脂质体包裹氯膦酸二钠剔除大鼠肝脏枯否细胞作用的研究

目的探讨脂质体包裹氯膦酸二钠剔除大鼠肝脏枯否细胞的作用。方法实验组大鼠给予脂质体包裹的氯膦酸二钠,对照组给予等量生理盐水。给药后不同时间点计数ED1、ED2阳性细胞数目;尾静脉注射印度墨汁判断枯否细胞吞噬碳素颗粒的情况;RT-PCR检测枯否细胞受体mRNA的表达情况。结果给药后2d大鼠肝脏吞噬碳素颗粒的枯否细胞消失,ED1、ED2阳性细胞基本消失,PCR检测不到肝脏枯否细胞受体mRNA的表达。给药后第8天ED1阳性细胞开始明显增多,至第11天其数目基本恢复正常。ED2阳性细胞至第11天开始增加,但平均每中倍视野仍仅有(4.8±1.7)个细胞,明显少于对照组的(17.7±2.1)个细胞,差异具有统计学意义(P0.01)。结论静脉单次注射脂质体包裹氯膦酸二钠至少在1周内能有效剔除肝脏枯否细胞。     

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.     

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.     

枯否细胞在肝移植脂肪供肝的缺血再灌注损伤中的作用研究进展

缺血再灌注损伤(ischemia-reperfusion injury,IRI)是肝移植的常见危险因素,与早期移植肝的无功能和功能障碍密切相关.当发生IRI时,会产生大量的氧自由基和炎症因子,引起肝脏损伤,枯否细胞(Kupffer cells,KCs)的激活与其密切相关.同时,由于肝移植供肝的需求不断增多,脂肪供肝在肝...     

Structural changes of Kupffer cells in rat liver following experimental thermal injury

Changes in plasma haemoglobin levels and morphology of Kupffer cells were studied in rats following lethal thermal injury. Severe haemolysis was observed immediately after thermal injury. The plasma haemoglobin levels rapidly increased to a maximum level 15 min after injury and then rapidly decreased with time. However, the values were still higher 5 h after injury than those found before injury. Soon after burning the Kupffer cells phagocytized not only circulating cell debris including erythrocyte membranes and degenerate leucocytes but also large amounts of haemoglobin. The degradation of phagocytized haemoglobin was relatively slow compared to that of other cell debris. This phagocytized haemoglobin is considered to inhibit the generation of bactericidal-free radicals and to depress Kupffer cell function. The number of Kupffer cells was markedly decreased 5 h after thermal injury, and probably relates to the persistent depletion of the reticuloendothelial system function which follows lethal thermal injury. In contrast to the decreased number of Kupffer cells, a small number of monocytic cells appeared in the sinusoidal spaces and adhered to the endothelial cells. These monocytic cells may transform into Kupffer cells.     

Inhibition of Kupffer cells reduced CXC chemokine production and liver injury

BACKGROUND: Cytokine production is a critical component of ischemia/reperfusion (IR) injury. In the liver, Kupffer cells produce cytokines and chemokines (i.e., cytokines with chemoattractant properties) that are important mediators in neutrophil recruitment and subsequent hepatocellular injury. Therefore, the role of Kupffer cells in chemokine production in hepatic IR injury was investigated. METHODS: Adult male C57BL/6 mice underwent 90 min of partial hepatic ischemia followed by various reperfusion times (i.e., 0, 1.5, 3, and 6 h). Gadolinium chloride (GC), which inhibits Kupffer cell activity, was administered to mice 48 and 24 h prior to ischemia. The control group received a corresponding volume of normal saline. Plasma levels of the cytokine macrophage inflammatory protein-2 (MIP-2), KC, and tumor necrosis factor (TNF)-alpha and liver mRNA were measured. Liver injury was assessed by plasma level of alanine transaminase (ALT) and histopathology. RESULTS: A reperfusion time-dependent liver injury occurred as indicated by increased levels of plasma ALT and histopathology. The injury was associated with increased plasma TNF-alpha, MIP-2, and KC and their hepatic mRNA expression and neutrophil infiltration into ischemic lobes of the liver. GC treatment significantly reduced the number of Kupffer cells as determined by the immunostained liver tissue sections. The extent of liver injury significantly decreased in GC-treated mice that were associated with decreased levels of plasma ALT, TNF-alpha, MIP-2, and KC and neutrophil infiltration. CONCLUSIONS: This study suggests that Kupffer cells are major contributors to cytokine production in hepatic IR and their modulation may serve as a potential target for therapeutic intervention.     

Kupffer cells infiltrate liver tissue early after ischemia-reperfusion and partial hepatectomy

Kupffer cells, ED2+macrophages of the liver, play an important role in liver damage and regeneration. It is proposed that Kupffer cells are stationary and regenerate after acute liver trauma by local proliferation. We analyzed their kinetics in three surgically relevant murine models of acute liver injury: partial liver resection, ischemia with reperfusion and sepsis. We found an early increase in ED2+cells after 0.5 h and a maximum after 12 h. These results suggest an infiltration of the cells early after the injury and a later local proliferation. These ED2+macrophages are localized predominantly periportally; nearly no macrophages are found pericentrally, except in the sepsis model. Therefore, a shifting of macrophages from portal to central seems to be unlikely, suggesting a hepatic zonation of homing factors.     

Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells

BACKGROUND: Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages. METHODS: An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-alpha were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-kappaB) was also analyzed using enzyme-linked immunosorbent assay. RESULTS: Dietary glycine significantly improved the survival rate of endotoxemic mice (P < .05), whereas serum alanine aminotransferase and TNF-alpha levels were significantly decreased at different time points (P < .05); IL-10 levels were increased (P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue (P < .05) was enhanced; and expression of TLR4 and TNF-alpha in liver tissue was downregulated (P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-alpha caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-kappaB DNA-binding activity was also significantly inhibited by glycine (P < .05, respectively). CONCLUSIONS: Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-alpha secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-kappaB/TNF-alpha may be a novel potential mechanism by which glycine inhibits KC activity.     

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。     

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.     

Kupffer cells in hepatocellular adenomas

Hepatocellular adenomas are usually visualized as defects on technetium-99m-sulfur colloid liver scans, a fact which has been attributed to the absence of phagocytic Kupffer cells in the tumors. To determine whether this is true, seven hepatocellular adenomas were subjected to immunoperoxidase staining for lysozyme, a marker of mononuclear phagocytes. The Kupffer cells were counted in the tumors and surrounding non-neoplastic liver. All hepatocellular adenomas studied were found to contain Kupffer cells. Three tumors had fewer Kupffer cells than the surrounding liver. Three had about the same number as the surrounding liver, and one had more Kupffer cells than the non-neoplastic liver. Thus, the lack of phagocytosis of colloid in liver scans is probably due to something other than a deficiency of Kupffer cells in the hepatocellular adenomas.     

The effects of the elimination of Kupffer cells in the isolated perfused liver from non‐heart‐beating rat

Abstract We examined the effect of elimination of Kupffer cells on the sinusoidal microcirculation in graft harvested from non‐heart‐beating donors (NHBD), focusing on the arachidonic acid cascade and cytokines. Cardiac arrest was induced by thoracotomy. Livers were harvested 30 min after thoracotomy and perfused by Krebs‐Henseleit bicarbonate buffer for 60 min after 6 h cold preservation. For the elimination of Kupffer cells, rats were pretreated liposome‐encapsulated dichloromethylene diphosphonate (KE group). Eicosanoids (TXB₂, 6‐keto‐PGF_1α, LTB₄) and cytokines (TNFα, IL‐1β) in the perfusate were measured. Histological examination was also carried out. In the KE group, the value of TXB₂ was suppressed completely and cytokines were reduced, and sinusoidal structures and hepatocytes were well protected. These results indicated that the elimination of Kupffer cells improved sinusoidal microcirculation in NHBD and liver transplantation using grafts from NHBD could be made to succeed by modulation of Kupffer cells.     

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

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.     

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.     

Physiological role of sinusoidal endothelial cells and Kupffer cells and their implication in the pathogenesis of liver injury

We reviewed the morphological characteristics and physiological functions of hepatic sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), both of which are major components of the hepatic sinusoid, and we showed the implication of these hepatic sinusoidal lining cells in the pathophysiology of the liver, based on our experimental studies. The most outstanding feature of SECs is that they are provided with numerous fenestrae, thereby allowing direct communication between the sinusoidal lumen and the space of Disse. Physiologically, SECs play a role in filtration function, endocytic function, and putative participation in the regulation of sinusoidal blood flow. As for KCs, they account for major portion of fixed macrophages in the entire body, and exhibit vigorous activity for phagocytosis, and produce many kinds of soluble mediators such as cytokines, prostanoids, oxygen radicals, and proteases. To determine whether these cells are implicated in pathophysiological processes in the liver we directed our attention to liver injury associated with sepsis and cold-preservation injury of liver tissue. In a septic rat model, we found that when KCs that included hepatic macrophages were activated, they released excess tissue-toxic mediators, probably leading to SEC damage. In the cold-preserved liver,we demonstrated that KCs were functionally activated and that the morphology of SECs was destroyed. When the liver was reperfused with plasma and a leucocyte suspension, hypercoagulability and increased leucocyte adherence occurred. In both experimental models, we demonstrated that KC blockade ameliorated the liver injury, and this was associated with the morphological improvement of SECs. Thus, we showed the pathogenetic implication of KCs and SECs, due possibly to microcirculatory disturbance in the hepatic sinusoid, and further emphasized the involvement of activated KCs in SEC impairment. Received for publication on Nov. 19, 1998; accepted on March 1, 1999