Localization and infrastructure of the Kupffer cells in orthotopically transplanted liver grafts in the rat

Abstract. Kupffer cells play an important role in the acceptance or rejection of liver grafts. We examined the ultrastructure of the Kupffer cells in transplanted rat livers, from an early to a late stage where the graft is accepted, using a DA-to-PVG combination. Two days after surgery, endocytic activity of the Kupffer cells had increased, as evidenced by worm-like structures and many endocytic vacuoles. There was often close apposition to the monocytes or lymphocytes. By day 4, infiltration of mononuclear cells into the sinusoids was readily noticeable. By day 7, several Kupffer cells had migrated into the space of Disse through the openings in endothelial linings. The number of Kupffer cells reached a maximum at 14 days. They were located mostly outside the sinusoid, adhering to the hepatocytes. At this point in time, however, the Kuppfer cells contained few endocytic vacuoles and phagolysosomes, in contrast to those at 2 days. The number and location of Kupffer cells became almost normal at 2 months. The present results indicate that Kupffer cells are highly activated before mononuclear cell infiltration becomes manifest in the sinusoid, and that when a rejecting reaction reaches a peak, they are usually located extrasinusoidally and show a morphologically immature profile.     

Kupffer cell and hepatocyte function in rat transplanted liver

The liver consists essentially of two compartments, parenchymal cells (PC) and non parenchymal cells (NPC) i.e. Kupffer cells, endothelial cells, fat storing cells and pit cells. PC remain after transplantation but NPC are eventually exchanged with host cells. Dynamic liver scintigraphy with albumin colloid, extracted by NPC, and IODIDA, extracted by PC, were tested to evaluate function as determined by clearance rates in these two cellular compartments. Experimental liver transplantation was performed in 15 syngeneic rats. Following transplantation, we performed dynamic liver scintigraphy with 0.5 ml 5 MBq ^99mTc-Nanocoll and 0.5 ml 20 MBq ^99mTc-IODIDA, 10 s per frame, 30 min for each examination. Percentage clearance rate, per minute was calculated from uptake curves over the liver. Uptake curves were nearly exponential and clearance rates could be estimated from a logarithmic plot of uptake versus time. The clearance rate was 25 ± 4% per min (mean ± SD) for NPC and 32 ± 15% per min for PC in controls. After liver transplantation it was 31 ± 7% per min for NPC and 30 ± 15% per min for PC. Dynamic liver scintigraphy with ^99mTc-Nanocoll and ^99mTc-IODIDA alloweds a separate assessment of the function of PC and NPC after experimental liver transplantation in rats.     

Methyl palmitate prevents Kupffer cell activation and improves survival after orthotopic liver transplantation in the rat

The purpose of this study was to determine whether prevention of Kupffer cell activation following orthotopic liver transplantation improves postoperative survival. First, particle phagocytosis by Kupffer cells was monitored continuously from the uptake of colloidal carbon by the perfused liver. Unstored livers took up carbon at rates of around 150 mg/g per hour, whereas storage for 24 h in Euro-Collins solution nearly doubled values to about 290 mg/g per hour. Treatment of rats with methyl palmitate, an inhibitor of phagocytosis by Kupffer cells, reduced carbon uptake to about one-third to one-half of control values in unstored and stored livers, respectively. Oxygen uptake, which was increased about 25% in stored and unstored livers by infusion of colloidal carbon, was only increased 5%–10% in both groups following treatment with methyl palmitate, suggesting that Kupffer cell activation was prevented by methyl palmitate. In livers transplanted after storage for 6 h in Euro-Collins solution (nonsurvival conditions), control rats survived only about 12 h, while treatment with methyl palmitate increased survival time significantly — more than threefold — to about 40 h. These data are consistent with the hypothesis that activation of Kupffer cells following cold ischemic storage and reperfusion is an early event involved in liver graft failure. Present address: Department of Trauma Surgery, University of Saarland, W-6650 Homburg/S., Federal Republic of Germany     

Methyl palmitate prevents Kupffer cell activation and improves survival after orthotopic liver transplantation in the rat

Abstract. The purpose of this study was to determine whetherprevention of Kupffercell activation following orthotopic liver transplantation improves postoperative survival. First, particle phagocytosis by Kupffer cells was monitored continuously from the uptake of colloidal carbon by the perfused liver. Unstored livers took up carbon at rates of around 150 mg/g per hour, whereas storage for 24 h in Euro-Collins solution nearly doubled values to about 290 mg/g per hour. Treatment of rats with methyl palmitate, an inhibitor of phagocytosis by Kupffer cells, reduced carbon uptake to about one-third to one-half of control values in unstored and stored livers, respectively. Oxygen uptake, which was increased about 25% in stored and unstored livers by infusion of colloidal carbon, was only increased 5%-10% in both groups following treatment with methyl palmitate, suggesting that Kupffer cell activation was prevented by methyl palmitate. In livers transplanted after storage for 6 h in Euro-Collins solution (nonsurvival conditions), control rats survived only about 12 h, while treatment with methyl palmitate increased survival time significantly - more than threefold - to about 40 h. These data are consistent with the hypothesis that activation of Kupffer cells following cold ischemic storage and reperfusion is an early event involved in liver graft failure.     

Kupffer cells participate in rejection following liver transplantation in the rat

Abstract Recognition of foreign antigens involves macrophages which release mediators such as immunoactive interleukins, and in the liver, the resident macrophages (Kupffer cells) are activated following transplantation. Therefore, we evaluated the hypothesis that Kupffer cells participate in the rejection reaction following transplantation. Orthotopic liver transplantation was performed between different syngenic rat strains. Livers from Lewis rats were stored in lactated Ringer's solution for 1 h to minimize cold ischemic injury and transplanted into PVG recipients. At 24 h postoperatively, transaminases (AST) were elevated to values around 2000 U/l, total bilirubin was increased to values around 20 μmol/l, and five of six rats died within 3 days. Macroscopic and histological examination showed large areas of necrosis without cellular infiltration, characteristic of rejection. When donor rats were treated with gadolinium chloride (GdCl₃, 10 mg/kg i.v. 24 h before storage of the liver) to inactivate the Kupffer cells, AST levels only rose to around 700 U/l, and the total bilirubin level was in the normal range (<4 μmol/l). Survival was improved significantly by GdCl₃, with five of seven rats surviving more than 1 month ( P < 0.05) and four of seven rats surviving for at least 100 days without immunosuppressive drug therapy. Rejection was not totally prevented, however, since the surviving rats had elevated AST and bilirubin levels, and cellular infiltration in portal areas along with proliferation of bile canaliculi was observed. These data are consistent with the hypothesis that Kupffer cells participate in mechanisms of early rejection following liver transplantation.     

Role of Kupffer cells in the survival after rat liver transplantation with long portal vein clamping times

Applying the orthotopic rat liver transplantation (ORLT) model, postoperative survival has been shown to be mainly dependent on the portal vein clamping time (PVCT). It was hypothesized that prolonged intestinal congestion was responsible for the activation of Kupffer cells (KC) with overproduction of TNF, secondary to splanchnic endotoxin accumulation and release on reperfusion. The role of KCs was directly investigated in the context of long PVCTs by eliminating them (using liposome-encapsulated dichloromethylene diphosphonate), by preventing their activation (using a calcium channel blocker, nisoldipine) and by inhibiting TNF production (using thalidomide). Livers from different groups of rats were transplanted following 24-h cold preservation in the UW solution with long PVCTs (from 18–21 min). KCs depletion, preservation with nisoldipine and pretreatment with thalidomide significantly improved survival in conditions using long PVCTs. KC depletion and nisoldipine preservation had no effect on liver enzymes or pathological findings while lung injury was significantly improved. The present data confirm that, in the context of ORLT with long PVCTs, KCs are directly responsible for the systemic endotoxin-like shock syndrome and their effect is mediated through overproduction of TNF. Received: 30 August 1999 Revised: 18 May 2000 Accepted: 12 September 2000     

Low-temperature fluorometric technique for evaluating the viability of rat liver grafts after simple cold storage

Time-dependent changes in the viability of rat liver graft during cold preservation with Euro-Collins solution were evaluated with NADH fluorometry. Correlation between the fluorometric analysis, 1-week survival rate after liver transplantation, and mitochondrial ATP synthesis activity in the early phase after transplantation was studied. Fluorometric study: Rat livers were preserved at 0°–4°C for 0–48 h in Euro-Collins solution and then reperfused for 15 min with oxygenated Krebs-Henseleit solution at 4°C. The amplitude (R x A) between the oxidized and the reduced steady-state NADH fluorometric trace and the velocity (R x V) of the trace were determined to evaluate the mitochondrial respiratory chain. The R x A and R x V remained at levels higher than 90% of control after 6-h preservation, while the R x A of the 9-h preservation group and the R x V of the 12-h preservation group decreased significantly compared with those of the control and the 6-h preservation group. Survival study: a 100% survival rate after transplantation was achieved in the 6-h preservation group, whereas the rates were 18.8% and 0% in the 9-and 12-h preservation groups respectively. These survival rates correlated closely with the time-dependent decrease of the fluorometric parameters. Study of mitochondrial phosphorylative activity and energy charge 3 h after transplantation: With fresh grafts, the decrease in hepatic energy charge after transplantation was reduced to 0.79 from the control value of 0.86 by a 30% increase in mitochondrial ATP synthesis ability. When the graft was preserved for 12 h, the energy charge dropped to 0.63 due to lack of the enhancement of ATP synthesis ability. The results of this study indicate a possibility of using fluorometric evaluation of the graft to predict post-transplantation mitochondrial ATP synthesis ability and survival rate.     

Quantitative measurements of collateral arterial blood flow in nonarterialized rat liver grafts

The early development of arterial blood flow in the grafted liver after orthotopic liver transplantation in the rat without reconstruction of the hepatic artery was studied. Arterial liver blood flow was measured on day 21 after transplantation with NEN-TRAC microspheres (size 15.5±0.1 m) and labelled with ¹⁰³Ru. The arterial liver blood flow in the grafted liver was 0.778±0.247 ml/min per gram for transplanted rats after 21 days. One day after transplantation, the blood flow was only 0.006±0.002 ml/min per gram. The results of this study demonstrate that there was no arterial blood flow on day 1 after transplantation, as expected, but that there was a high arterial blood flow in the transplanted liver by day 21. This was also supported by the angiographic findings. The early development of arterial blood flow via collaterals may account for the excellent results that we and others have attained in orthotopic liver transplantation without rearterialization in the rat.     

Kupffer细胞在肝移植缺血再灌注损伤中的双重作用

Kupffer细胞足定居于肝内的巨细胞,在月十移植缺血再灌注损伤中发挥着重要的作用,门静脉恢复血流后刺激Kupffer细胞激活,释放活性氧族、多种炎性介质和细胞因子,对肝脏造成损伤.另一方面又可上调HO-1的表达,保护肝脏缺血再灌注损伤,因此,Kupffer细胞在肝移植缺血再灌注损伤中发挥着双重效应.     

大鼠移植肝内Kupffer细胞分离及对T细胞增殖的抑制效应

目的:建立分离、纯化和培养大鼠肝脏Kupffer细胞(KC)的方法,并观察KC对同种反应性T细胞增殖的影响。方法:采用在体原位肝脏灌注结合密度梯度离心技术,进行肝移植受体大鼠肝脏KC分离、纯化和培养。在体外实验中进一步观察KC在MLR(混合淋巴细胞反应)体系中对同种反应性T细胞增殖程度的影响。结果:KC得率为(1.1±0.2)×107/肝,平均存活率为(93.5±1.8)%,纯度≥90%,ED-2( )。培养24h可见大量细胞伸展生长,呈现典型的星形或多边形。在MLR体系中加入未经辐照的KC后,反映T细胞增殖程度的每分钟脉冲数值明显降低,且接受联合免疫治疗并长期存活的D、E两组所获得的KC对T细胞增殖有着更为明显的抑制作用。结论:本实验成功建立了分离、纯化和培养大鼠KC的方法。KC对同种反应性T细胞增殖具有抑制作用,尤其从接受联合免疫治疗后长期存活受体大鼠所获得的KC抑制作用更为明显。     

Liver glycogen in fasted rat livers does not improve outcome of liver transplantation

Controversy exists over how the nutritional condition of the donor liver affects transplant outcome. Some studies suggest that livers from fasted animals (liver glycogen-depleted) are more readily injured than livers from fed animals. Our previous study suggested the opposite, i.e., livers from donors fasted for 4 days were significantly more viable on orthotopic liver transplantation. Fasting may decrease the sensitivity of the liver to an inflammatory response or block Kupffer cell activation following transplantation. Thus, long-term fasting may be beneficial for reasons unrelated to liver glycogen content. In this study we attempted to separate out the roles of fasting and liver glycogen in liver transplant outcome by fasting donors for 2 days and then feeding them only glucose to elevate liver glycogen. Rats (Brown Norway) were fed (standard diet), fasted (4 days), or fasted 2 days and then fed glucose (in water) for 2 days. Livers were preserved for either 30 or 44 h in UW solution and transplanted. Four-day fasting of the donor improved the survival rate in liver transplantation (50%–100% in 30-h cold storage, 29%–83% in 44-h cold storage). However, feeding glucose for 2 days to fasted animals caused a decrease in survival in this series of transplants (40% in 30-h cold storage, 0% in 44-h cold storage). In the glucose-fed group, liver glycogen was 240% of that in the control group. This suggests that the presence of a high concentration of liver glycogen is not beneficial to the preserved and transplanted rat liver.     

大鼠肝移植缺血再灌注后Kupffer细胞CD14基因及蛋白的表达

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14基因及蛋白的表达,探讨其在再灌注损伤中的作用.方法分离培养大鼠肝移植缺血再灌注后0（对照组）、2、6、12 h（IR组）的Kupffer细胞,用逆转录聚合酶联反应（RT-PCR）检测Kupffer细胞CD14 mRNA的表达,用免疫印迹检测CD14蛋白合成,用酶联免疫吸附试验（ELISA）法测定培养上清TNF-α的分泌量.然后在上述时间点的细胞培养液中加入抗CD14抗体（anti-CD14组）,观察CD14抗体对TNF-α分泌的影响.结果再灌注后Kupffer细胞CD14 mRNA、蛋白以及TNF-α随观察时间点呈逐步上升趋势（与对照组相比,P＜0.01）.应用抗CD14抗体后,TNF-α表达较IR组明显降低（P＜0.01）.结论再灌注后Kupffer细胞CD14基因及蛋白的表达明显升高,TNF-α的合成和分泌也明显增强;抗CD14单抗能明显抑制TNF-α的产生;CD14在介导Kupffer细胞激活和肝移植缺血再灌注损伤中可能起重要作用.     

A study of liver regeneration using fetal rat liver tissue transplanted into the spleen

The liver morphology of fetal hepatic tissue transplanted into an ectopic location was investigated over one year period. Fetal liver fragments prepared from a maternal rat on the 18th or 19th day of pregnancy were injected into the splenic parenchyma of syngeneic rats using a 21 gauge needle. Histologically, the fetal liver did not essentially show any apparent lobular architecture or cord structure. The transplanted fetal hepatic tissues survived and formed hepatic cords in the spleen instead of undergoing degeneration and necrosis. Three characteristic features became complete during the 4 weeks following transplantation, namely; clumps of hepatocytes with obvious hepatic cords and sinusoids, markedly proliferating bile ducts and proliferating individual hepatocytes. Macroscopic nodules of the hepatocytes on the spleen were seen at about 6 months after transplantation. When the differentiation of the transplanted fetal hepatic tissue was compared with the development of a normal neonatal liver after birth, it was delayed by only about one week, while there was no proliferation of bile ducts in the normal neonatal liver. This experimental model provides a useful system for investigating liver regeneration and the mechanism of cell growth.     

人枯否细胞在同种肝移植免疫中作用机制初步探讨

目的 探讨肝脏枯否细胞(KC)在肝移植后早期免疫反应中的可能作用.方法 将KC和(或)异体PBMC共培养,收集细胞上清液,培养结束时分别收获培养的KC和PBMC.检测HLA-G在细胞表面的表达;测定上清液中NO、IFN-γ、IL-10和TGF-β1的浓度;MTT试验观察KC对淋巴细胞增殖的影响.结果 实验组及对照组中KC和PBMC表面均未检测到HLA-G的表达.与不含KC实验组相比,含KC实验组中,NO、IL-10和TGF-β1的产量显著升高,而IFN-γ呈相对偏低趋势;对照组中未能检测到IL-10和IFN-γ的分泌,仅含KC的对照组中含少量NO及TGF-β1,且显著低于实验组.MTT实验发现,不含KC实验组OD值显著高于含KC实验组及对照组.结论 体外KC接触异体PBMC后早期,各细胞膜表面均无HLA-G表达,但参与了NO及Th2/Th3样细胞因子的分泌调节,并能抑制淋巴细胞增殖反应,可能促进肝脏移植早期免疫耐受的形成.     

Generation of lipid free radicals by adherent leukocytes from transplanted rat liver

The production of free radicals in blood correlates with primary nonfunction of transplanted livers, but the source of the free radicals is unknown. The purpose of this study was to determine if adherent leukocytes in the transplanted liver are responsible for the radicals detected in blood. First, a new method to harvest adherent leukocytes from the liver without enzymatic digestion was developed and characterized by transplanting livers from ethanol-treated rats, which increases primary nonfunction, and from saline-treated controls. Free radicals were then detected in isolated leukocytes using the spin-trapping technique and electron spin resonance (ESR) spin spectroscopy. Livers were perfused with a balanced salt solution (200 ml), followed by a Ca^{2 +} -free solution containing EGTA and heparin (400 ml). Perfusion with Ca^{2 +} -free buffer removed greater than 90 % of all adherent leukocytes from saline-treated livers and nearly 80 % of all leukocytes from fatty livers without removing Kupffer cells. Transplanted fatty livers from rats given ethanol contained significantly more adherent leukocytes (5.0 × 10⁷ cells/liver) than grafts from control donors (3.2 × 10⁷ cells/liver) and almost double the number of adherent neutrophils and monocytes. Moreover, adherent white blood cells from transplanted livers produced the same three free radical species that have been detected previously in blood; however, cells from ethanol-treated livers produced about five times more radical adducts. These data show that adherent white blood cells produce free radicals that are important in the mechanism of primary graft nonfunction. Received: 20 October 1997 Received after revision: 13 March 1998 Accepted: 30 March 1998     

核因子κB圈套寡核苷酸减轻大鼠移植肝缺血/再灌注损伤的作用和机制

目的探讨抑制枯否（Kupffer）细胞核因子κB（Nuclearfactor-kappaB，NF-κB）活性对减轻大鼠移植肝缺血／再灌注损伤（IRI）的作用和机制。方法建立大鼠肝移植缺血／再灌注损伤模型。实验分正常对照组、缺血／再灌注组和圈套寡核苷酸组，每组均为8只大鼠。圈套寡核苷酸组于移植术前2d经供者尾静脉注入120μg脂质体包裹的NF-κB圈套寡核苷酸。移植再灌注后2h，取各组受者移植肝分离枯否细胞。凝胶迁移变动分析法（EMSA）检测枯否细胞NF-κB蛋白结合活性，逆转录聚合酶链法（RT—PCR）观察枯否细胞肿瘤坏死因子α（TNF—α）和白细胞介素6（IL-6）mRNA的表达，同时观察肝组织病理及肝功能变化。结果缺血／再灌注组移植肝再灌注后2h，枯否细胞NF-κB活性及TNF-α、IL-6 mRNA表达量较对照组明显升高（P〈0．01）。光镜下肝细胞大量变性、坏死，伴有肝血窦明显淤血，血清丙氨酸转氨酶（ALT）和胆红素总量（TBIL）较对照组明显升高（P〈0．01）。相反，圈套寡核苷酸组枯否细胞NF-κB活性及细胞因子mRNA表达与缺血／再灌注组相比明显下降（P〈0．01），移植肝未见明显病理组织学改变，肝功能明显改善。结论NF-κB圈套寡核苷酸能高效抑制枯否细胞NF-κB活性，并抑制其下游有害细胞因子的产生，从而减轻缺血／再灌注损伤对移植肝的打击和损害。     

Influence of liver transplantation and cyclosporin on bile secretion —an experimental study in the rat

Bile secretion is reduced after liver transplantation. It has been suggested that this is due either to the effect of cyclosporin or to the damage to the liver graft during preservation and reperfusion. The aim of this study was to explore the influence of cyclosporin as well as of liver transplantation on bile secretion. Bile flow was studied in an experimental model in the rat. In syngeneic liver-transplanted animals, the bile flow was increased compared to the bile flow in the control group (1.29±0.09 ml/h vs 0.66±0.03 ml/h; P<0.01), mainly due to an increased bile acid-independent flow (0.76 ml/h vs 0.50 ml/h; P<0.01). The findings in the livertransplanted rats contrasted with those in a group of nontransplanted animals treated with cyclosporin. Cyclosporin treatment resulted in a reduced bile acid-independent fraction (0.37 ml/h vs 0.50 ml/h, P<0.05) of the bile flow, although no biochemical signs of hepatotoxicity were present. This reduction in the bile acid-independent fraction could, however, not be demonstrated when cyclosporin was given to a group of liver-transplanted rats, although a reduced total bile flow was recorded in the 1st hour measurements. In contrast to previous studies, we found that the cyclosporin vehicle (Cremophor EL), when administered chronically, induced a higher bile flow than that in the control rats. This effect was not seen in the transplanted rats. Our findings in this experimental rat model indicate that cyclosporin will influence and reduce bile secretion and bile acid secretion even if no other signs of liver dysfunction are present. On the other hand, the preservation and reperfusion in this model resulted in an increased bile flow, while bile acid secretion remained constant.     

Synergistic effects of nafamostat mesilate rinse and Kupffer cell blockade for rat liver preservation

Abstract  We investigated the efficacy of a new rinse solution containing nafamostat mesilate (NM) (a serine protease inhibitor) for liver preservation with modulation of Kupffer cell function. Orthotopic liver transplantation (OLT) was performed in male Lewis rats after 24 h of cold storage in University of Wisconsin organ preservation solution. After OLT, survival was determined, together with assays of blood chemistry, tissue NM metabolites, and histology 3 h after OLT. NM rinse was found to have a cytoprotective effect on liver parenchymal cells, based on enzyme data showing that NM rinse reduced the release of serum alanine aminotransferase significantly in comparison with saline rinse ( P < 0.05). However, the effect was not sufficient to improve the survival rate. In contrast, when the donor was treated with gadolinium chloride 24–30 h before graft harvest, NM rinse improved the survival rate to around 80 % compared with 25 % for saline. The assay of NM metabolites in grafted liver tissue showed that pretreatment of the donor rats with GdCl₃ delayed the degeneration of NM in the liver tissue. These data demonstrate that NM rinse and Kupffer cell blockade exert synergistic effects, leading to increased survival after cold-preserved liver transplantation.     

Kupffer细胞在梗阻性黄疸致肝损害作用中的研究进展

在梗阻性黄疸(obstructive jaundice,OJ)的病理过程中,肝脏是最容易受到损害的器官。梗阻性黄疽致肝损害的机制是复杂多样的。Kupffer细胞作为肝脏内的巨噬细胞,参与了梗阻性黄疸致肝损害的诸多环节。梗阻性黄疸致肝损害的首要病理因素是内毒素血症(Endotoxemia)的形成。当人血的内毒素浓度达到一定程度后就可激活Kupffer细胞,被激活的Kupffer细胞不仅可产生大量的炎性因子导致肝损害,而且还加剧内毒素血症的形成、参与肝脏炎性反应、氧化应激等病理过程来损害肝细胞。