Iron overload impairs pro-inflammatory cytokine responses by Kupffer cells

AIM: The aim of the present study was to determine the effect of chronic iron overload on Kupffer cell cytokine production. METHODS: Kupffer cells were isolated from rats that were fed either a control or iron-supplemented diet for 12 months. Cytokine mRNA and protein levels were determined by using a ribonuclease protection assay and ELISA, respectively. RESULTS: Baseline levels of tumor necrosis factor-alpha, transforming growth factor-beta1, interleukin-6 and granulocyte macrophage colony stimulating factor were similar in iron-loaded and control Kupffer cells. Following the addition of lipopolysaccharide to control cells, tumor necrosis factor-alpha, interleukin-1alpha and interleukin-6 mRNA levels increased. Tumor necrosis factor-alpha mRNA and protein levels were reduced by 40 and 60%, respectively, in iron-loaded cells compared with controls following the addition of lipopolysaccharide. Interleukin-6 mRNA levels in iron-loaded Kupffer cells were also reduced. Granulocyte macrophage colony stimulating factor mRNA levels remained unchanged in controls, but were significantly elevated in iron-loaded cells. Tumor growth factor-beta1 mRNA and protein levels were similar in control and iron-loaded cells. CONCLUSION: Deposition of iron in Kupffer cells in chronic dietary iron overload results in an impaired pro-inflammatory cytokine response to lipopolysaccharide. Our observations may have relevance to the altered immune function observed in chronic iron-overload syndromes.     

库普弗细胞在非酒精性脂肪性肝病中的作用

库普弗细胞(KC)是定居于肝内的单核-巨噬细胞,其表面存在多种受体,可被多种配体激活,通过产生细胞因子、炎症介质和活性氧簇等在多种肝损伤的发病过程中起重要作用。近年的研究显示KC也参与非酒精性脂肪性肝病的发病机制,在胰岛素抵抗、氧应激和炎症过程中起重要作用,因此控制KC的活化将有助于减轻或阻止非酒精性脂肪性肝病的的发展。     

Kupffer细胞在非酒精性脂肪性肝病发病机制中的作用

Kupffer细胞是肝脏的非实质细胞,具有分泌细胞因子及吞噬、免疫等功能,并与多种肝脏疾病如肝损伤、肝纤维化、肝硬化等密切相关。近年来,国内外学者对其在非酒精性脂肪性肝病（NAFLD）发生机制中的作用给予高度重视。本文从脂质代谢、信号蛋白变化、活性氧、抗原递呈等方面对Kupffer细胞在NAFLD发病机制中的作用进行综述。     

库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响

目的 研究库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响。方法 封闭和不封闭BALB／c小鼠库普弗细胞(从尾静脉注射GdCl3或生理盐水27ml／kg体重)48h后，夹闭肠系膜上动脉1h后松夹，复制肠缺血再灌注模型。运用流式细胞术和酶联免疫法，分别检测缺血前、缺血60min、再灌注30min、60min肝细胞凋亡情况和血清肿瘤坏死因子的变化。结果 结果表明，肠缺血60min、再灌注30min、60min时，肝细胞凋亡数增多，血清肿瘤坏死因子水平逐渐升高；封闭库普弗细胞后，肝细胞凋亡数增多更显著，血清肿瘤坏死因子水平在同时间点上无显著差异。结论 库普弗细胞功能状态的变化对肠缺血再灌注时肝损伤有重要影响。     

CD14 expression and Kupffer cell dysfunction in non-alcoholic steatohepatitis: superparamagnetic iron oxide-magnetic resonance image and pathologic correlation

Background and Aim: Kupffer cell (KC) function and CD14 expression contributes to pathogenesis of non‐alcoholic steatohepatitis (NASH). However, these relationships remain unclear. We investigated the relationship of KC function with superparamagnetic iron oxide‐enhanced magnetic resonance imaging (SPIO‐MRI), histopathological severity of NASH, and number of CD14‐positive KCs in NASH. Methods: This retrospective study included 32 patients (24 with NASH and eight with simple steatosis) who had previously undergone SPIO‐MRI with T2‐weighted gradient‐recalled echo sequence. All subjects were diagnosed pathologically and were evaluated for necroinflammation grade, fibrosis stage, and number of CD14‐positive KCs. Patients with NASH and simple steatosis were compared by using the Mann–Whitney test to determine differences in percent reduction of liver‐to‐muscle signal intensity ratio (reduction‐%LMR), as a surrogate parameter of KC function, and number of CD14‐positive KCs. Kruskal–Wallis test and Pearson's correlation coefficient were used to analyze relation among reduction‐%LMR, histopathological severity and number of CD14‐positive KCs. Results: There were statistically significant differences in reduction‐%LMR and number of CD14‐positive KCs between NASH and simple steatosis patients (Mann–Whitney test, P < 0.001 for all comparisons). Reduction‐%LMR decreased with an increase in necroinflammation grade or fibrosis stage. The number of CD14‐positive KCs increased with an increase in necroinflammation grade and fibrosis stage (Kruskal–Wallis test, both, P < 0.001). A high correlation was seen between number of CD14‐positive KCs and reduction‐%LMR (Pearson r = 0.81; P < 0.001). Conclusions: KC phagocytic function evaluated with SPIO‐MRI correlated with histopathological severity and number of CD14‐positive KCs. These results support the concept that KC phagocytic dysfunction contributes to the pathogenesis of NASH.     

Adiponectin prevents progression of steatohepatitis in mice by regulating oxidative stress and Kupffer cell phenotype polarization

Aim:  We reported previously that hypoadiponectinemia enhances hepatic oxidative stress and accelerates progression of nonalcoholic steatohepatitis (NASH) in mice. However, the precise mechanism and preventive effects of adiponectin on NASH remain unclear. The aim of this study was to examine the effects of adiponectin on steatohepatitis using adiponectin-knockout (KO) mice and adenovirus-mediated adiponectin expression system.
Methods:  We used male KO mice and C57BL6/J (WT) mice fed methionine choline-deficient (MCD)-diet as a steatohepatitis model. Liver histology, hepatic oxidative stress markers, and hepatic gene expression levels were investigated. In addition, Hepa 1–6 cells, a mouse liver cell line, were cultured with or without recombinant adiponectin, and gene expressions were investigated by real-time RT-PCR.
Results:  After 2-week feeding of MCD diet, hepatic steatosis was enhanced and plasma alanine aminotransferase elevated in KO mice than in WT mice. In KO mice liver, thiobarbituric acid reactive substances increased, glutathione levels decreased, and mRNA expression levels of antioxidant enzymes (catalase, superoxide dismutase-1) downregulated. Adenovirus-mediated adiponectin expression prevented these changes in KO mice. Moreover, Kupffer cell infiltration was enhanced and mRNA levels of anti-inflammatory M2 macrophage markers (interleukin-10, arginase-1) were decreased in KO mice liver. In the in vitro study, adiponectin significantly increased catalase gene expression in Hepa 1–6 cells.
Conclusions:  Lack of adiponectin enhanced, and adiponectin administration prevented steatohepatitis progression in mice. These changes were due to the anti-oxidative effects of adiponectin, and its effects on Kupffer cells recruitment and phenotype polarization. Augmentation of adiponectin effects could be a useful preventive approach for NASH progression.     

Role of Kupffer cells in the outgrowth of colorectal cancer liver metastases

Colorectal cancer is one of the commonest malignancies in the "developed" world. The liver constitutes the main host organ for its distant metastases which, when present, augur a bad prognosis for the disease. Kupffer cells (KCs) are macrophages that constantly reside within the liver and form an effective first line defence against multiple harmful agents which reach the hepatic sinusoids via the portal circulation. KCs remove chemical compounds and dead or damaged cells, eliminate bacteria and protect against invading tumour cells. They may play a crucial tumouricidal role, exerting cytotoxic and cytostatic functions through the release of multiple cytokines and chemokines. Subsequently, colorectal metastasising cells are destroyed either by KC-performed phagocytosis or via the stimulation of other immune cells which migrate into the sinusoids and act accordingly. On the contrary, KC products, including cytokines, growth factors and matrix-degrading enzymes may promote liver metastasis, supporting tumour cell extravasation, motility and invasion. Current research aims to exploit the antineoplastic properties of KCs in new therapeutic approaches of colorectal cancer liver metastasis. Numerous agents, such as the granulocyte macrophage-colony stimulating factor, interferon gamma, muramyl peptide analogues and various antibody based treatments, have been tested in experimental models with promising results. Future trials may investigate their use in everyday clinical practice and compare their therapeutic value with current treatment of the disease.     

库普弗细胞内毒素受体CD14在大鼠肝损伤中的变化

目的动态观察四氯化碳(CCl4)诱导大鼠肝损伤过程中库普弗细胞膜上脂多糖(LPS)受体CD14表达变化及其在细胞因子分泌中的作用。方法以CCl4皮下注射诱导大鼠肝损伤。采用联合酶灌注消化、不连续密度梯度离心法分离不同时期大鼠肝脏库普弗细胞。将库普弗细胞与不同浓度内毒素孵育6h,收集培养上清并抽提细胞RNA。ELISA检测细胞培养上清中肿瘤坏死因子(TNF)α水平。RTPCR法检测库普弗细胞CD14mRNA表达。偶氮基质显色法测定大鼠血浆内毒素水平。结果经CCl4处理4周和6周,大鼠库普弗细胞在体外培养时TNFα基础分泌量明显高于正常大鼠(P<0.05)。在LPS刺激下,CCl4处理2、4和6周库普弗细胞的TNFα分泌水平明显增加(P<0.05),呈浓度依赖方式。CCl4组大鼠库普弗细胞CD14mRNA表达从2周开始增加,6周时最明显,8周时恢复至正常水平。大鼠外周血内毒素浓度在肝损伤过程中升高。结论在CCl4诱导的大鼠慢性肝损伤早期过程中,库普弗细胞内毒素受体CD14表达上调,对内毒素敏感性增加。库普弗细胞是肝脏早期炎症反应中的一个重要效应细胞。     

脂联素及其受体在酒精性肝病中的作用

脂联素是脂肪细胞特异分泌的一种脂肪因子,具有抗炎、促进脂肪酸氧化、减轻胰岛素抵抗等作用,在酒精性肝病患者及动物模型中,脂联素水平均下降,提示脂联素在酒精性肝病的发病中可能起重要作用,进一步研究其具体作用将为酒精性肝病的防治提供新的思路.     

The role of adiponectin in the pathogenesis and treatment of non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) is recognized as the most common type of chronic liver disease in Western countries and the leading cause of cryptogenic cirrhosis. Insulin resistance (IR) is a key factor in the pathogenesis of NAFLD, the latter being considered as the hepatic component of IR or metabolic syndrome (MetS). Although the pathogenesis of NAFLD is not fully elucidated, a complex interaction between adipokines and cytokines produced by adipocytes and/or inflammatory cells infiltrating adipose tissue appears to play a crucial role in MetS and NAFLD. Adiponectin is the most abundant and adipose‐specific adipokine. In the liver, adiponectin acts through the activation of 5‐AMP‐activated protein kinase and peroxisome proliferator‐activated receptor‐α pathways and inhibition of toll‐like receptor‐4 mediated signalling. There is an evidence that adiponectin decreases hepatic and systematic IR and attenuates liver inflammation and fibrosis. Adiponectin generally predicts steatosis grade and severity of NAFLD, but it remains to be addressed to what extent this is a direct effect or related to the presence of more severe IR. Although there is no proven pharmacotherapy for the treatment of NAFLD, recent therapeutic strategies have focused on the indirect upregulation of adiponectin through the administration of various therapeutic agents and/or lifestyle modifications. Weight loss, through diet, lifestyle changes and/or medications including orlistat, sibutramine, rimonabant or bariatric surgery, increase adiponectin and may improve liver histology. Insulin sensitizers, including pioglitazone and rosiglitazone, and lipid‐lowering agents, including statins and fibrates, also upregulate adiponectin and ameliorate liver histology. The wider use of new treatment approaches appears to signal the dawn of a new era in the management of NAFLD. In this adiponectin‐focused review, the pathogenetic role and the potential therapeutic benefits of adiponectin in NAFLD are systematically analysed.     

Serum procollagen-III-peptide: Failure to reflect the extent of hepatic fibrosis

Abstract In order to test the hypothesis that serum levels of the amino terminal propeptide of type III procollagen (PPCP III) reflect hepatic fibrosis, we have studied PPCP III levels in 30 patients with genetic haemochromatosis (GH), a disease which is characterized by progressive fibrosis without significant inflammation or necrosis. Patients with alcoholic liver disease and chronic hepatitis were included as comparative diseases in which fibrosis occurs concurrently with inflammation and necrosis. Of 13 GH cases with cirrhosis, four (30%) had normal serum PPCP III levels, while of 17 GH cases without cirrhosis, two (12%) had elevated levels. The mean serum concentrations of the cirrhotic and non-cirrhotic GH groups were not significantly different when patients with excessive alcohol consumption (> 80 g/day) were excluded from the GH groups. In 29 subjects with alcoholic liver disease, serum PPCP III correlated significantly with both fibrosis ( P < 0.01) and necrosis ( P < 0.02) but not with inflammation. In 23 subjects with chronic hepatitis, PPCP III levels correlated significantly with inflammation when assessed histologically ( P < 0.01) or as reflected by serum AST ( P < 0.01), but not with fibrosis or necrosis. Furthermore, the correlation between PPCP III and inflammation was not strengthened when the three features (inflammation, necrosis and fibrosis) were combined into a single variable. We conclude that elevated PPCP III levels in chronic liver disease do not reflect solely the extent of fibrosis but are also influenced by inflammation and necrosis and are thus of limited clinical value in predicting hepatic histopathology.     

脂联素在肝纤维化发生中的作用

脂联素（ADN）具有多种生物学作用。近年研究表明其可抑制肝纤维化的发展。一些动物实验显示ADN基因敲除（ADN-KO）鼠易发生纤维化,可能与ADN具有抗氧化应激及抗炎作用有关。肝星状细胞（HSC）在肝纤维化中起核心作用,ADN可抑制HSC增殖、迁移,促进其凋亡。临床研究报道ADN与慢性肝病的纤维化进展有关,为肝纤维化的无创性诊断提供新的方向。进一步分析ADN的抗肝纤维化机制有重要意义。     

脂联素与非酒精性脂肪肝病

脂联素(adiponectin,ADP)作为一种新发现的脂肪激素,主要是由脂肪细胞分泌的,在肥胖者、糖尿病及非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)患者中血清ADP水平低于正常.ADP有胰岛素增敏作用,与胰岛素抵抗相关;能够使肝脏肿瘤坏死因子(tumor necrosis factor-alpha,TNF-α)分泌下降,减少肝中脂肪堆积和炎症介质作用,对ADP的研究将为探讨NAFLD的发病机制及治疗方案提供新的线索.     

CD14 expression on Kupffer cells during the course of carbon tetrachloride‐mediated liver injury

OBJECTIVE: To investigate the expression of CD14 on Kupffer cells during the course of carbon tetrachloride (CCl₄)‐mediated liver injury and its role in the activation of Kupffer cells. METHODS: Rats were administered CCl₄ twice weekly for up to 8 weeks. Kupffer cells were isolated from normal and CCl₄‐treated rats by the combined ‘collagenase‐pronase’ perfusion method, discontinuous density gradient centrifugation. On the day after isolation, the cells were incubated with RPMI‐1640 containing varying doses of lipopolysaccharide (LPS) for 6 h. Supernatants were then collected for measuring the concentration of tumor necrosis factor‐alpha (TNF‐α) by enzyme‐linked immunosorbent assay (ELISA). The expression of CD14 mRNA on Kupffer cells were determined by RT‐PCR. The plasma concentrations of endotoxin were determined by chromogenic substrate Limulus amebocyte lysate assay. RESULTS: Basic TNF‐α production of Kupffer cells isolated from CCl₄‐treated rats at 4 and 6 weeks was significantly higher than that of normal (P < 0.05). Following LPS stimulation the production of TNF‐α was markedly increased in Kupffer cells from the 2‐, 4‐ and 6‐week treatment groups (P < 0.05). Moreover, LPS‐induced TNF‐α production was dose‐dependent. CD14 mRNA expression on Kupffer cells isolated from CCl₄‐treated rats was elevated following 2 weeks of CCl₄ administration and the maximum elevation occurred at 6 weeks. Gene expression was decreased in Kupffer cells after 8 weeks of CCl₄ treatment. CCl₄ administration elicited extensive changes in liver morphology, including steatosis, inflammation and necrosis. The plasma concentrations of endotoxin of CCl₄ ‐ treated rats were increased during the time of liver injury. CONCLUSION: Up‐regulation of CD14 expression in Kupffer cells during CCl₄‐mediated chronic liver injury indicates cell activation and that they are more sensitive to LPS stimulation. Kupffer cells are critical effector cells in the early stage of liver injury.     

Alcoholic hepatitis: The pivotal role of Kupffer cells

Kupffer cells play a central role in the pathogenesis of alcoholic hepatitis (AH). It is believed that alcohol increases the gut permeability that results in raised levels of serum endotoxins containing lipopolysaccharides (LPS). LPS binds to LPS-binding proteins and presents it to a membrane glycoprotein called CD14, which then activates Kupffer cells via a receptor called toll-like receptor 4. This endotoxin mediated activation of Kupffer cells plays an important role in the inflammatory process resulting in alcoholic hepatitis. There is no effective treatment for AH, although notable progress has been made over the last decade in understanding the underlying mechanism of alcoholic hepatitis. We specifically review the current research on the role of Kupffer cells in the pathogenesis of AH and the treatment strategies. We suggest that the imbalance between the pro-inflammatory and the anti-inflammatory process as well as the increased production of reactive oxygen species eventually lead to hepatocyte injury, the final event of alcoholic hepatitis.     

Early studies on the safety and efficacy of thalidomide for symptomatic inflammatory bowel disease

BACKGROUND AND AIM: Thalidomide is clinically effective in the treatment of graft versus host disease in bone marrow transplantation and aphthous ulceration in HIV infection. It appears to exert a selective effect on tumor necrosis factor-alpha (TNF-alpha) production. Tumor necrosis factor-alpha is implicated in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to assess the efficacy and safety of thalidomide in symptomatic IBD. METHODS: Eleven patients (nine males, mean age 33 years, range 20-77 years) with chronic inflammatory bowel disease (six Crohn's disease (CD), four ulcerative colitis (UC), one indeterminate colitis (IC)) who were symptomatic despite standard medical therapy were administered a daily dose of thalidomide for 12 weeks in an open-labeled protocol. Their response was assessed by using clinical, colonoscopic, histological, and immunological methods. RESULTS: Two patients withdrew at 3 weeks because of mood disturbances. Of the remaining nine patients, eight (five CD, two UC and one IC) had a marked clinical response, while one patient with CD had no response. The mean stool frequency decreased from 4.3 to 2.3 per day (P = 0.0012), and the stool consistency increased from 2.1 to 1.2 (P = 0.02). The mean Crohn's Disease Activity Index decreased from 117 to 48 (P = 0.0008). Endoscopic inflammatory and histological grade, C-reactive protein and erythrocyte sedimentation rate (ESR) all decreased significantly (P = 0.011, P = 0.03, P = 0.023 and P = 0.044, respectively). However, the serum TNF-alpha levels did not change. Side-effects included mild sedation, xerostomia and skin dryness in all, constipation in three, and minor abnormalities in nerve conduction in one patient. CONCLUSION: These data strongly suggest that thalidomide is an effective short-term treatment for symptomatic IBD.