Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model

AIM： TO investigate Kupffer cell dynamics and phagocytic activity, using a rat nonalcoholic steatohepatitis （NASH） model.
METHODS： Male F344 rats were fed either a control diet or a choline-deficient L-amino aciddefined （CDAA） diet, followed by contrast enhanced ultrasonography （CEUS） using Levovist. The uptake of latex beads by the Kupffer cells was determined by fluorescent microscopy. The status of the Kupffer cells was compared between the two groups, using the immunohistochemical staining technique.
RESULTS： After 4 or more wk of the CDAA diet, CEUS examination revealed a decrease in the signal intensity, 20 min after intravenous Levovist. Fluorescent microscopic examination showed that the uptake of latex beads by the Kupffer cells was reduced at week 1 and 2 in the study group, compared with the controls, with no further reduction after 3 wk. Immunohistochemical staining revealed no significant difference in the Kupffer cell counts between the control group and the CDAA group.
CONCLUSION： CEUS examination using Levovist demonstrated reduced contrast effect and phagocytic activity in the liver parenchymal phase, although the Kupffer cell numbers were unchanged, indicating reduced phagocytic function of the Kupffer cells in the rat NASH model. We believe that CEUS examination using Levovist is a useful screening modality, which can detect NASH in fatty liver patients.     

Iron Chelator Deferoxamine Reduces Preneoplastic Lesions in Liver Induced by Choline-Deficient L-Amino Acid-Defined Diet in Rats

The aim of this study was to investigate whetheran iron chelator, deferoxamine (DFO) can prevent lipidperoxidation, resulting in reduced liver injury as wellas reducing preneoplastic lesions induced by a choline-deficient L-amino acid-defined(CDAA) diet. CDAA diet administration resulted in anincreased serum ALT level (367 ± 58) after twoweeks, but simultaneous DFO treatment for two weeksreduced this elevation of ALT as well asmalondialdehyde (MDA) production in the liver. Feedingrats a CDAA diet for 12 weeks led to the development ofsevere liver fibrosis and preneoplastic lesions detectedas enzymealtered lesions. DFO treatment preventedthe expression of activated stellate cells, resulting inthe reduction of liver fibrosis as well as reducing thedevelopment of preneoplastic lesions. These results indicate that iron chelation can reducethe development of preneoplastic lesions in a CDAA dietmodel.     

The peroxisome proliferator-activated receptor-gamma agonist, pioglitazone, inhibits fat accumulation and fibrosis in the livers of rats fed a choline-deficient, l-amino acid-defined diet.

Administration of a choline-deficient, l-amino acid-defined (CDAA) diet to rats causes steatohepatitis, hepatic fibrosis, and hepatocellular carcinoma, a pathology similar to that observed in non-alcoholic steatohepatitis (NASH). The aim of this study was to evaluate if a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, pioglitazone (PGZ), could ameliorate CDAA diet-induced fatty liver and cirrhosis. Rats were fed a CDAA diet for 1 week and were given the CDAA diet for an additional week with or without PGZ (2-week model). Also, after administration of the CDAA diet for 12 weeks, rats were administered the CDAA diet for an additional 4 weeks with or without PGZ (16-week model). The CDAA diet, administered for either one or 12 weeks, induced fatty liver or cirrhosis with up-regulation of hepatic PPAR-gamma expression, respectively. In the 2-week model, rats treated with PGZ for 1 week demonstrated significantly lower hepatic triglyceride content and serum levels of tumor necrosis factor-alpha. In the 16-week model, treatment for 4 weeks with PGZ ameliorated hepatic fibrosis with a decrease in the expression of procollagen, alpha-smooth muscle actin, and transforming growth factor-beta1 in comparison to rats without PGZ. These results suggest that PPAR-gamma agonist is a potential therapeutic modality to treat NASH.     

Angiotensin II type 1 receptor antagonist prevents hepatic carcinoma in rats with nonalcoholic steatohepatitis

Background

Angiotensin II type 1 receptor blockers (ARBs) have been reported to attenuate hepatic fibrosis in non-alcoholic steatohepatitis (NASH). However, it is uncertain whether ARBs prevent hepatocarcinogenesis in NASH even after hepatic fibrosis has developed.

Methods

Male Wistar rats were fed with a choline-deficient, l-amino acid-defined (CDAA) diet for 24 weeks, and then fed with the CDAA diet with telmisartan (2 mg/kg/day), a novel ARB, or vehicle for another 24 weeks. The liver histology and the expression of genes and proteins related to angiogenesis were investigated.

Results

The 24-week CDAA diet induced liver cirrhosis. The 48-week CDAA diet exacerbated liver cirrhosis, and developed hepatocellular carcinoma (HCC) in 54.6 % of the rats concurrently with increases of hypoxia-inducible factor-1α (HIF-1α) protein and vascular endothelial growth factor (VEGF) mRNA, which are potent angiogenic factors in the liver. Telmisartan inhibited hepatic fibrosis and preneoplastic lesions and prevented the development of HCC along with inducing decreases in HIF-1α protein and VEGF mRNA.

Conclusions

These data indicated that telmisartan may prevent hepatocarcinogenesis through the inhibition of hepatic angiogenesis even after liver cirrhosis has been established.     

Preventive effect of cyclosporin A on experimentally induced acute liver injury in rats

Abstract: We examined the effect of cyclosporin A (CsA) on the pathogenesis of acute experimental liver injury in rats induced by injection of heat-killed Propionibacterium acnes (P. acnes) and subsequent injection of lipopolysaccharide (LPS). Pretreatment with CsA significantly reduced serum alanine aminotransferase (ALT), serum tumor necrosis factor-α (TNF-α) production, without changing the TNF-α mRNA level in the liver, and plasma interferon-γ (IFN-γ), following LPS injection in this model. Twenty-four-hour mortality was also markedly improved, from 100% in the P. acnes plus LPS group to 0% in the CsA-pretreated group. Although direct addition of CsA to isolated hepatic macrophages from P. acnes-pretreated rats did not prevent the production of TNF-α and active oxygen species, isolated hepatic macrophages from P. acnes plus CsA-pretreated rats significantly reduced their production in response to the addition of LPS. These results suggest that CsA protects against P. acnes plus LPS-induced acute liver injury, not by direct inhibition of hepatic macrophage activation, but by indirect prevention of hepatic macrophage activation, presumably related to the reduction in plasma IFN-γ levels.     

HIF-1α对非酒精性脂肪性肝病小鼠肝组织的影响*

目的 探讨缺氧诱导因子1α(HIF-1α)在非酒精性脂肪性肝病(NAFLD)进展中的作用。方法 将30只雄性C57BL/6J小鼠随机分为对照组、高脂饮食模型组和蛋氨酸-胆碱缺乏饮食(MCD)模型组,每组10只,建立非酒精性脂肪性肝炎(NASH)模型,分别取肝组织行组织病理学检查,采用缺氧探针检测肝组织缺氧程度,采用Real-time PCR法检测肝组织HIF-1α mRNA水平,采用Western blot法检测肝组织HIF-1α蛋白表达。另取小鼠肝星状细胞JS-1细胞,分为对照组、腺病毒载体组和HIF-1α ShRNA腺病毒载体感染组,采用MTT法检测细胞活性,采用Real-time PCR法检测细胞1型胶原(COL1)和3型胶原(COL3)、TNF-α、IL-1β和TGF-β1 mRNA水平。结果 缺氧探针染色显示高脂饮食模型组和MCD模型组小鼠肝组织染色阳性率分别为85%和78%,显著高于对照组的7%(P<0.05);高脂饮食模型组和MCD模型组小鼠肝组织HIF-1α mRNA水平分别为对照组小鼠的2.1倍和1.6倍(P<0.05),HIF-1α蛋白表达水平也相应地增强;HIF-1α ShRNA腺病毒载体感染细胞活力仅为对照组的37%(P<0.05),细胞COL1、COL3、TNF-α、IL-1β、TGF-β1 mRNA水平也显著降低(P<0.05)。结论 HIF-1α能够促进小鼠NAFLD进展,抑制HIF-1α表达可能能延缓NAFLD进展,值得进一步研究。     

Inhibition of renin-angiotensin system attenuates liver enzyme-altered preneoplastic lesions and fibrosis development in rats

BACKGROUND/AIMS: It is suggested that the renin-angiotensin system (RAS) is involved in tumor development and fibrogenesis. The aim of the present study was to examine the effect of RAS inhibition on the liver enzyme-altered preneoplastic lesions and fibrosis development. METHODS: The effects of the clinically used angiotensin-I converting enzyme inhibitor (ACE-I), perindopril (PE), on two different rat model of liver carcinogenesis models induced separately by diethylnitrosamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet were studied. This CDAA model was also used to elucidate the effect of PE on liver fibrosis development. RESULTS: The immunohistochemical evaluation revealed that the glutathione S-transferase placental form (GST-P), and gamma-glutamyltransferase (GGT)-positive preneoplastic foci significantly decreased in the livers of the PE-treated groups. In CDAA-induced liver fibrosis model, PE revealed a marked inhibitory effect of liver fibrosis development. The hepatic hydroxyproline, serum fibrosis markers, alpha-smooth muscle actin (alpha-SMA) immunopositive cells in number, and alpha-(III) pro-collagen mRNA expression were significantly suppressed by PE treatment. These inhibitory effects of PE were achieved even at a clinically comparable dose (2 mg/kg per day). CONCLUSIONS: These results suggested that the RAS is involved in liver carcinogenesis and fibrosis development.     

枯否氏细胞在大鼠非酒精性脂肪性肝炎发病中的作用

目的：探讨枯否氏细胞大鼠非酒精性脂肪性肝炎（non-alcoholic steatohepatitis,NASH)发病中的作用,方法：19只雄性SD大鼠随机分为模型组（10只）和正常组（9只）,分别预高脂肪饮食和标准饮食饲养12周,HE梁色观察肝细胞切片病理学改变,透射电镜和溶菌酶免疫组织化学染色观察枯否氏细胞的数量和形态。结果：模型组大鼠均出现肥胖,高脂血症伴肝细胞大泡性脂肪变,小叶内炎症细胞浸润和坏死,与正常相比,模型组肝小叶内枯否氏细胞数显著增加,并呈活化状态,模型组枯否氏细胞变化与其肝病理学改变相一致,结论：高脂饮食大鼠肝脏枯否氏细胞增多,并可能与其脂肪性肝炎的发病有关。     

Hypoadiponectinemia accelerates hepatic tumor formation in a nonalcoholic steatohepatitis mouse model

BACKGROUND/AIMS: Adipose tissue produces a number of adipocytokines, including adiponectin, leptin, and tumor necrosis factor-alpha. Obesity, which is associated with low plasma adiponectin levels, is an independent risk factor for various liver diseases including nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the effects of adiponectin on the progression of NASH to cirrhosis and tumor formation using adiponectin-knockout (KO) mice. METHODS: Using a choline-deficient L-amino acid-defined (CDAA) diet-induced mouse NASH model, liver histology and oxidative stress markers were investigated in KO and wild-type (WT) mice. RESULTS: Hepatic steatosis was enhanced to a greater extent in KO mice, compared to WT mice after a 1-week CDAA diet. After 24 weeks, 6 out of 14 KO mice developed liver cirrhosis and hepatic tumors, whereas the 15 WT mice showed only simple steatosis. In KO mice, hepatic cytochrome P450 2E1 levels were upregulated, and markers of oxidative stress (thiobarbituric acid reactive substances, 8-hydroxydeoxyguanosine-positive cells) were significantly increased compared with WT mice. CONCLUSIONS: Our results indicate that lack of adiponectin enhances the progression of hepatic steatosis, fibrosis, and hepatic tumor formation in an animal model of NASH. Hypoadiponectinemia in obesity could be a risk factor for NASH-related hepatic tumor formation.     

枯否细胞在大鼠非酒精性脂肪性肝炎发病中的作用

目的　探讨枯否细胞在大鼠非酒精性脂肪性肝炎（non-alcoholic steatohepatitis,NASH）发病中的作用。方法　19只雄性SD大鼠随机分为模型组（10只）和正常组（9只）,分别予高脂肪饮食和标准饮食饲养12周。HE染色观察肝组织切片病理学改变,透射电镜和溶菌酶免疫组织化学染色观察枯否细胞的数量和形态。结果　模型组大鼠均出现肥胖、高脂血症伴肝细胞大泡性脂肪变、小叶内炎症细胞浸润和坏死。与正常组相比,模型组肝小叶内枯否细胞数显著增加,并呈活化状态;模型组枯否细胞变化与其肝病理学改变相一致。结论　高脂饮食大鼠肝脏枯否细胞增多,并可能与其脂肪性肝炎的发病有关。     

IFN-γ and TNF-α secretion by CD4+ and CD8+ TCRαβ+ T-cell clones derived early after allogeneic bone marrow transplantation

Abstract: Secretion of the potentially antileukaemic cytokines IFN-γ and TNF-α was investigated for CD4 ⁺ and CD8 ⁺ TCRαβ⁺ T-cell clones derived from 4 leukaemia patients 3–6 weeks after allogeneic BMT. We investigated cytokine secretion in response to the activation signal accessory cells + phytohaemagglutinin + Interleukin 2. All clones derived after BMT were capable of IFN-γ and TNF-α secretion, and both for CD4⁺ (n = 96) and CD8⁺ (n = 8) T cells quantities of IFN-γ and TNF-α were significantly correlated with one another. When comparing the overall results for posttransplant and normal T-cell clones derived from 2 bone marrow donors (n = 65), both CD4⁺ and CD8⁺ TCRαβ⁺ T-cell clones showed increased IFN-γ production, and CD4⁺ but not CD8⁺ clones showed a decreased TNF-α secretion. The results suggest that noncytotoxic T cells derived after allogeneic BMT can produce IFN-γ and TNF-α and may thus be capable of mediating antileukaemic effects.     

疏肝祛瘀通络降浊法对大鼠非酒精性脂肪性肝炎的保护作用

目的探讨非酒精性脂肪性肝炎大鼠内毒素性肝损伤机制及中药对其影响。方法用喂饲高脂饮食的方法建立非酒精性脂肪性肝炎大鼠模型。4周后用疏肝祛瘀通络降浊法分小、中、大剂量进行治疗,12周后处死测定血脂、ALT、内毒素(ET)、肿瘤坏死因子(TNF-α)和白细胞介素(IL-1β)的含量;免疫组化法观察肝组织CD14和核转录因子(NF-κB)的表达;RT-PCR检测脂多糖结合蛋白(LBP)和4型Toll样受体(TLR-4)mRNA的表达。同期正常饮食饲养大鼠作对照。结果第12周时,模型组大鼠腹主动脉血清内毒素水平较正常组明显升高,有显著性差异;中剂量治疗组大鼠血清ET、TNF-α、IL-1β水平明显低于模型组,差异有显著意义。模型组大鼠肝组织CD14阳性细胞数量明显增多,主要分布于肝窦内,部分呈灶型聚集;与模型组相比,中剂量治疗组大鼠肝组织CD14阳性细胞数量明显减少。模型组可见少量细胞核染色的NF-κB阳性细胞散在分布于汇管区。模型组肝组织LBPmRNA和TLR-4mRNA表达均明显上调,与正常组比较差异均有显著意义;中剂量治疗组大鼠肝组织LBPmRNA和TLR-4mRNA表达均较模型组明显下调,且有显著性差异。结论疏肝祛瘀通络降浊法对非酒精性脂肪肝有疗效,可能与其降低血清内毒素水平和下调肝组织内毒素相关受体表达继而减轻炎症性肝损害有关。     

脂多糖预处理对非酒精性脂肪性肝炎的影响

目的 探讨脂多糖(lipopolysaccharide,LPS)预处理对非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)的影响.方法 选取24只成年雄性Wistar大鼠,分为正常对照组、NASH组和LPS预处理组.NASH组饲以高糖高脂饲料;LPS预处理组饲料同肝损伤组,隔日皮下注射LPS 0.5 mg/kg;正常对照组饲以普通饲料;所有大鼠自由进食与饮水.于实验第9周末处死大鼠.制备肝组织切片,计数浸润肝组织的淋巴细胞;测定血浆内毒素水平和丙氨酸转氨酶(ALT)活性、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)含量.结果 NASH组血浆内毒素水平比正常对照组高;LPS预处理组血浆ALT水平、肝组织淋巴细胞计数与NASH组比较均显著降低;血浆TNF-α水平LPS预处理组与NASH组比较明显降低,而血浆IL-10则相反,差异有统计学意义(P＜0.05).肝组织切片HE染色结果显示,LPS预处理组与NASH组比较,肝细胞内脂肪空泡小、数量少,脂肪变性明显减轻.结论 小剂量LPS预处理与减轻NASH密切相关.其机制可能是LPS预处理影响了细胞因子的释放,导致Th1/Th2细胞因子失衡,Th1向Th2偏离,可能诱导了宿主的免疫耐受,表现为肝组织损伤减轻.

Abstract：

Objective To investigate the changes of Th1/Th2 cytokines and its relationship with lipopolysaccharide (LPS) in pretreatment of relieving nonalcoholic steatohepatitis (NASH).Methods The 24 male Wistar rats were randomly divided into 3 groups: normal control group, liver injury group and LPS pretreatment group. The rats were given normal diet in normal control group,high-sucrose and high-fat diet both in liver injury group and in LPS pretreatment group, and the rats in LPS pretreatment group were given hypodermic injection of LPS 0. 5 mg/kg every other day. The level of plasma endotoxin (ET), activity of alanine aminotransferase (ALT), content of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were determined. At the end of week 9, the rats were executed, and the liver tissue slices were prepared to investigate hepatic pathologic change by hematoxylin and eosin (HE) staining.Results The level of plasma ET was significantly higher in liver injury group than in normal control group. The level of plasma ALT and infiltrating lymphocytes in liver tissue were significantly lower in LPS pretreatment group than in liver injury group. The level of plasma TNF-α was significantly lower in LPS pretreatment group compared with liver injury group.In contrast, the level of plasma IL-10 was higher (P＜0. 05). Histology with HE staining showed that hepatocyte steatosis was obviously relieved with smaller lipid droplet in LPS pretreatment group than in liver injury group. Conclusions LPS pretreatment can alleviate high-sucrose and high-fat induced NASH. The disequilibrium of Th1/Th2 cytokines may be an important part of mechanism.     

模拟失重对大鼠实验性胃溃疡氧化应激状态的影响

目的探讨模拟失重对乙酸诱导的大鼠实验性胃溃疡氧化应激状态的影响及可能机制。方法 32只SD大鼠随机分为4组,即尾部悬吊7 d组、尾部悬吊14 d组和相应的同步对照组。采用乙酸烧灼法制备大鼠慢性胃溃疡模型,造模后第3天悬吊组大鼠采用尾悬吊法建立模拟失重动物模型。游标卡尺检测胃溃疡面积,生化比色法测定大鼠血清中丙二醛(MDA)含量,超氧化物岐化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)活性。结果与对照7 d组相比,悬吊7 d组大鼠溃疡面积显著增大(t=5.661,P<0.01);与对照14 d组比较,悬吊14 d组溃疡面积显著增大(t=4.233,P<0.01),血清MDA含量及SOD活性显著增高(t=2.641,P<0.05,t=5.758,P<0.01);与悬吊7 d组比较,悬吊14 d组溃疡面积显著减小(t=3.805,P<0.01),血清MDA含量及SOD活性显著增高。血清GSH-PX活性差异无统计学意义(P<0.05)。结论模拟失重可加重溃疡氧化应激反应,延迟溃疡愈合。     

Expression of tumor necrosis factor-α gene during allograft rejection following rat liver transplantation

Abstract: Background/Aims: Tumor necrosis factor-α (TNF-α) is believed to play a role in hepatic allograft rejection. However, the specific cellular population responsible for TNF-α production during hepatic allograft rejection is not known. Circulating monocyte-macrophage cells are the primary systemic sources of TNF-α. In the liver, Kupffer cells are the main producers of TNF-α. In this study, we determined which cells are involved in TNF-α production during allograft rejection after orthotopic liver transplantation. Methods: In situ hybridization was used to identify cells with TNF-α mRNA in the liver. Immunohistochemical staining with ED2 and ED3 was used to differentiate between cellular types (Kupffer cells versus infiltrating monocytes). To detect DNA fragmentation in liver cells, TdT-mediated biotin-dUTP nick-end labeling (TUNEL) was done. Studies were performed in the rat liver transplant model using rejecting (ACI to LEW) and non-rejecting (ACI to ACI) donor/recipient combinations. Results: In the control group, cells with TNF-α mRNA were rarely observed. In the rejection group, TNF-α mRNA was observed in mononuclear cells that were mainly within the vessels of the portal region and occasionally in the sinusoids. The cells with the signals for TNF-α mRNA were ED2-negative and ED3-positive. DNA fragementation was observed in hepatocytes as well as infiltrating mononuclear cells. Conclusions: The main producer of TNF-α may be infiltrating mononuclear cells such as monocyte-macrophage cells rather than Kupffer cells during allograft rejection after liver transplantation. Circulating monocyte-macrophages may play a role in the control of allograft rejection.     

The role of oxidative stress in NASH and fatty liver model.

The aim of this study was to investigate whether oxidative stress is related to the development of liver injury and an iron chelator, deferoxamine (DFO) can prevent lipid peroxidation resulting in reduced liver injury as well as reduce preneoplastic lesions induced by a choline-deficient l-amino acid-defined (CDAA) diet. CDAA diet administration resulted in an increased serum ALT level after one week. Hepatocytes in rat liver fed a CDAA diet showed malondialdehyde (MDA) accumulation. But simultaneous DFO treatment for one week reduced this elevation of ALT as well as MDA accumulation in the liver. Feeding rats a CDAA diet for 14 weeks led to the development of severe liver fibrosis and preneoplastic lesions detected as enzyme-altered lesions. DFO treatment also prevented the expression of activated stellate cells, resulting in the reduction of liver fibrosis as well as reducing the development of preneoplastic lesions. These results indicate that iron chelation can reduce the development of preneoplastic lesions in a CDAA diet model.     

Therapeutic effects of cytokine modulator Y-40138 in the rat alcoholic liver disease model

Background and Aim: Inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interferon‐gamma (IFN‐γ), induce liver injury in the rat alcoholic liver disease (ALD) model. Y‐40138 is known to suppress the pro‐inflammatory cytokines and augment the anti‐inflammatory cytokines. We investigated whether or not Y‐40138 may be effective as a novel immunotherapy in the rat ALD model. Methods: Male Wistar rats were fed Lieber‐DeCarli ethanol liquid diet. The effects of Y‐40138 treatment in the ALD models were assessed by analyzing the serum and the liver tissues. Results: The serum levels of alanine aminotransferase (ALT), TNF‐α, and IFN‐γ, and the liver levels of TNF‐α and IFN‐γ were significantly higher in the ethanol‐fed group than in the pair‐fed group. The immunohistochemistry of the liver TNF‐α and 4‐hydroxynonenal (4HNE), and the expressions of TNF‐α and IFN‐γ mRNA were increased, too. The gene expressions of interleukin‐10 (IL‐10) in the ethanol‐fed group were suppressed as compared with the pair‐fed group. The serum triglyceride (TG) and liver TG were increased, and Oil Red O and α‐smooth muscle actin (α‐SMA) staining showed greater expression by ethanol‐fed feeding. After administration of Y‐40138, enzyme linked immunosorbent assay and real‐time polymerase chain reaction of the liver showed that the increased TNF‐α and IFN‐γ were suppressed, and that IL‐10 was augmented. Moreover, ethanol‐induced lipid accumulation in the liver was suppressed by administering Y‐40138. Conclusions: Y‐40138 decreased the inflammation, fibrosis, oxidative stress, and lipid synthesis, and augmented the anti‐inflammatory cytokines of the liver. These results indicate that the multiple cytokine production modulator, Y‐40138, is a promising novel therapy for ALD.     

Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells

The production of interleukin-8 (CINC: cytokine-induced neutrophil chemo-attractant) from different cell populations in the rat liver was studied and cells related to the initiation of CINC production in lipopolysaccharide (LPS)-injected endotoxaemic rats were characterized. Sinusoidal endothelial cells (16.4 ± 10.6 ng/mL) produced significantly higher amounts of CINC in 24 h primary cultures compared with hepatocytes (0.9 ± 0.9 ng/mL; P < 0.05) and Kupffer cells (6.5 ± 5.1 ng/mL; P < 0.05). Lipopolysaccharide, tumour necrosis factor-α (TNF-α), and interleukin-1α (IL-1α) stimulated different liver cell populations to produce CINC; LPS mainly stimulated Kupffer cells, TNF-α stimulated hepatocytes and IL-1α stimulated all three types of cells. Intraperitoneal injection of LPS (4 mg/kg) caused CINC accumulation in non-parenchymal cells of the rat liver within 1 h of injection, as shown by immunohistochemical staining. In contrast, CINC-positive hepatocytes were not seen until 3 h after injection of LPS. Ethanol was not a direct inducer of CINC production by rat hepatocytes in vitro. These findings strongly suggest that non-parenchymal liver cells, including sinusoidal endothelial cells, are the main source of CINC. Our data also suggest that during endotoxaemia, CINC production is initiated by non-parenchymal cells and this is followed by production from hepatocytes.