Division of Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Pediatric Liver Care Center, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Division of Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Pediatric Liver Care Center, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
J Hepatol. 2014 Feb;60(2):370-6. doi: 10.1016/j.jhep.2013.09.021. Epub 2013 Oct 2.
BACKGROUND & AIMS: Biliary atresia represents obstructive cholangiopathy in infants progressing rapidly to cirrhosis and end-stage liver disease. Activated NK cells expressing Nkg2d have been linked to bile duct injury and obstruction by establishing contact with cholangiocytes. To define the mechanisms used by cytotoxic cells, we investigated the role of perforin and granzymes in a neonatal mouse model of rotavirus (RRV)-induced biliary atresia.
We used complementary cell lysis assays, flow cytometric analyses, quantitative PCRs and in vivo systems to determine the mechanisms of bile duct epithelial injury and the control of the tissue phenotype in experimental biliary atresia.
RRV-infected hepatic NK and CD8 T cells increased the expression of perforin and injured cholangiocytes in short-term culture in a perforin-dependent fashion. However, the loss of perforin in vivo delayed but did not prevent the obstruction of bile ducts. Based on the increased expression of granzymes by perforin-deficient cytotoxic cells in long-term cytolytic assays, we found that the inhibition of granzymes by nafamostat mesilate (FUT-175) blocked cholangiocyte lysis. Administration of FUT-175 to perforin-deficient mice after RRV infection decreased the development of jaundice, minimized epithelial injury, and improved long-term survival. However, the inhibition of granzymes alone in wild-type mice was not sufficient to prevent the atresia phenotype in newborn mice. In infants with biliary atresia, hepatic Granzymes A and B mRNA, but not Perforin, increased at the time of portoenterostomy.
Perforin and granzymes have complementary roles mediating epithelial injury by NK and CD8 T cells. The prevention of experimental biliary atresia can only be achieved by inhibiting both granules.
先天性胆道闭锁是一种婴儿期进行性进展的胆管梗阻性疾病，可迅速发展为肝硬化和终末期肝病。表达 Nkg2d 的活化 NK 细胞已被证明通过与胆管细胞建立接触而导致胆管损伤和梗阻。为了确定细胞毒性细胞使用的机制，我们在轮状病毒（RRV）诱导的先天性胆道闭锁新生鼠模型中研究了穿孔素和颗粒酶的作用。
我们使用互补细胞溶解测定、流式细胞分析、定量 PCR 和体内系统来确定胆管上皮损伤的机制和实验性胆道闭锁中组织表型的控制。
RRV 感染的肝 NK 和 CD8 T 细胞增加了穿孔素依赖性方式的短时间培养中胆管细胞的穿孔素表达和损伤。然而，体内穿孔素的缺失延迟但并未阻止胆管的阻塞。基于长期细胞溶解测定中穿孔素缺陷细胞毒性细胞中颗粒酶表达的增加，我们发现 FUT-175（奈莫司他）抑制颗粒酶可阻止胆管细胞溶解。在 RRV 感染后给予穿孔素缺陷小鼠 FUT-175 可减少黄疸的发展，最大限度地减少上皮损伤，并改善长期生存。然而，在野生型小鼠中单独抑制颗粒酶不足以预防新生小鼠的闭锁表型。在先天性胆道闭锁婴儿中，肝颗粒酶 A 和 B mRNA 但不是穿孔素在门腔吻合术时增加。
穿孔素和颗粒酶通过 NK 和 CD8 T 细胞发挥介导上皮损伤的互补作用。只有通过抑制两种颗粒才能预防实验性胆道闭锁。
