Mechanism of liver ischemia-reperfusion injury with monoclonal antibodies of adhesin molecules and its application to organ transplantation

本研究旨在确定中性粒细胞（PMNs）是否参与肝缺血/再灌注和内毒素血症下的细胞损伤。采用肝热缺血/再灌注- mate Wistar大鼠，用动脉钳封堵供应肝正中叶和左外侧叶的血管90 min。闭塞前5分钟，静脉注射抗ICAM-1 （IA29）、CD11a （WT-1）、CD18 （WT-3）或PBS单克隆抗体（mAb）作为安慰剂。再灌注后，用组织学方法检测肝组织中pmn的浸润及ICAM-1的表达。为了检验单抗治疗的效果，以肝三磷酸腺苷（ATP）和丙二醛（MDA）水平作为肝细胞损伤的指标。再灌注后24小时内，pmn数量持续增加。肝组织中ICAM-1的表达在再灌注后4小时增强。单抗可抑制PM . More NS的浸润6小时，ATP的再合成6、12和24小时，再灌注后12小时降低肝脏MDA水平。然后，这些单抗提高了全肝缺血(90min) /再灌注大鼠的存活率。内毒素-脂多糖（LPS：大肠杆菌）腹腔注射ICR小鼠，同时静脉注射lmg/kg抗白细胞粘附分子mAb（抗cd11a: KAB，抗cd11b: MI/70，抗cd18， C17/16，抗icam -1: cat -1，抗lecam -1: MEL-14）， 30mg/kg甲基强龙（MP）作为常规休克剂，或PBS作为安慰剂。安慰剂组小鼠给药后48小时存活率为36%（20/55）。抗cd11a、antid18、抗lecam -1和MP治疗的生存率分别为70（7/10）、62（8/13）、64（7/11）和100%（11/11）。另一方面，抗cd11b和抗icam -1对生存率无显著影响。使用抗CD18单抗的FACS分析显示，粒细胞表面CD18的表达在30分钟内增加了12倍，MP在LPS处理后3小时内显著抑制CD18的表达。LPS处理4 h后，小鼠肝脏MDA含量由0.50（未处理小鼠）上升至2.46 nmol/mg蛋白，抗cd18 mAb和MP分别将其抑制至1.80和1.41 nmol/mg蛋白。在肝脏缺血/修复和内毒素血症的情况下，白细胞介导了氧化损伤引起的肝细胞损伤。这些单克隆抗体可以作为预防此类损伤的治疗剂。少

The present study is to determine whether neutrophils (PMNs) contribute to the cellular injuries under hepatic ischermia/reperfusion and endotoxemia.Hepatic warm ischemia/reperfusion-Mate Wistar rats were used and the vessels supplying the median and the left lateral hepatic lobes were occuluded with arterial clamp for 90 min. Five minutes before the occulusion, monoclonal anitibody (mAb) against ICAM-1 (IA29), CD11a (WT-1), CD18 (WT-3) or PBS as the placebo was administered intravenously. After reperfusion, the infiltration of PMNs and the expression of ICAM-1 in the liver were examined histologically. To examine the effect of mAb treatment, hepatic adenosine triphosphate (ATP) and malondialdehyde (MDA) levels were determined as indices of hepatic cellular injury. The number of accumulated PMNs increased continuously up to 24 hours after reperfusion. The expression of ICAM-1 in the liver was enhanced 4 houres after reperfusion. Treatment with the mAbs suppressed the infiltration of PM … More NS by 6 hours, ATP resynthesis by 6,12 and 24 hours, and reduced hepatic MDA level by 12 hours after reperfusion. Then, these mAbs increased the survival rate of rats receiving total hepatic ischemin (90min) / reperfusion.Endotoxemia-Lipopolysaccharide (LPS : E.coli) was administered to ICR mice intraperitoneally, and lmg/kg antileukocyte adhesion molecule mAb (anti-CD11a : KAB,anti-CD11b : MI/70, anti-CD18 ; C17/16, anti-ICAM-1 : KAT-1, anti-LECAM-1 : MEL-14), 30mg/kg methylprednisolone (MP) as a conventional agent for shock, or PBS as the placebo was administered intravenously, simultancously. In the placebo group, the survival rate of mice 48 houres after LPS administration was 36% (20/55). Treatment with anti-CD11a, antiCD18, anti-LECAM-1 and MP increased the survival rate to 70 (7/10), 62 (8/13), 64 (7/11) and 100% (11/11), respectively. ON the other hand, anti-CD11b and anti-ICAM-1 had no significant effect on the survial rate. FACS analysis using the anti-CD18 mAb revealed that the expression of CD18 on the surfaces of granulocytes increased 12 folds within 30 min, and MP suppressed it significantly for up to 3 hours after LPS administration. The hepatic MDA content increased from 0.50 (untreated mice) to 2.46 nmol/mg protein 4 hours after LPS administration, and anti-CD18 mAb and MP suppressed it to 1.80 and 1.41 nmol/mg protein, respectively.Leukocytes are concluded to mediate significant roles for oxidative injury causing hapatic cellular damages under hepatic ischemia/reparfuion and endotoxemia. These mAbs can be therapeutic agents preventing such injuries. Less