 microM）处理暴露于异种血清的PAEC，与仅用人血清处理的PAEC相比，显著（P < 0.01）降低了白细胞黏附。异种血清可能通过核因子κB激活促进白细胞与内皮细胞相互作用的这一发现，可能与设计旨在延长异种移植存活时间的未来治疗策略相关。
