Tomiyama Koji, Ikeda Atsushi, Ueki Shinya, Nakao Atsunori, Stolz Donna B, Koike Yasushi, Afrazi Amin, Gandhi Chandrashekhar, Tokita Daisuke, Geller David A, Murase Noriko
Department of Surgery, Thomas E Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA.
Hepatology. 2008 Nov;48(5):1608-20. doi: 10.1002/hep.22482.
Proinflammatory responses play critical roles in hepatic ischemia/reperfusion (I/R) injury associating with liver transplantation (LTx), and carbon monoxide (CO) can effectively down-regulate them. Using wild-type (WT) to enhanced green fluorescent protein (EGFP)-transgenic rat LTx with 18-hour cold preservation in University of Wisconsin solution, this study analyzed the relative contribution of donor and host cells during early posttransplantation period and elucidated the mechanism of hepatic protection by CO. CO inhibited hepatic I/R injury and reduced peak alanine aminotransferase levels at 24 hours and hepatic necrosis at 48 hours. Abundant EGFP(+) host cells were found in untreated WT liver grafts at 1 hour and included nucleated CD45(+) leukocytes (myeloid, T, B, and natural killer cells) and EGFP(+) platelet-like depositions in the sinusoids. However, reverse transcription polymerase chain reaction (RT-PCR) analysis of isolated graft nonparenchymal cells (NPCs) revealed that I/R injury-induced proinflammatory mediators [for example, tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS)] were not up-regulated in purified CD45(+) cells of donor or host origin. Instead, TNF-alpha and IL-6 messenger RNA (mRNA) elevation was exclusively seen in isolated CD68(+) cells, whereas iNOS mRNA up-regulation was seen in hepatocytes. Nearly all CD68(+) cells at 1 hour after LTx were EGFP(-) donor Kupffer cells, and CO efficiently inhibited TNF-alpha and IL-6 up-regulation in the CD68(+) Kupffer cell fraction. When graft Kupffer cells were inactivated with gadolinium chloride, activation of inflammatory mediators in liver grafts was significantly inhibited. Furthermore, in vitro rat primary Kupffer cell culture also showed significant down-regulation of lipopolysaccharide (LPS)-induced inflammatory responses by CO.
These results indicate that CO ameliorates hepatic I/R injury by down-regulating graft Kupffer cells in early postreperfusion period. The study also suggests that different cell populations play diverse roles by up-regulating distinctive sets of mediators in the acute phase of hepatic I/R injury.
促炎反应在与肝移植（LTx）相关的肝缺血/再灌注（I/R）损伤中起关键作用，而一氧化碳（CO）可有效下调这些反应。本研究采用野生型（WT）至增强型绿色荧光蛋白（EGFP）转基因大鼠肝移植，并在威斯康星大学溶液中进行18小时冷保存，分析了移植后早期供体和宿主细胞的相对贡献，并阐明了CO的肝保护机制。CO抑制肝I/R损伤，降低24小时时的谷丙转氨酶峰值水平以及48小时时的肝坏死程度。在未处理的WT肝移植1小时时发现大量EGFP（+）宿主细胞，包括有核CD45（+）白细胞（髓样细胞、T细胞、B细胞和自然杀伤细胞）以及窦状隙中的EGFP（+）血小板样沉积物。然而，对分离的移植非实质细胞（NPCs）进行逆转录聚合酶链反应（RT-PCR）分析显示，I/R损伤诱导的促炎介质[例如肿瘤坏死因子α（TNF-α）、白细胞介素-6（IL-6）和诱导型一氧化氮合酶（iNOS）]在供体或宿主来源的纯化CD45（+）细胞中并未上调。相反，TNF-α和IL-6信使核糖核酸（mRNA）升高仅在分离的CD68（+）细胞中可见，而iNOS mRNA上调在肝细胞中可见。肝移植后1小时时，几乎所有CD68（+）细胞均为EGFP（-）供体库普弗细胞，CO可有效抑制CD68（+）库普弗细胞部分中TNF-α和IL-6的上调。当用氯化钆使移植的库普弗细胞失活时，肝移植中炎症介质的激活受到显著抑制。此外，体外大鼠原代库普弗细胞培养也显示CO可显著下调脂多糖（LPS）诱导的炎症反应。
这些结果表明，CO通过在再灌注早期下调移植的库普弗细胞来改善肝I/R损伤。该研究还表明，在肝I/R损伤急性期，不同细胞群体通过上调不同组别的介质发挥不同作用。
