Tumor necrosis factor alpha in systemic scleroderma

The authors present the review of the literature on the role of tumor necrosis factor alpha (TNF alpha) in scleroderma systematica (SS). It is shown that TNF alpha participates in activation of vascular endothelium, regulation of immune response and metabolism of the connective tissue by modulation of fibroblastic function. SS patients exhibit a systemic and local rise of TNF alpha content. This rise contributes to SS progression, development of fibrosing alveolitis and skin fibrous alterations in Raynaud's syndrome.     

肿瘤坏死因子a促进人冠状动脉内皮细胞的凋亡

背景:研究表明冠状动脉内皮细胞凋亡参与了动脉粥样硬化的发生、发展过程。目的:观察肿瘤坏死因子a对人冠状动脉内皮细胞的诱导损伤作用。方法:取对数生长期的人冠状动脉内皮细胞c-12221,分别加入含0(对照),200,400,600mg/L肿瘤坏死因子a的培养液,采用MTT检测细胞增殖率变化,Hoechst 33258/PI双染观察凋亡细胞形态变化,Annexin V-FITC和PI双染流式检测细胞凋亡率变化,高内涵活细胞成像系统检测细胞线粒体膜电位变化。结果与结论:肿瘤坏死因子a呈剂量依赖性抑制人冠状动脉内皮细胞的增殖。Hoechst 33258/PI染色观察可见凋亡细胞染色质凝集、细胞核碎裂成碎片等典型细胞凋亡的特征性变化,不同质量浓度肿瘤坏死因子a组细胞凋亡率高于对照组(P<0.05),线粒体膜电位低于对照组(P<0.05),且呈剂量依赖性。表明肿瘤坏死因子a呈剂量依赖性促进人冠状动脉内皮细胞凋亡,抑制其增殖,作用机制与线粒体凋亡通路有关。     

Formation of endothelium-derived relaxing factor in porcine kidney epithelial LLC-PK1 cells: an intra- and intercellular messenger for activation of soluble guanylate cyclase.

Oxytocin, bradykinin, melittin and A23187 increased cyclic GMP levels through activation of soluble guanylate cyclase in cultured porcine kidney epithelial cells, LLC-PK1. NG-monomethyl-L-arginine, an inhibitor of endothelium-derived relaxing factor/nitric oxide formation, decreased both basal and stimulated levels of cyclic GMP in a concentration-dependent manner. L-Arginine, but not D-arginine, augmented basal as well as stimulated levels of cyclic GMP and prevented the inhibition induced by NG-monomethyl-L-arginine. Similar effects of L-arginine were also observed with L-argininamide, L-arginine ethyl ester, L-arginine methyl ester and the dipeptide L-arginyl-L-aspartic acid. NG-monomethyl-L-arginine did not affect cyclic GMP accumulation induced by sodium nitroprusside, an activator of soluble guanylate cyclase, and atrial natriuretic factor, an activator of particulate guanylate cyclase. Stimulatory effects of oxytocin, glyceryl trinitrate, sodium nitroprusside, bradykinin, melittin and A23187 on cyclic GMP accumulation were enhanced with superoxide dismutase and diminished with oxyhemoglobin. However, atrial natriuretic factor-induced cyclic GMP accumulation was not affected. Furthermore, endothelium derived relaxing factor-like activity was detected in the conditioned medium from LLC-PK1 cells stimulated with oxytocin. Based on these data, we conclude that endothelium-derived relaxing factor is produced in this cell type and participates in the regulatory mechanism of cyclic GMP formation as an intra- and intercellular messenger for activation of soluble guanylate cyclase.     

Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.     

组织因子刺激体外培养人脐静脉内皮细胞肿瘤坏死因子α、白细胞介素1的表达

目的:组织因子介导了细胞凝集、组织损伤、血栓形成,从而参与"凝血-炎症-血栓形成"网络。组织因子是否为一种独立的前炎症介质介导炎症反应,刺激人脐静脉内皮细胞使之分泌肿瘤坏死因子α、白细胞介素1。方法:实验于2004-09/2006-02在贵阳医学院组织工程与干细胞实验中心(省级重点实验室)完成。利用重组的组织因子刺激体外培养的人脐静脉内皮细胞,并设立空白对照组、0.2mg/L组织因子刺激组和0.8mg/L组织因子刺激组,用ELISA检测不同组织因子刺激浓度在同一刺激时间(6h)其炎症介质(肿瘤坏死因子α、白细胞介素1)的表达分泌情况。结果:①组织因子可以刺激人脐静脉内皮细胞使之分泌肿瘤坏死因子α,0.2mg/L组织因子刺激组与0.8mg/L组织因子刺激组肿瘤坏死因子α水平高于空白对照组(P<0.05);0.8mg/L组织因子刺激组肿瘤坏死因子α水平高于0.2mg/L组织因子刺激组(P<0.05)。②与空白对照组相比较,0.2mg/L组织因子刺激组白细胞介素1水平未升高,0.8mg/L组织因子刺激组略有升高,但差异无显著性意义(P>0.05)。结论:组织因子可以作为一种前炎症介质介导炎症反应,使内皮细胞肿瘤坏死因子α表达增强。     

Tumor necrosis factor alpha is involved in mouse growth and lymphoid tissue development

Tumor necrosis factor alpha (TNF-alpha), a major mediator of inflammation, also possesses a wide pleiotropism of actions, suggesting its involvement in physiological conditions. TNF-alpha mRNA is present in mouse embryonic tissues and also in fetal thymus and spleen. Repeated injections of a monospecific polyclonal rabbit anti-mouse TNF-alpha antibody in mice, starting either during pregnancy or at birth, led to a severe but transient growth retardation, already present at birth, reaching a 35% decrease in body weight at 3 wk, with complete recovery at 8 wk. The insulin growth factor I (IGF-I) blood levels were decreased to about 50%; growth hormone release and other endocrine functions were unaltered. A marked atrophy of the thymus, spleen, and lymph nodes was also observed, with lymphopenia and impaired development of T and B cell peripheral lymphoid structures. The pathways involving TNF-alpha in IGF-I release and early body growth are probably distinct from those by which TNF-alpha participates in early development of lymphoid tissues, where its low physiological release may contribute to enhance lymphoid cell expansion.     

Neuromodulatory effects of atrial natriuretic factor are independent of guanylate cyclase in adrenergic neuronal pheochromocytoma cells

This study tests the hypothesis that atrial natriuretic factor (ANF) and C-ANF(4-23)-NH2 (C-ANF) augment cGMP generation and inhibit both cAMP generation and depolarization-induced catecholamine release in nerve growth factor treated pheochromocytoma cells by a pertussis toxin (PTX)-sensitive mechanism. Synthetic rat ANF(99-126) and the clearance receptor antagonist C-ANF (10(-12)-10(-9) M) inhibited basal and 5 microM vasoactive intestinal peptide (VIP)-induced cAMP generation in a concentration-dependent manner. These actions of ANF and C-ANF were blocked by 12-18 h pretreatment with PTX (100 ng/ml), suggesting ANF receptor coupling to adenylate cyclase via an inhibitory guanine nucleotide-binding protein. Both ANF (10(-11)-10(-9) M) and C-ANF (10(-11)-10(-8) M) also inhibited K(+)-induced catecholamine release in a concentration-dependent manner. ANF (10(-11)-10(-8) M) increased cGMP generation in a concentration-dependent manner but C-ANF did not. The accumulation of cGMP in response to ANF was not altered by treatment with PTX. Therefore, PTX dissociated the increased concentrations of cGMP from the ANF-mediated depression of evoked catecholamine release. C-ANF also dissociated elevations in cGMP concentrations from an ANF-mediated attenuation of evoked catecholamine release. The results of this study indicate that ANF inhibits adrenergic neurotransmission independent of guanylate cyclase.     

肿瘤坏死因子α刺激人脐静脉内皮细胞线粒体偶联因子6表达及其分子机制

背景:线粒体偶联因子6作为一种新的血管活性肽,参与了许多生理功能调节. 目的:探讨人脐静脉内皮细胞在肿瘤坏死因子α刺激下线粒体偶联因子6的表达及其分子机制.设计、时间及地点:分组对照观察,实验于2006-06/2007-03在南方医科大学附属珠江医院完成.材料:健康产妇足月分娩的脐带来源于南方医科大学附属珠江医院产科(产妇均签署知情同意书);肿瘤坏死因子α为北京邦定泰克生物有限公司产品;鼠抗人线粒体偶联因子6单克隆抗体为美国凤凰药业公司产品.方法:应用肿瘤坏死因子α(100～250 μg/L)刺激体外培养的人脐静脉内皮细胞.主要观察指标:以流式细胞术检测线粒体偶联因子6在细胞膜上的表达,以反转录-聚合酶链反应方法检测线粒体偶联因子6 mRNA不同时间的表达变化,以蛋白质印迹分析检测细胞核中N F -κB不同时间的表达变化,同时检测胞浆中IκBα的含量变化.结果: 肿瘤坏死因子α刺激人脐静脉血管内皮细胞膜表面线粒体偶联因子6表达增加,线粒体偶联因子6 mRNA表达增强,刺激1 h后线粒体偶联因子6 mRNA表达最强,1.5 h后回到原来水平,核内核因子κB含量在6 h明显增加,之后一直持续高水平状态,而胞浆中IκBα含量在6 h明显减少,之后一直持续低水平状态.结论:肿瘤坏死因子α刺激人脐静脉血管内皮细胞后,迅速活化核因子κB,启动线粒体偶联因子6的转录,线粒体偶联因子6 mRNA的表达增加,线粒体偶联因子6合成增加,导致线粒体偶联因子6在细胞膜表面表达增加.     

Tumor necrosis factor alpha stimulates the growth of the clonogenic cells of acute myeloblastic leukemia in synergy with granulocyte/macrophage colony-stimulating factor

TNF-alpha has been shown to antagonize the proliferative effects of growth factors present in crude conditioned media from PHA-stimulated leukocytes or cell lines on the clonogenic cells of acute myeloblastic leukemia (AML) (19,21). In the present study, we investigated the responses of AML blasts to TNF-alpha in the presence of defined growth factors (recombinant granulocyte/macrophage-CSF [rGM-CSF], recombinant granulocyte-CSF [rG-CSF], rIL-3, and rIL-1) and under conditions described for autocrine stimulation (32). While TNF-alpha antagonized the stimulatory effects of G-CSF and IL-3 on blast progenitors, TNF-alpha did not affect blast colony formation in the presence of IL-1. Unexpectedly, TNF-alpha significantly enhanced blast proliferation in the presence of GM-CSF. Further, TNF-alpha also acted synergistically with an endogenous source of growth stimulatory signal to promote proliferation of blast clonogenic cells. Thus, on human leukemic cells, TNF-alpha appears to be a molecule that is at least bifunctional, having the ability to either support or inhibit cell proliferation, depending on the other growth factors present. It is postulated that the proliferative response of blast progenitors to TNF-alpha under conditions that favor autocrine stimulation may represent one property that allows the cells to escape from negative regulation and proliferate in AML.     

Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis, and mitogenicity in mesangial proliferative glomerulonephritis.

Mesangial injury and cell proliferation are frequent findings in various glomerular diseases in man. Previous studies have demonstrated that basic fibroblast growth factor (bFGF) is a potent mesangial cell mitogen in vitro. To further elucidate the role of bFGF in rat mesangial cell (RMC) proliferation, we examined whether RMC synthesize bFGF in vitro and whether bFGF is involved in mesangial proliferation in vivo. Cultured RMC expressed bFGF protein (23, 21.5, and 18 kD forms) and bFGF mRNA, and released biologically active bFGF into the culture medium after antibody- and complement-mediated injury. Normal rat glomeruli in vivo contained no detectable bFGF mRNA, but bFGF protein (23 and 21.5 kD) could be demonstrated, which immunolocalized to the mesangium. Glomerular bFGF decreased markedly during the acute phase of glomerulonephritis induced by anti-Thy 1.1 antibody, compatible with mesangial bFGF release after complement-mediated mesangiolysis. During the subsequent mesangial proliferative phase, glomerular bFGF protein and mRNA increased above normal. Intrarenal infusion of heparin did not affect the bFGF immunostaining of glomeruli at this stage, indicating a predominantly intracellular localization of the bFGF. The capability of bFGF to mediate proliferation in the anti-Thy 1.1 model was further supported by experiments in which intravenous bFGF given 24 h after a subnephritogenic dose of anti-Thy 1.1 antibody led to a 4.9- to 5.1-fold increase in glomerular cell proliferation (with > 60% of the cells identified as mesangial cells by double immunolabeling). No such increase was observed in normal rats injected with bFGF. These data show that mesangial cells produce and release bFGF after injury and that bFGF is mitogenic for injured mesangial cells in vivo. Release of mesangial cell bFGF thus may be an important mechanism involved in the initiation of mesangial cell proliferation in vivo.     

Macrophage colony stimulating factor expression in human cardiac cells is upregulated by tumor necrosis factor-α via an NF-κB dependent mechanism

INTRODUCTION: Macrophage colony stimulating factor (M-CSF) is a key factor for monocyte and macrophage survival and proliferation. M-CSF has been implicated in cardiac healing and repair after myocardial infarction. METHODS AND RESULTS: We show by immunohistochemistry and Western blotting analysis that M-CSF protein is present in human heart tissue. Cultured human adult cardiac myocytes (HACM) and human adult cardiac fibroblasts (HACF) isolated from human myocardial tissue constitutively express M-CSF. When HACM and HACF were treated with tumor necrosis factor-alpha (TNF-alpha) M-CSF protein production and M-CSF mRNA expression, determined by ELISA or by using RT-PCR, respectively, was significantly increased. To determine a possible role of nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) in M-CSF regulation, blockers to both pathways and an adenovirus overexpressing a dominant negative (dn) form of IkappaB kinase 2 (IKK2) were used. Only the NF-kappaB blocker dimethylfumarate and the dn IKK2, but not januskinase inhibitor-1 (JNK-I), were able to block the TNF-alpha-induced increase in M-CSF production in these cells, suggesting that the induction of M-CSF through TNF-alpha is mainly dependent on the activation of the NF-kappaB pathway. The monocyte activation marker CD11b was significantly increased after incubating U937 cells with conditioned medium from HACM or HACF as determined by FACS analysis. CONCLUSIONS: Our in vitro data taken together with our immunohistochemistry data suggest that human cardiac cells constitutively express M-CSF. This expression of M-CSF in the human heart and its upregulation by TNF-alpha might contribute to monocyte and macrophage survival and differentiation.     

慢病毒介导可溶性肿瘤坏死因子受体1在小鼠骨髓未成熟树突状细胞中的表达(英文)

背景:肿瘤坏死因子α是介导树突状细胞成熟的重要细胞因子之一,可溶性肿瘤坏死因子受体1与其结合可阻断肿瘤坏死因子α的作用,维持树突状细胞于不成熟状态,诱导免疫耐受.目的:构建含有人sTNFR1的慢病毒表达载体,观察其在未成熟树突状细胞中的表达.方法:以人外周血单个核细胞总RNA为模板,RT-PCR扩增出sTNFR1基因片段,亚克隆至慢病毒转移质粒pXZ208,通过IRES连接eGFP报告基因,建立双顺反子慢病毒转移质粒,命名为pXZ9-sTNFR1,DNA测序鉴定.采用脂质体转染293 FT细胞,根据报告基因eGFP测定病毒滴度.采用小剂量粒-巨噬细胞集落刺激因子+白细胞介素4体外培养扩增C57BL/6小鼠骨髓来源树突状细胞.培养第5天,以pXZ9-sTNFRl重组慢病毒上清感染未成熟树突状细胞,RT-PCR检测感染后sTNFRl转录,Westernblot法检测sTNFR1蛋白表达,观察sTNFR1基因修饰及脂多糖刺激后树突状细胞的表型特征.结果与结论:成功构建重组质粒pXZ9-sTNFR1,转染293 FT细胞24 h后观察到eGFP表达,病毒滴度在10~6U/L以上.RT-PCR显示pXZ9-sTNFR1感染的未成熟树突状细胞sTNFR1呈阳性表达,Western blot检测到sTNFR1蛋白存在于感染后未成熟树突状细胞和培养上清中.培养第5天的树突状细胞低表达CD40、CD86、CD80和MHCⅡ类分子,脂多糖刺激后,高表达MHCⅡ类分子和CD40、CD80、CD86分子,显示出成熟型树突状细胞表型特征,sTNFR修饰的树突状细胞MHC Ⅱ类分子和CD40、CD80、CD86分子表达水平无变化.提示:①成功构建了负载sTNFR1基因片段及含eGFP报告基因的慢病毒载体,获得了高滴度的重组慢病毒颗粒.②经慢病毒高效转导的未成熟树突状细胞sTNFR1 mRNA及蛋白稳定地表达,可以保护未成熟树突状细胞不被外源性脂多糖刺激活化,维持树突状细胞于非成熟状态.     

Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells

Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-alpha) mRNA and protein levels by real-time quantitative PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. We studied the effects on the DNA binding of NF-kappaB and specificity protein 1 (Sp1) by an ELISA. Non-CF cells express significantly lower TNF-alpha mRNA and protein levels than an isogenic CF cell line. In CF cells, AZM treatment causes a 30% reduction of TNF-alpha mRNA levels (P < 0.05) and a 45% decrease in TNF-alpha secretion (P < 0.05), reaching approximately the levels of the untreated isogenic non-CF cells. In CF cells, NF-kappaB and Sp1 DNA binding activities were also significantly decreased (about 45 and 60%, respectively; P < 0.05) after AZM treatment. Josamycin, a macrolide lacking clinically described anti-inflammatory effects, was ineffective. Finally, AZM did not alter the mRNA expression levels of interleukin-6, a proinflammatory molecule not differentially expressed in CF and isogenic non-CF cells. The results of our study support the anti-inflammatory activities of this macrolide, since we show that AZM reduced the levels of TNF-alpha and propose inhibitions of NF-kappaB and Sp1 DNA binding as possible mechanisms of this effect.     

肿瘤坏死因子α基因多态性对强直性脊柱炎发病易感性和临床病变程度的影响

背景肿瘤坏死因子α基因多态性与强直性脊柱炎发生与发展的关系是近年强直性脊柱炎遗传学研究的热点.目的探讨肿瘤坏死因子α基因启动子-238和-308位点多态性对强直性脊柱炎发病易感性和病变程度的影响.设计病例-对照观察.单位第二军医大学长海医院风湿免疫科.对象随机选择1999-01/2003-12在第二军医大学长海医院风湿免疫科就诊的强直性脊柱炎患者108例,各对象间均无血缘关系.男女比为5.31;年龄13～71(30±12)岁,根据骶髂关节破坏程度X射线片评估为Ⅰ～Ⅳ级.另从解放军上海血站(长海医院)随机选取100名健康献血者作为对照,年龄19～56(33±9)岁,男女比为4.91,参与者均知情同意.方法每例参与者取外周血,加乙二胺四乙酸A抗凝后进行肿瘤坏死因子-α基因启动子的聚合酶链反应扩增和纯化;对聚合酶链反应产物测序,并用Chromas 1.62软件展示分析DNA测序结果.②以X射线骶髂关节片分级和肿瘤坏死因子α-308位点(G/C)和(G/A)的对应值评估其对疾病程度的影响.主要观察指标以DNA直接测序法对-238和-308位点进行基因分型,并分析基因类型与疾病临床表现之间的关系.结果①肿瘤坏死因子α-238G/G基因型和-238G/A基因型强直性脊柱炎组有106例(98.1%)和2例(1.9%),正常对照组有95例(95.0%)和5例(5.0%),两组间比较无显著性差异.②肿瘤坏死因子α-308.1.1(G/G)基因型和-308.1.2(G/A)基因型强直性脊柱炎组有89例(82.4%)和19例(17.6%),正常对照组中有85例(85.0%)和14例(14.0%).两组比较亦无显著性差异.③根据骶髂关节X射线分级判定疾病严重程度与肿瘤坏死因子α-308位点G/G和G/A基因型之间的关系强直性脊柱炎患者X射线片Ⅰ级、Ⅱ级、Ⅲ级和Ⅳ级时,(G/G)型为3/35/40/11例,(G/A)型为1/12/6/0例.两组差异比较意义显著(x2GMH=4 77,P＜0.05).结论在观察对象中未发现肿瘤坏死因子α基因启动子-238和-308位点多态性与强直性脊柱炎发病易感性相关,但根据骶髂关节损害程度X射线分级说明肿瘤坏死因子α基因启动子-308位点的多态性对强直性脊柱炎的严重程度有影响.     

High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells.

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.