Adaptation to bacterial lipopolysaccharide controls lipopolysaccharide-induced tumor necrosis factor production in rabbit macrophages.

These experiments provide an explanation for the observation that two intravenous injections of lipopolysaccharide (LPS) spaced 5 h apart in rabbits cause tumor necrosis factor/cachectin (TNF) levels to rise in the blood only after the first LPS injection. Herein we show that treatment of elicited peritoneal exudate rabbit macrophages (PEM) with two doses of LPS given 9 h apart results in a marked reduction in TNF production by the second LPS exposure. This state of hyporesponsiveness is a result of adaptation to LPS, is induced by LPS concentrations that are 1,000-fold less than required to induce TNF production (picograms vs. nanograms), is characterized by a decrease in LPS-induced TNF mRNA without any change in TNF mRNA half-life, is not changed by including indomethacin in cultures, and is specific for LPS since LPS-adapted cells display a TNF response to heat-killed Staphylococcus aureus that is at least as good as that observed in control PEM.     

Lead enhances lipopolysaccharide and tumor necrosis factor liver injury.

Lead markedly augments the lethality of endotoxin lipopolysaccharide (LPS) in rats. In this model of LPS toxicity, the liver is severely injured. Much of the tissue injury produced by LPS is thought to be mediated by the cytokine tumor necrosis factor (TNF). Tumor necrosis factor recently has been speculated to be a mediator of several models of liver injury such as that produced by galactosamine. To investigate the possible role of TNF in the lead-enhanced LPS toxicity model, we administered doses of lead acetate (15 mg/kg), LPS (100 micrograms/kg), or TNF (6.25 x 10(6) U/kg) that produced minimal changes in liver enzymes. However, when lead was administered simultaneously with either LPS or TNF, serum aspartate transaminase, alanine transaminase, alkaline phosphatase, glutamyl transpeptidase, and plasma triglyceride levels were markedly increased. Lead + LPS treatment increased both peak serum TNF concentrations and TNF "area under the curve" as compared with LPS alone. We conclude that lead not only enhances LPS lethality but also LPS liver injury. Furthermore, lead enhances TNF liver injury and increases LPS-stimulated serum TNF levels. These data suggest that the lead-enhanced LPS model offers a system for studying TNF-induced liver injury.     

Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice.

To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 microM), 4'-chlorodiazepam was the most effective agent, clonazepam was the least effective agent, and midazolam had an effect intermediate between those of the other two ligands. The peripheral benzodiazepine receptor ligands had a dose-dependent suppressive effect, and the 50% inhibitory concentrations were 0.01 microM for 4'-chlorodiazepam and 5 microM for midazolam. Concomitant use of PK 11195 (10 microM), an antagonist of the peripheral benzodiazepine receptor, reversed this suppressive effect with 4'-chlorodiazepam (10 microM) or midazolam (10 microM). PK 11195 showed this antagonistic effect in a dose-dependent manner. Intravenous 4'-chlorodiazepam (5 mg/kg of body weight) significantly suppressed LPS (100-micrograms)-induced TNF activity of sera (2 h postchallenge with LPS) from thioglycolate-treated mice. The present findings suggest that the peripheral benzodiazepine receptor plays an important role in modulating LPS-induced TNF activity in mouse macrophages.     

Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)alpha production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNF alpha production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNF alpha production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNF alpha production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNF alpha production. We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.     

Role of tumor necrosis factor in sepsis and acute lung injury

TNF is a small protein secreted by activated monocytes and macrophages that mediates the in vivo effects of endotoxin. When injected into experimental animals, TNF reproduces the picture of septic or endotoxin shock. In addition, antibodies to TNF protect animals against the deleterious effects of IV injections of either LPS or live bacteria. Specifically, the available evidence suggests that TNF may be necessary for the organ injury and failure seen in sepsis. However, TNF probably is not the final common pathway to shock and tissue injury. Inhibition of cyclooxygenase is protective from the lethal effects of both LPS and TNF infusion, suggesting that prostanoids play an important, and perhaps more proximal role in the generation of tissue injury. In addition, TNF is produced and cleared from the blood-stream within a short period of time after an LPS stimulus, suggesting that TNF sets into motion a chain of events that may be self-perpetuating even in the absence of further TNF stimulus. In the near future, the treatment of sepsis may involve the administration of antibodies both to TNF and to LPS. Cyclooxygenase inhibitors should also begin to play a role in the therapy of sepsis. In the more distant future it is likely that we will be able to manipulate the state of activation of genes that code for TNF to exert some control over its production and secretion. It is perhaps within our grasp to finally reduce the morbidity and mortality of this lethal condition.     

Hemoglobin infusion augments the tumor necrosis factor response to bacterial endotoxin (lipopolysaccharide) in mice.

OBJECTIVE: To determine whether cell-free hemoglobin augments the inflammatory cascade, as detected by production of tumor necrosis factor (TNF) elicited by bacterial endotoxin (lipopolysaccharide [LPS]). DESIGN: In vivo and ex vivo study, using a mouse model of sepsis. SETTING: Animal research facility SUBJECTS: Female Swiss Webster mice. INTERVENTIONS: For the in vivo experiments, an LD50 dose (500 microg) of Escherichia coli LPS was injected intraperitoneally into mice. Cell-free crosslinked hemoglobin (60 mg/mouse) or saline was administered intravenously 10 hrs before or coincident with LPS. For the ex vivo experiments, hemoglobin (60 mg/mouse) or saline was administered intravenously to mice, and, 10 hrs later, hepatic Kupffer cells, peripheral blood mononuclear cells, or peritoneal macrophages were isolated. MEASUREMENTS AND MAIN RESULTS: Intravenous infusion of hemoglobin either 10 hrs before or coincident with intraperitoneal LPS resulted in a peak of plasma TNF that was greater than in control mice administered LPS only. Cultured Kupffer cells, isolated from mice that had received hemoglobin in vivo 10 hrs before cell collection, produced more TNF in response to LPS in vitro than cells from normal mice. A trend toward greater TNF production in vitro by peripheral blood mononuclear cells obtained from hemoglobin-treated mice also was observed. Enhanced sensitivity to LPS was not observed with cultured peritoneal macrophages from mice that had received hemoglobin. CONCLUSIONS: Intravenous hemoglobin increased the sensitivity of hepatic macrophages to subsequent stimulation by LPS. This effect may contribute to the increased mortality that we have observed in animals that have received both LPS and hemoglobin.     

大鼠严重烫伤并发心肌损害时肿瘤坏死因子的变化

目的：探讨炎症介质在严重烧伤后心肌损害中的作用。方法：采用大鼠３０％ 体表面积Ⅲ度烫伤模型,随机分为对照组（１０ 只）和烫伤组（５０ 只）,于烫伤后１、３、６、１２ 和２４ 小时检测大鼠血浆肿瘤坏死因子（ＴＮＦ）和肌钙蛋白Ｔ（ＴｎＴ）及心肌组织中ＴＮＦ的含量。结果：烫伤后６ 小时血浆ＴＮＦ水平〔（３．３８±０．９０）μｇ／Ｌ〕较正常对照组〔（１．０８±０．０１）μｇ／Ｌ〕显著升高（Ｐ＜ ０．０１）,１２ 小时达峰值〔（８．０２±１．０５）μｇ／Ｌ〕,伤后２４ 小时〔（６．４４±１．４３）μｇ／Ｌ〕虽有所下降,但仍显著高于正常对照组（Ｐ＜ ０．０１）。心肌组织ＴＮＦ含量伤后１２ 小时〔（２．１５±０．０９）ｎｇ／ｍ ｇ〕显著高于正常对照组〔（０．８８±０．０１）ｎｇ／ｍ ｇ,Ｐ＜ ０．０１〕。血浆ＴＮＦ水平与反映心肌损伤的敏感指标血浆ＴｎＴ的变化密切相关。结论：炎症介质ＴＮＦ在烧伤后心肌损害的发生发展中起重要作用     

肿瘤坏死因子α在肢体缺血再灌注损伤软骨中的表达

背景:肢体缺血再灌注损伤的研究起步相对较晚,且主要集中在对肢体骨骼肌、神经等软组织的研究,对肢体缺血再灌注后关节软骨的损伤目前国内外却较少报道.目的:观察肿瘤坏死因子a在肢体缺血再灌注损伤时不同时间段内软骨中的表达变化.方法:将SD大鼠随机分为假手术组、缺血6 h组、缺血6 h再灌注1,3,7,14,30,60 d组.建立大鼠肢体缺血再灌注损伤模型.分别于缺血再灌注后相应时间点处死动物,切取左胫骨平台内侧软骨组织做苏木精-伊红染色观察软骨的组织形态学变化,免疫组织化学检测软骨中肿瘤坏死因子α表达水平的变化.结果与结论:早期(7 d内)软骨组织学光镜下改变不明显;后期损伤加重,可见软骨变薄,排列紊乱,表面不完整,偶见软骨缺损.假手术组肿瘤坏死因子a阳性表达较弱,缺血6 h组阳性表达开始增强,再灌3,7 d组达到高峰,随后阳性表达下降;再灌60 d组与假手术组相比差异仍有显著意义(P<0.05).结果表明,肢体缺血再灌注可导致关节软骨的损伤.肢体缺血再灌注损伤时,关节软骨中肿瘤坏死因子a增加在软骨损伤中发挥重要作用.     

Interleukin 1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition.

In addition to activating T and B lymphocytes, interleukin 1 (IL-1) induces several hematologic and metabolic changes typical of host responses to infection and injury. We now report a new biological property, namely, the induction of hypotension. Rabbits given a single intravenous injection of recombinant human IL-1-beta (5 micrograms/kg) rapidly developed decreased systemic arterial pressure, which reached the lowest levels after 50-60 min and slowly returned to pre-IL-1 values after 3 h. Associated with the hypotension, systemic vascular resistance and central venous pressure fell, while cardiac output and heart rate increased. These responses were prevented by ibuprofen given 15 min before the IL-1. A bolus injection of IL-1 followed by a 2-h infusion sustained the hypotension and was associated with leukopenia and thrombocytopenia. Ibuprofen given at the mid-point of the infusion reversed the changes in all hemodynamic parameters, but had no effect on the leukopenia or thrombocytopenia. Tumor necrosis factor (TNF) also induced a shock-like state in rabbits. When the dose of IL-1 or TNF was reduced to 1 microgram/kg, no hemodynamic changes were observed; however, the combination of these low doses of both cytokines resulted in a profound shock-like state including histological evidence of severe pulmonary edema and hemorrhage. Pretreatment with ibuprofen prevented the hemodynamic, leukocyte, and platelet changes induced by the low-dose cytokine combination, and ameliorated the pulmonary tissue damage. These results demonstrate that IL-1, like TNF, possesses the ability to induce hemodynamic and hematological changes typical of septic shock, and that the combination of IL-1 and TNF is more potent than either agent alone. These effects seem to require cyclooxygenase products, and suggest that intravenous cyclooxygenase inhibitors may be of therapeutic value in patients with IL-1/TNF-mediated shock.     

山莨菪碱干预急性肺损伤家兔肺泡巨噬细胞中核因子κB和肿瘤坏死因子αmRNA的表达

目的:观察山莨菪碱对创伤性急性肺损伤家兔肺泡巨噬细胞中核因子κB活化和下游基因肿瘤坏死因子αmRNA表达的影响。方法:实验于2004-03/2005-02在解放军第四军医大学西京医院检验科临床分子生物学实验中心完成。将健康成年家兔72只抽签法随机分为3组(n=24):①山莨菪碱组:于撞击台上行胸壁撞击(冲击速度5.7m/s,力75.8N/cm2,冲撞能量约204J)后,立即静脉注射大肠杆菌脂多糖50μg/kg,建立创伤性急性肺损伤模型,造模后立即予以山莨菪碱2mg/kg静注,随后以1mg/(kg·h)静脉维持。②急性肺损伤组:造模同山莨菪碱组,造模后以等量生理盐水静注和维持。③正常对照组:不做胸壁撞击,静注等量生理盐水代替脂多糖,其余处理同急性肺损伤组。各组动物分别于模型建立后1,2,3,4h放血处死6只,留取支气管肺泡灌洗液,分离肺泡巨噬细胞,分别用凝胶电泳迁移率改变分析法和半定量反转录-聚合酶链反应法检测肺泡巨噬细胞中核因子κB的活性变化及肿瘤坏死因子αmRNA的表达水平,以相对吸光度表示。结果:67只兔进入结果分析。①核因子κB的活性:急性肺损伤组于模型建立后1～4h内均明显高于正常对照组(P<0.01),在2h达到高峰,其相对吸光度值,是同期正常对照组的8倍;山莨菪碱组高于正常对照组(P<0.01),显著低于急性肺损伤组(P<0.01),其中2h降幅最大,达30.19%。②肿瘤坏死因子αmRNA的表达水平:急性肺损伤组在模型建立后1h其表达即开始上升,两三小时达到高峰,3h最高,其相对吸光度值,是同期正常对照组的5倍,4h其表达较前有所下降,但仍明显高于正常对照组(P<0.01);山莨菪碱组各时间的表达显著低于急性肺损伤组(P<0.01,0.05),最大降幅出现在2h,达34.48%。结论:①机体遭受创伤及脂多糖刺激后,肺泡巨噬细胞被激活导致核因子κB的活化是肺泡巨噬细胞大量释放肿瘤坏死因子α等前炎细胞因子的关键环节。②山莨菪碱可能是通过抑制肺泡巨噬细胞核中核因子κB活性,在基因转录水平抑制其介导的一系列细胞因子和炎症递质的表达(肿瘤坏死因子α等),阻断细胞因子和炎症递质的失控性释放来实现对急性肺损伤的治疗作用的。     

山莨菪碱干预急性肺损伤家兔肺泡巨噬细胞中核因子κB和肿瘤坏死因子αmRNA的表达

目的：观察山莨菪碱对创伤性急性肺损伤家兔肺泡巨噬细胞中核因子κB活化和下游基因肿瘤坏死因子αmRNA表达的影响。 方法：实验于2004-03／2005-02在解放军第四军医大学西京医院检验科临床分子生物学实验中心完成。将健康成年家兔72只抽签法随机分为3组（n=24）：①山莨菪碱组：于撞击台上行胸壁撞击（冲击速度5．7m／s，力75．8N／cm^2，冲撞能量约204J）后，立即静脉注射大肠杆菌脂多糖50μg／kg，建立创伤性急性肺损伤模型，造模后立即予以山莨菪碱2mg／kg静注，随后以1mg／（kg&;#183;h）静脉维持。②急性肺损伤组：造模同山莨菪碱组，造模后以等量生理盐水静注和维持。③正常对照组：不做胸壁撞击，静注等量生理盐水代替脂多糖，其余处理同急性肺损伤组。各组动物分别于模型建立后1，2，3，4h放血处死6只，留取支气管肺泡灌洗液，分离肺泡巨噬细胞，分别用凝胶电泳迁移率改变分析法和半定量反转录-聚合酶链反应法检测肺泡巨噬细胞中核因子κB的活性变化及肿瘤坏死因子αmRNA的表达水平，以相对吸光度表示。 结果：67只兔进入结果分析。①核因子κB的活性：急性肺损伤组于模型建立后1-4h内均明显高于正常对照组（P〈0．01），在2h达到高峰，其相对吸光度值，是同期正常对照组的8倍；山莨菪碱组高于正常对照组（P〈0．01），显著低于急性肺损伤组（P〈0．01），其中2h降幅最大，达30．19％。②肿瘤坏死因子αmRNA的表达水平：急性肺损伤组在模型建立后1h其表达即开始上升，两三小时达到高峰，3h最高，其相对吸光度值，是同期正常对照组的5倍，4h其表达较前有所下降，但仍明显高于正常对照组（P〈0．01）；山莨菪碱组各时间的表达显著低于急性肺损伤组（P〈0．01，0．05），最大降幅出现在2h，达34．48％。 结论：①机体遭受创伤及脂多糖刺激后，肺泡巨噬细胞被激活导致核因子κB的活化是肺泡巨噬细胞大量释放肿瘤坏死因子α等前炎细胞因子的关键环节。②山莨菪碱可能是通过抑制肺泡巨噬细胞核中核因子κB活性，在基因转录水平抑制其介导的一系列细胞因子和炎症递质的表达（肿瘤坏死因子α等），阻断细胞因子和炎症递质的失控性释放来实现对急性肺损伤的治疗作用的。     

Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor.

We have developed a rat model of ischemic bowel necrosis associated with shock by injection of platelet-activating factor (PAF) or a combination of PAF and endotoxin. Recent investigations have shown that tumor necrosis factor (TNF) also induces shock and necrosis of the gastrointestinal tract. The morphological changes of TNF-induced bowel lesions are indistinguishable from those caused by PAF. The mechanism of TNF-induced bowel necrosis is unclear. In the present study, we have shown that (a) TNF caused PAF production in bowel tissue; (b) the effects of TNF and LPS on PAF production in the intestine are additive; (c) TNF and LPS are synergistic in inducing bowel necrosis; and (d) TNF-induced bowel necrosis is due to PAF release and can be prevented by pretreatment with PAF antagonists.     

注射肿瘤坏死因子-α对大鼠脑缺血再灌注的影响

背景一些研究提示肿瘤坏死因子-α预注射对小鼠局灶性脑缺血可产生保护作用,脑缺血耐受与血浆肿瘤坏死因子-α水平增高有关.另一方面,肿瘤坏死因子-α是与脑卒中有关的一个有害细胞因子,抗肿瘤坏死因子-α循环抗体对再灌注损伤起保护作用.目的探讨脑缺血再灌注前、后不同时期注射肿瘤坏死因子-α对大鼠脑缺血灶的影响,是保护作用还是毒性作用.设计随机对照实验.单位哈尔滨医科大学附属第二医院神经科.材料实验于2002-01/2002-04在哈尔滨医科大学动物实验中心完成;选择健康雄性Wistar大鼠120只,随机将大鼠分为8组预注射肿瘤坏死因子-α 0.05,0.5,1.0μg及磷酸盐缓冲液对照组,后注射肿瘤坏死因子-α 0.05,0.5,1.0μg及磷酸盐缓冲液对照组,每组15只.方法制备大鼠大脑中动脉局灶性脑缺血模型.缺血前48 h或缺血2 h再灌注后,在大鼠小脑延髓池内分别注射不同剂量肿瘤坏死因子-α(0.05 μg,0.5 μg,1.0μg)或磷酸盐缓冲液.①各组取8只大鼠,于脑缺血2 h再灌注22 h,麻醉下处死取脑,制备TTC切片,用计算机图像分析系统测定每个脑片面积及梗死面积,然后计算梗死体积百分比.②各组取7只大鼠,于缺血2 h再灌注22 h后,麻醉下处死取脑,行HE、GFAP和ICAM-1免疫组化染色,用显微镜和计算机图像分析系统分析组织病理和免疫组化切片,计算胶质纤维酸性蛋白阳性细胞数目及每侧半球细胞内黏附分子-1阳性血管数.主要观察指标①各组大鼠脑梗死体积百分比.②各组大鼠脑组织病理学观察.③各组大鼠脑组织胶质纤维酸性蛋白和细胞间黏附分子-1蛋白表达情况.结果120只大鼠全部进入结果分析.①脑梗死体积百分比肿瘤坏死因子-α 0.5 μg预注射组及1.0μg预注射组脑梗死体积减小,0.5μg预注射组脑梗死体积百分比减小70.9%,1.0 μg预注射组减小66.5%;肿瘤坏死因子-α 0.5 μg后注射及1.0 μg后注射组脑梗死体积增加,0.5 μg后注射组脑梗死体积百分比增加22.3%,1.0 μg后注射组增加46.7%.肿瘤坏死因子-α 0.5 μg预注射组与1.0 μg预注射组间比较无显著差异(P＞0.05),肿瘤坏死因子-α 0.5 μg后注射组与1.0 μg后注射组间比较有显著差异(P＜0.05).②病理变化肿瘤坏死因子-α0.5 μg预注射组及1.0 μg预注射组脑组织变性坏死程度减轻;肿瘤坏死因子-α0.5 μg后注射及1.0 μg后注射组脑组织变性坏死程度加重.③胶质纤维酸性蛋白和细胞间黏附分子-1蛋白的表达肿瘤坏死因子-α0.5 μg预注射组及1.0 μg预注射组胶质纤维酸性蛋白和细胞间黏附分子-1蛋白表达减少(P均＜0.05);肿瘤坏死因子-α 0.5μg后注射及1.0 μg后注射组胶质纤维酸性蛋白和细胞间黏附分子-1蛋白表达增加(P均＜0.05);肿瘤坏死因子-α 0.5 μg预注射组与1.0 μg预注射组间比较无显著差异(P均＞0.05),肿瘤坏死因子-α 0.5 μg后注射组与1.0 μg后注射组间比较有显著差异(P均＜0.05).结论①肿瘤坏死因子-α预注射对脑缺血再灌注损伤有神经保护作用,该作用与星形胶质细胞的修复作用无关,而与细胞间黏附分子-1表达减少有关.②在脑缺血再灌注后给肿瘤坏死因子-α则加重脑缺血,该作用与细胞间黏附分子-1表达增加有关.③脑缺血前或脑缺血后给予肿瘤坏死因子-α决定了它的作用效果,这两种作用都有剂量依赖性.     

T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6

This study investigates the molecular mechanisms underlying the induction of and protection from T cell activation-associated hepatic injury. When BALB/c mice were given a single intravenous injection of concanavalin A (Con A) (> or = 0.3 mg/mouse), they developed acute hepatic injury as assessed by a striking increase in plasma transaminase levels within 24 h. Histopathologically, only the liver was injured while moderate infiltration of T cells and polymorphonuclear cells occurred in the portal areas and around the central veins. The induction of hepatic injury was dependent on the existence as well as the activation of T cells, as untreated BALB/c nu/nu mice or BALB/c mice pretreated with a T cell-specific immunosuppressive drug, FK506, failed to develop disease. Significant increases in the levels of various cytokines in the plasma were detected before an increase in plasma transaminase levels. Within 1 h after Con A injection, tumor necrosis factor (TNF) levels peaked, this being followed by production of two other inflammatory cytokines, interleukin 6 (IL-6) and IL-1. Passive immunization with anti-TNF but not with anti-IL-1 or anti-IL-6 antibody, conferred significant levels of protection. Moreover, administration of rIL-6 before Con A injection resulted in an IL-6 dose-dependent protection. A single administration of a given dose of rIL-6 completely inhibited the release of transaminases, whereas the same regimen induced only 40-50% inhibition of TNF production. More than 80% inhibition of TNF production required four consecutive rIL-6 injections. These results indicate that: (a) TNFs are critical cytokines for inducing T cell activation-associated (Con A-induced) hepatitis; (b) the induction of hepatitis is almost completely controlled by rIL-6; and (c) rIL-6 exerts its protective effect through multiple mechanisms including the reduction of TNF production.     

注射肿瘤坏死因子-α对大鼠脑缺血再灌注的影响

背景：一些研究提示肿瘤坏死因子-α预注射对小鼠局灶性脑缺血可产生保护作用，脑缺血耐受与血浆肿瘤坏死因子-α水平增高有关。另一方面，肿瘤坏死因子-α是与脑卒中有关的一个有害细胞因子，抗肿瘤坏死因子-α循环抗体对再灌注损伤起保护作用。目的：探讨脑缺血再灌注前、后不同时期注射肿瘤坏死因子-α对大鼠脑缺血灶的影响，是保护作用还是毒性作用。设计：随机对照实验。单位：哈尔滨医科大学附属第二医院神经科。材料：实验于2002-01／2002-04在哈尔滨医科大学动物实验中心完成；选择健康雄性Wistar大鼠120只，随机将大鼠分为8组：预注射肿瘤坏死因子-α0．05，0．5，1．0μg及磷酸盐缓冲液对照组，后注射肿瘤坏死因子-α0．05，0．5，1．0μg及磷酸盐缓冲液对照组，每组15只。方法：制备大鼠大脑中动脉局灶性脑缺血模型。缺血前48h或缺血2h再灌注后，在大鼠小脑延髓池内分别注射不同剂量肿瘤坏死因子-α（0．05μg，0．5μg，1．0μg）或磷酸盐缓冲液。①各组取8只大鼠，于脑缺血2h再灌注22h，麻醉下处死取脑，制备TFC切片，用计算机图像分析系统测定每个脑片面积及梗死面积，然后计算梗死体积百分比。②各组取7只大鼠，于缺血2h再灌注22h后，麻醉下处死取脑，行HE、GFAP和ICAM-1免疫组化染色，用显微镜和计算机图像分析系统分析组织病理和免疫组化切片，计算胶质纤维酸性蛋白阳性细胞数目及每侧半球细胞内黏附分子-1阳性血管数。主要观察指标：①各组大鼠脑梗死体积百分比。②各组大鼠脑组织病理学观察。③各组大鼠脑组织胶质纤维酸性蛋白和细胞间黏附分子-1蛋白表达情况。结果：120只大鼠全部进入结果分析。①脑梗死体积百分比：肿瘤坏死因子-α0．5μg预注射组及1．0μg预注射组脑梗死体积减小，0．5μg预注射组脑梗死体积百分比减小70．9％，1．0μg预注射组减小66．5％；肿瘤坏死因子-α0．5μg后注射及1．0μg后注射组脑梗死体积增加，0．5μg后注射组脑梗死体积百分比增加22.3％，1．0μg后注射组增加46．7％。肿瘤坏死因子-α0．5μg预注射组与1．0μg预注射组间比较无显著差异（P〉0．05），肿瘤坏死因子-α0．5μg后注射组与1．0μg后注射组间比较有显著差异（P〈0．05）。②病理变化：肿瘤坏死因子-α0．5μg预注射组及1．0μg预注射组脑组织变性坏死程度减轻；肿瘤坏死因子-α0．5μg后注射及1．0μg后注射组脑组织变性坏死程度加重。③胶质纤维酸性蛋白和细胞间黏附分子-1蛋白的表达：肿瘤坏死因子-α0．5μg预注射组及1．0μg预注射组胶质纤维酸性蛋白和细胞间黏附分子-1蛋白表达减少（P均〈0．05）；肿瘤坏死因子-α0．5μg注射及1．0μg后注射组胶质纤维酸性蛋白和细胞间黏附分子-1蛋白表达增加（P均〈0．05）；肿瘤坏死因子-α0．5μg预注射组与1．0μg预注射组间比较无显著差异（P均〉0．05），肿瘤坏死因子-α0．5μg后注射组与1．0μg后注射组间比较有显著差异（P均〈0．05）。结论：④肿瘤坏死因子-α预注射对脑缺血再灌注损伤有神经保护作用，该作用与星形胶质细胞的修复作用无关，而与细胞间黏附分子-1表达减少有关。②在脑缺血再灌注后给肿瘤坏死因子-α则加重脑缺血，该作用与细胞间黏附分子-1表达增加有关。③脑缺血前或脑缺血后给予肿瘤坏死因子-α决定了它的作用效果，这两种作用都有剂量依赖性。     

Stimulation of alpha 7 cholinergic receptors inhibits lipopolysaccharide-induced neutrophil recruitment by a tumor necrosis factor alpha-independent mechanism

The cholinergic nervous system controls inflammation by inhibiting the release of proinflammatory cytokines such as tumor necrosis factor (TNF) alpha from lipopolysaccharide (LPS)-stimulated macrophages. The key endogenous mediator of this so-called cholinergic anti-inflammatory pathway is acetylcholine, the principal neurotransmitter of the vagus nerve, which specifically interacts with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit TNF-alpha production. We here investigated the capacity of the selective alpha7 cholinergic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) to inhibit LPS-induced inflammatory responses in mice in vivo. To this end, mice received an intraperitoneal injection of LPS (from Escherichia coli, 200 microg) preceded by GTS-21 (4 mg/kg) or vehicle. GTS-21 strongly inhibited LPS-induced TNF-alpha release into the peritoneal cavity and the circulation. In addition, GTS-21 attenuated the influx of neutrophils into peritoneal fluid upon administration of LPS. This inhibitory effect on neutrophil recruitment by GTS-21 was independent of its effect on TNF-alpha release, considering that etanercept, a potent TNF-alpha-blocking protein containing the extracellular domain of the p75 TNF-alpha receptor, did not influence LPS-induced neutrophil influx either in the presence or in the absence of GTS-21 treatment. GTS-21 did not reduce the local secretion of macrophage inflammatory protein 2 and keratinocyte-derived cytokine, suggesting that altered concentrations of these neutrophil-attracting chemokines did not contribute to GTS-21-induced inhibition of neutrophil migration. These data identify a novel anti-inflammatory effect of chemical alpha7 cholinergic receptor stimulation that is independent from its capacity to inhibit TNF-alpha production.     

Radioimmunoassay of tumor necrosis factor in serum

We present a double-antibody radioimmunoassay for determination of the concentration of tumor necrosis factor (TNF) in serum. TNF in serum competes with a fixed amount of 125I-labeled TNF for the binding sites of specific rabbit antibodies. The bound TNF is precipitated with Sepharose-bound anti-rabbit IgG, then centrifuged, and the radioactivity of the pellets is counted. The detection limit of the assay is 7 ng/L (B0-3 SD). Bound radioactivity in the range of 10% to 90% of the B0 counts corresponds to TNF concentrations of 26 to 10,000 ng/L. Of 40 sera from healthy subjects, 21 (53%) contained TNF concentrations greater than 7 ng/L (range 8-40 ng/L). Some patients with parasitic or neoplastic disease and patients with septic shock had highly increased TNF values. Three of the 14 sera (21%) from patients with rheumatoid arthritis had TNF concentrations greater than 40 ng/L.