Regulation of Human Monocyte Functions by Acute Ethanol Treatment: Decreased Tumor Necrosis Factorα, Interleukin-lβ and Elevated Interleukin-10, and Transforming Growth Factor-β Production

We and others have previously shown that even acute ethanol exposure has the capacity to modulate immune functions, particularly monocyte functions. Herein, we tested the hypothesis that acute ethanol treatment inhibits inflammatory, while increasing inhibitory cytokine production in human blood monocytes that, in tum, could contribute to the overall immune abnormalities seen after alcohol use. Our data show that in vitro treatment of blood monocytes with a physiologically relevant dose of alcohol (W mM) results in significantly decreased induction of tumor necrosis factor-α (TNFα) and interleukin (IL)-lβ by bacterial stimulation of either Gram-positive [staphylococcal enterotoxin B (SEB), 1 μg/ml of SEB] or Gram-negative [lipopolysaccharide (LPS), 1 μg/ml of LPS] origin both at the protein and mRNA levels. In contrast, acute ethanol treatment induces monocyte production of mediators with immunoinhibitory potential, including transforming growth factor-β and IL-10. We further show that ethanol not only induces monocyte/macrophage (Mø) IL-10 and transforming growth factor-β, but even augments bacterial (both LPS and SEB) stimulation-induced production of both of these cytokines. IL-10 is a potent inhibitor of Mø TNFα production. We found that ethanol-induced elevation in Mø IL-10 levels contributes to the decreased Mø TNFα production to bacterial challenge in eth-anol-exposed Mø. However, mRNA levels for TNFα are downregu-lated as early as 1.5 hr after ethanol treatment, suggesting that ethanol likely has an IL-10 independent, direct effect on early signaling events of TNF α induction.     

Effect of Alcohol on the Secretion of Tumor Necrosis Factor-α by Macrophages in the Presence of Rat Serum

Background It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To determine the pathophysiological roles of macrophages in alcoholic liver disease, we examined the effect of ethanol on TNF-α secretion of rat Kupffer cells, alveolar macrophages, and peritoneal macrophages in the presence or absence of LBP.
Methods Kupffer cells, alveolar macrophages, and peritoneal macrophages were isolated from male Sprague Dawley rats. After the preculture in the medium containing 0, 10, 50, and 100 mmol/liter of ethanol, TNF-α secretion by these cells incubated with 100 ng/ml of endotoxin in the presence or absence of LBP (1% rat serum) was determined.
Results In the absence of LBP, an addition of ethanol to the medium suppressed TNF-α secretion of alveolar macrophages. Kupffer cells and peritoneal macrophages were less affected. Addition of LBP led to marked enhancement (7- to 24-fold) of TNF-α secretion of macrophages either with or without ethanol in the medium. Although ethanol tended to suppress TNF-α secretion of these cells, alveolar macrophages were less affected in the presence of LBP.
Conclusions Serum LBP enhances the secretion of TNF-α by macrophages. Alveolar macrophages may be important for excessive production of TNF-α in chronic alcoholics with endotoxemia.     

细菌脂多糖诱导人外周血单核细胞肿瘤坏死因子α的合成及核因子-κB的活化

为研究细菌脂多糖对人外周血单核细胞肿瘤坏死因子α合成和核因子-kB活化的影响，采用密度梯度离心法分离人外周血单核细胞，经细菌脂多糖刺激后，应用酶联免疫吸附法检测肿瘤坏死因子α水平随刺激剂量和时间的变化，应用免疫细胞化学方法观察核因子-Kb在单核细胞的激活随刺激时间的变化。发现核因子-kB在接受刺激15min-1h之间，随刺激时间的延长逐渐从胞质向胞核转移，1h达高峰。细菌脂多糖呈浓度依赖性（1-1000μg/L）诱导肿瘤坏死因子α合成，肿瘤坏死因子α在刺激后1h出现，6h达到高峰，其合成出现在核因子-kB的活化后。以上提示细菌脂多糖通过激活单核细胞内核因子-kB来诱导炎性细胞因子肿瘤坏死因子α的合成。     

Effect of Prostaglandin E Receptor Subtype EP4 Selective Agonist on the Secretion of Tumor Necrosis Factor-α by Macrophages in Acute Ethanol-Loaded Rats

Background: It is suggested that endotoxin and proinflammatory cytokines play an important role in the development and progression of alcoholic liver disease. Recently, a prostaglandin receptor subtype EP4 agonist with cytoprotective effect has been developed. We examined the efficacy of an EP4 agonist ONO-AE1-437 on tumor necrosis factor-α (TNF-α) secretion of Kupffer cells, splenic macrophages, and alveolar macrophages in acute ethanol-loaded rats.
Methods: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from control and acute ethanol-loaded rats (5 mg/g body weight of ethanol, intraperitoneally). After the preculture in the medium that containing 0, 0.1, 1, 10, or 100 nmol/liter of ONO-AE1-437, TNF-α secretion of these cells stimulated by 100 ng/ml of endotoxin was determined for 3 hr.
Results: The amount of TNF-α secreted from alveolar macrophages was largest in both the control and the acute ethanol-loaded rats. Acute ethanol load enhances TNF-α secretion of splenic macrophages. The addition of ONO-AE1-437 significantly inhibited TNF-α secretion of Kupffer cells and splenic macrophages in both the control and the acute ethanol-loaded rats. Alveolar macrophages were less affected.
Conclusions: An EP4 agonist ONO-AE1-437 suppresses excess TNF-α secretion from macrophages and seems promising for future trial in patients with severe alcoholic hepatitis.     

Alveolar Macrophage Release of Tumor Necrosis Factor-α in Chronic Alcoholics without Liver Disease

Alcohol is an immunosuppressive drug, and chronic abuse has been associated with increased susceptibility to a variety of infections, including bacterial pneumonia and tuberculosis. Alveolar macrophages are the resident phagocytes of the lung and play a central role in lung host defenses against infection ranging from direct antibacterial activity to the release of proinflammatory cytokines such as tumor necrosis factor-α (TNFα). TNFα, in particular, plays a key role in the development of the early inflammatory response. In this study, we investigated the effects of chronic alcohol consumption on alveolar macrophage release of TNFα in vitro. We prospectively studied lipopolysaccharide (LPS)-stimulated release of TNFα from alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF) in 22 alcoholic (18 smokers, 4 nonsmokers) and 7 nondrinking healthy volunteers (3 smokers, 4 nonsmokers). The total number of cells recovered by bronchoalveolar lavage (BAL) and their differential distribution were not significantly different in alcoholics versus controls (43 ± 8 × 10⁶ and 39 ± 13 × 10⁶, respectively). However, the total number of cells recovered from BALF was significantly higher in smokers (51 ± 8 × 10⁶) than in nonsmokers (19 ± 5 × 10⁸). Spontaneous (basal) release of TNFα by alveolar macrophages was the same in alcoholics and controls. In contrast, LPS-stimulated release of TNFα was significantly suppressed in alcoholics compared with that of controls (1343 ± 271 vs. 3806 ± 926 U TNF/ml/10⁶ cells, respectively, p < 0.015). When controlled for smoking, LPS-stimulated TNFα production was suppressed in alcoholic nonsmokers (563 ± 413 U TNF/ml/10⁶) compared with control nonsmokers (5113 ± 1264 U TNF/ml/10⁶). LPS-stimulated TNFα production was also less in control smokers (2063 ± 386 U TNF/ml/10⁶ cells) than in control nonsmokers (5113 ± 1264 U TNF/ml/10⁶ cells). There was no difference in TNFα production between smoking alcoholics and smoking control subjects. We conclude that chronic alcohol consumption significantly suppresses LPS-stimulated alveolar macrophage production of TNFα. This effect is obscured if the subject also smokes. Because TNFα production is an important element in host defense, this may explain, in part, the susceptibility of chronic alcohol abusers to a variety of infections.     

Augmentation of Ethanol-Induced Cell Damage and Activation of Nuclear Factor-κB by Annexin IV in Cultured Cells

Background: We previously reported that the amount of annexin IV expression was increased in culture cells by exposure to ethanol. Here, to investigate the physiologic role of annexin IV, we analyzed ethanol-induced cytotoxicity and nuclear factor (NF)-κB activity by using annexin IV-overexpressed cells.
Methods: Annexin IV overexpression was performed by transfection of expression vector, in which annexin IV complementary DNA was ligated, to culture cells (rat glioma C6 cells and human neuroblastoma SH-SY5Y cells). Ethanol-induced cytotoxicity was assayed by measuring the mitochondrial enzyme (dehydrogenase) activity or trypan blue exclusion. NF-κB activity was measured by electrophoretic mobility shift assay with a κB-oligonucleotide probe.
Results: Ethanol-induced cytotoxicity was increased by overexpression of annexin IV in both C6 cells and SH-SY5Y cells. Annexin IV overexpression augmented ethanol-induced NF-κB activation.
Conclusions: Ethanol-induced increase in annexin IV expression might amplify ethanol-induced cytotoxicity via NF-κB activation.     

肝细胞核因子与肝细胞癌关系的研究进展

肝细胞核因子（HNFs）是一类在肝脏优势表达的转录因子,可调控多种重要肝细胞功能基因表达,在肝脏发育以及肝细胞分化、功能维持中起关键作用。近年来,越来越多的证据显示HNFs家族成员与肝细胞癌（HCC）的发生、发展密切相关。许多研究表明HCC细胞和组织中HNFs表达下调,利用基因工程手段上调HCC细胞中的特定HNF表达,可抑制肿瘤细胞增殖,诱导其向成熟肝细胞分化,为HCC的治疗提供了新的思路。     

Secondary alveolar echinococcosis in lymphotoxin-α and tumour necrosis factor-α deficient mice: exacerbation of Echinococcus multilocularis larval growth is associated with cellular changes in the periparasitic granuloma

The availability of mice carrying a deletion of LT-alpha and tumour necrosis factor (TNF)-alpha genes enabled us to investigate the role of the TNF during alveolar echinococcosis. We compared the growth rate of Echinococcus multilocularis in LT-alphaTNF-alpha +/+ mice to that of mice having either no or only one LT-alphaTNF-alpha functionnal allele. LT-alphaTNF-alpha -/- mice harboured a significantly higher parasite burden than did the other two populations at 5, 10, and 15 weeks of infection, and they did not survive thereafter. Liver metacestodes removed from these mice were alive and the dehydrogenase activities of peritoneal metacestodes were decreased. Liver lesions regressed in most wild-type mice. Indeed, dead parasites were cordoned by granulomas containing numerous macrophages and lymphocytes leading to focal liver fibrosis at an early stage of infection. In contrast, most of LT-alphaTNF-alpha -/- mice harboured metacestodes interspersed with leucocytes, realising purulent abscesses with secondary extensive irregular fibrosis at a late stage of infection. Heterozygous mice had behavioural characteristics intermediate between homozygous mutants and wild-type mice. Levels of E. multilocularis-specific delayed-type hypersensitivity and serum antibodies were slightly decreased in LT-alphaTNF-alpha -/- mice. This study shows that TNF-alpha and/or LT-alpha genes play an essential role in the immune protection mechanisms against E. multilocularis at the site of infection.     

CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells Have Divergent Effects on Intestinal Inflammation in IL-10 Gene-Deficient Mice

The regulatory effect of murine CD4⁺CD25⁺ T-cells in vivo appears to be dependent on the secretion of IL-10. The lack of IL-10 in the IL-10 gene-deficient mouse has a profoundly negative effect on the mouse’s regulation of the response to intestinal bacteria, resulting in severe enterocolitis. We investigated the effect of neonatal injection with wild-type CD4⁺CD25⁺ T-cells on the intestinal immune response in IL-10 gene-deficient mice. At the time of analysis, 8–15 weeks later, all mice demonstrated an increased, antigen-stimulated systemic response. However, the intestinal response was divergent with about half of the mice developing an intestinal inflammation with a high injury score, the other half demonstrating a remarkable reduction in injury score with a marked decrease in intestinal IFNγ release. Our data demonstrate that CD4⁺CD25⁺ T-cells can be activated in IL-10 gene-deficient mice and that this stimulation under stringent conditions has the potential to reduce intestinal inflammation.     

Thalidomide Enhances the Anti-Tumor Activity of Standard Chemotherapy in a Human Melanoma Xenotransplatation Model

It has been demonstrated that thalidomide's anti-angiogenic properties result in clear anti-tumor activity in a number of human malignancies. We studied thalidomide in a human melanoma severe combined immunodeficiency mouse xenotransplantation model. Thalidomide as a single agent showed a significant tumor reduction of 46% compared with the control group. Thalidomide combined with dacarbazine treatment markedly enhanced the anti-tumor effect of chemotherapy and showed a significant tumor reduction relative to the dacarbazine-only group (61%) and even more tumor reduction (74%) compared with the control group. We also measured clearly reduced levels of tumor necrosis factor-α in the thalidomide-treated group. A significantly lower microvessel density was encountered in the thalidomide treatment groups (thalidomide alone or combined with DTIC), underscoring the anti-angiogenic effect of thalidomide as a single agent as well as in combination with chemotherapy in this model. In line with these results, we observed a nearly 3-fold increase of apoptosis for the combination of thalidomide and DTIC compared with the rate of apoptotic cells in DTIC-only-treated melanoma xenotransplants. These data underline the rationale for combining dacarbazine—a cytotoxic agent—and thalidomide—an anti-angiogenic cytostatic agent—as a promising strategy for the treatment of melanoma.     

Herbal medicine 'Sho-saiko-to' induces tumour necrosis factor-α and granulocyte colony-stimulating factor in vitro in peripheral blood mononuclear cells of patients with hepatocellular carcinoma

‘Sho-saiko-to’ (TJ-9) is a Japanese herbal medicine that is commonly administered to patients with chronic viral liver disease in order to improve their overall physical condition and to prevent the development of liver cancer. The present in vitro study demonstrated that, by adding TJ-9 to cell cultures, there were dose-dependent increases in production levels of tumour necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) in peripheral mononuclear cells of patients with hepatocellular carcinoma accompanied by liver cirrhosis. Increases in the production of TNF-α and G-CSF in control cell cultures exposed to different herbal medicines were low, and this indicates the specificity of the responce increases in production of these cytokines to TJ-9. TNF-α and G-CSF are known to play important roles in the biological defence mechanism. Administration of TJ-9 may, therefore, be beneficial for patients afflicted with intractable liver diseases because it could mildly induce these cytokines.     

睾酮及氟他胺对雄兔动脉粥样硬化斑块稳定性与血清炎症因子的调节

目的探讨睾酮和氟他胺对动脉粥样硬化斑块稳定性与血清炎症因子的调节作用。方法主动脉病理切片行HE染色和Masson氏三色染色。血浆睾酮采用Advia Centaur免疫检测系统测定,血清肿瘤坏死因子和白细胞介素6采用放射免疫测定,血清可溶性细胞间粘附分子1和基质金属蛋白酶2应用酶联免疫吸附测定法检测。结果雄兔去睾丸后血浆睾酮水平显著降低,补充十一酸睾酮(每2周6mg/kg)使血浆睾酮恢复到正常水平,而且十一酸睾酮这一作用不受雄激素受体拮抗剂氟他胺的影响。去睾丸雄兔补充十一酸睾酮显著减小动脉粥样斑块的面积和内膜厚度,并使斑块纤维帽增厚、胶原含量增加。然而,同时补充雄激素受体拮抗剂氟他胺使去睾丸雄兔动脉粥样斑块的面积和内膜厚度增加,并使斑块纤维帽厚度减低、胶原含量下降。与假手术高脂喂养雄兔比较,去睾丸雄兔血清肿瘤坏死因子、白细胞介素6、可溶性细胞间粘附分子1和基质金属蛋白酶2显著升高;去睾丸雄兔补充十一酸睾酮后,血清肿瘤坏死因子、白细胞介素6、可溶性细胞间粘附分子1和基质金属蛋白酶2较单纯去睾丸雄兔明显降低;去睾丸雄兔同时补充氟他胺与十一酸睾酮,血清肿瘤坏死因子、白细胞介素6、可溶性细胞间粘附分子1和基质金属蛋白酶2水平再次显著升高。结论睾酮与氟他胺可以调节雄兔动脉粥样硬化斑块进展与斑块的稳定性,并影响其血清炎症因子水平。     

Suppression of macrophage interleukin-12 and tumour necrosis factor-α production in mice infected with Toxocara canis

Toxocara canis induces a predominantly Th2 type response with enhanced amounts of interleukin (IL)-4 and reduced amounts of interferon (IFN)-gamma in infected mice. In this study, we investigated the macrophage function of T. canis-infected mice from the standpoint of cytokine production. C3H/HeN mice were infected with T. canis by oral administration of embryonated eggs. Ten days after infection, macrophages were obtained from spleen and peritoneal cavity, were cultured with lipopolysaccharide, and cytokines in the culture supernatant were evaluated with enzyme-linked immunosorbent assay. Macrophages from T. canis-infected mice produced IL-1 and IL-6 equivalent to macrophages from normal mice. The production of IL-10 and tumour growth factor (TGF)-beta was enhanced, but IL-12 and tumour necrosis factor (TNF)-alpha production was diminished. The addition of IFN-gamma, anti-IL-10 antibody, anti-TGF-beta antibody or indomethacin did not restore the production of IL-12 and TNF-alpha by macrophages from T. canis-infected mice. Furthermore, the stimulation of normal macrophages with T. canis antigen in vitro induced IL-1alpha, IL-6, IL-10 and TGF-beta, but not IL-12 and TNF-alpha. These results indicate that cytokine producing pattern of macrophages is altered by T. canis-infection, and this altered macrophage function may play an important role in the modification of the immune response to T. canis.     

The Role of cAMP in Ethanol-Regulated β-Endorphin Release from Hypothalamic Neurons

We have previously shown that ethanol acutely stimulates immuno-reactive β-endorphin (IR-β-EP) release from hypothalamic neurons, whereas chronic administration of ethanol desensitizes these neurons. In the study reported herein, the role of the intracellular cAMP system in the ethanol-regulated IR-β-EP release from hypothalamic cells in primary cultures was investigated. Acute treatment with ethanol or with the cAMP analog, dibutyryl cAMP, revealed that both agents stimulate the release of IR-β-EP from the hypothalamic cells. Combined treatment of ethanol and the cAMP analog produced a synergistic effect on IR-β-EP release. Treatment with ethanol and a cAMP-elevating agent, forskolin, increased cAMP levels in cultured hypothalamic cells. However, prior exposure to ethanol reduced the cAMP-elevating responses of these neurons to ethanol and forskolin. These results indicate that the stimulatory and adaptive responses of IR-β-EP neurons to ethanol may involve the cAMP system.     

麝香保心丸对过氧化氢诱导人脐静脉内皮细胞凋亡及炎症因子表达的影响

目的探讨麝香保心丸对过氧化氢诱导人脐静脉内皮细胞增殖凋亡及炎症因子表达的影响。方法体外胶原酶消化法培养人脐静脉内皮细胞,5-溴脱氧尿嘧啶核苷渗入法测定细胞增殖活性,dUTP缺口末端标记法测定内皮细胞的凋亡,逆转录-聚合酶链反应法测定炎症因子单核细胞趋化蛋白1、白细胞介素6、核因子-κB-P65 mRNA表达水平,W estern b lotting测定内皮细胞核因子-κB-P65的蛋白水平。结果 (1)与对照组相比,过氧化氢组内皮细胞活性明显降低(P<0.05),麝香保心丸药物血清呈浓度依赖性抑制过氧化氢诱导内皮细胞损伤(P<0.05),与对照血清组相比,药物血清1 g组内皮细胞活性显著升高(P<0.05);(2)过氧化氢组内皮细胞大量凋亡,1 g麝香保心丸血清组只有少量细胞凋亡;(3)与对照组相比,过氧化氢组单核细胞趋化蛋白1、白细胞介素6、核因子-κB-P65 mRNA表达水平明显增高(P<0.05),麝香保心丸药物血清组呈浓度依赖性抑制上述指标的表达,1 g麝香保心丸血清组有明显降低,而对照血清组单核细胞趋化蛋白1、白细胞介素6、核因子-κB-P65 mRNA表达无明显差别(P>0.05);(4)与对照...     

The Production of Tumor Necrosis Factor-α by Macrophages in Rats With Acute Alcohol Loading

Background: It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To study the pathophysiological roles of macrophages in alcoholic liver diseases, we examined the production of TNF-α by rat Kupffer cells, splenic macrophages, and alveolar macrophages with acute alcohol loading in the presence or absence of LBP.
Methods: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from male Wistar rats given 5 mg/g body weight of ethanol intraperitoneally after an hour. The production of TNF-α by these cells incubated with endotoxin 100 ng/ml in the presence or absence of LBP (1% rat serum) was determined.
Results: Acute alcohol loading did not affect the production of TNF-α by Kupffer cells. With acute alcohol loading, splenic macrophages tended to produce more TNF-α. Alveolar macrophages produced more TNF-α than Kupffer cells, and although the production of TNF-α by alveolar macrophages tended to be suppressed by acute alcohol loading, the production of TNF-α by alveolar macrophages still remained high in the presence of rat serum.
Conclusions: Splenic macrophages and alveolar macrophages may be related to excessive production of TNF-α in acute alcoholics with endotoxemia.     

Effect of tauro-α-muricholate and tauro-β-muricholate on oestradiol-17β-glucuronide-induced cholestasis in rats

The effect of tauro-β-muricholate (βMC-tau) and tauro-α-muricholate (αMC-tau) on oestradiol-17β-glucuronide (E₂17G)-induced cholestasis was compared with that of taurourso-deoxycholate (UDC-tau) in rats. Like UDC-tau, αMC-tau and βMC-tau infused at the rate of 0.2 μmol/min per 100 g bodyweight (BW) completely inhibited the cholestasis induced by E₂17G infused at the rate of 0.06 μmol/min per 100 g BW for 20 min. These findings indicate that βMC-tau and αMC-tau are useful in protecting against various types of experimental cholestasis, as well as against bile acid-induced cholestasis.