Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an "atypical apoptosis" characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.     

Toll-like receptor 9-induced type I IFN protects mice from experimental colitis

Experimental colitis is mediated by inflammatory or dysregulated immune responses to microbial factors of the gastrointestinal tract. In this study we observed that administration of Toll-like receptor 9 (TLR9) agonists suppressed the severity of experimental colitis in RAG1-/- but not in SCID mice. This differential responsiveness between phenotypically similar but genetically distinct animals was related to a partial blockade in TLR9 signaling and defective production of type I IFN (i.e., IFN-alpha/beta) in SCID mice upon TLR9 stimulation. The addition of neutralization antibodies against type I IFN abolished the antiinflammatory effects induced by TLR9 agonists, whereas the administration of recombinant IFN-beta mimicked the antiinflammatory effects induced by TLR9 agonists in this model. Furthermore, mice deficient in the IFN-alpha/beta receptor exhibited more severe colitis than wild-type mice did upon induction of experimental colitis. These results indicate that TLR9-triggered type I IFN has antiinflammatory functions in colitis. They also underscore the important protective role of type I IFN in intestinal homeostasis and suggest that strategies to modulate innate immunity may be of therapeutic value for the treatment of intestinal inflammatory conditions.     

Extensive cell death in thymocytes in colon 26-induced cachectic mice

Extensive atrophy has been reported to occur in the thymus in a cancer-burden state but the mechanisms of this atrophy have not been fully elucidated. We investigated changes in the thymus in tumour-bearing mice inoculated with two subclones of the murine colon 26 adenocarcinoma cell line: clone 5 (non-cachectic) and clone 20 (cachectic). In clone 20 mice, body weights and thymocyte numbers decreased significantly compared with controls. Flow cytometric analysis of the thymocytes demonstrated that the frequency of single positive cells (CD4+ CD8- and CD4- CD8+) was significantly increased and that of double positive cells (CD4+ CD8+) was significantly decreased in clone 20 mice and, to a lesser extent, in clone 5 mice compared with controls. Serum levels of interleukin 6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly elevated. These results suggested that thymocyte apoptosis was accelerated in the cancer-cachectic state, and increased GM-CSF might be partly responsible for thymic atrophy.     

Fas and tumor necrosis factor receptor-mediated cell death: similarities and distinctions

Fas antigen and two tumor necrosis factor receptors (TNFR), p55 and p75, are implicated in the triggering of cell death upon stimulation by natural ligands and specific monoclonal antibodies. However, the relative efficiency of each receptor, the mechanisms that regulate their function and the signaling pathways they employ, remain to be elucidated. In this study, fusion proteins, composed of the extracellular domain of CD40 and the intracellular and transmembrane domains of Fas, TNFRp55 and TNFRp75, were stably expressed in a human melanoma cell line that is deficient in Fas and TNFR expression. Transfectants were stimulated by a soluble recombinant form of the CD40 ligand gp39, and the effect on cell viability determined. Engagement of all three fusion proteins by the gp39 ligand induced lethal signals, but the rate at which cell death occurred was distinct. Fas-derived signals were observed to have the most rapid effect, killing most cells within hours of stimulation, whereas TNFRp55- and TNFRp75-associated signals resulted in cell death within 2-3 d after engagement by ligand. It is interesting to note that optimal cell killing by all three fusion proteins was dependent on a critical, low to intermediate, cell surface expression level. High levels of fusion protein expression, on the other hand, were associated with inhibition of cell death. Our results provide a model to study Fas and TNFR-mediated cell death and suggest a novel mechanism for the regulation of death signals triggered by members of the TNFR family.     

P2 receptor antagonist trinitrophenyl-adenosine-triphosphate protects hippocampus from oxygen and glucose deprivation cell death

In this work, we mainly used the organotypic model of rat hippocampus to demonstrate the protective role of the P2 receptor antagonist trinitrophenyl-adenosine-triphosphate (TNP-ATP) during oxygen/glucose deprivation. Among the P2X receptors that TNP-ATP specifically blocks, mainly P2X1 seems to be involved in the processes of cell damage after oxygen/glucose deprivation. P2X1 receptor is strongly and transiently up-regulated in 24 h after an ischemic insult on structures likely corresponding to mossy fibers and Schaffer collaterals of CA1-3 and dentate gyrus. Furthermore, P2X1 receptor is down-regulated by pharmacological treatment with TNP-ATP, which is also found neuroprotective against ischemic cell death. Morphological studies conducted through immunofluorescence and confocal analysis in primary organotypic, in dissociated cultures, and in adult rat in vivo demonstrated the neuronal colocalization of P2X1 protein with neurofilament light chain and neuronal nuclei immunoreactivity in myelinated and unmyelinated fibers of both granular and pyramidal neurons. In conclusion, with this work, we proved the neuronal distribution of P2X1 receptor in hippocampus, and we presented evidence for a potential disadvantageous role of its expression during the path of in vitro ischemia.     

Dehydroepiandrosterone protects mice from endotoxin toxicity and reduces tumor necrosis factor production.

Recent reports have demonstrated an immunomodulating activity of dehydroepiandrosterone (DHEA) different from that described for glucocorticoids. The present study was designed to test DHEA's activity in endotoxic shock and to investigate its effect on endotoxin-induced production of tumor necrosis factor (TNF). Mortality of CD-1 mice exposed to a lethal dose of lipopolysaccharide (LPS; 800 micrograms per mouse) was reduced from 95 to 24% by treatment with a single dose of DHEA, given 5 min before LPS. LPS administration resulted in high levels of TNF, a response that was significantly blocked by DHEA, both in vivo and in vitro. DHEA treatment also reduced LPS-induced increments in serum corticosterone levels, a parameter considered not to be mediated by TNF. In another experimental model, mice sensitized with D-galactosamine, followed by administration of recombinant human TNF, were subjected to 89% mortality rate, which was reduced to 55% in DHEA-treated mice. These data show that DHEA protects mice from endotoxin lethality. The protective effect is probably mediated by reduction of TNF production as well as by effecting both TNF-induced and non-TNF-induced phenomena.     

T cell receptor-dependent cell death of T cell hybridomas mediated by the CD30 cytoplasmic domain in association with tumor necrosis factor receptor-associated factors

CD30 is a member of the tumor necrosis factor superfamily and a surface marker for Hodgkin's disease. Normal activated T cells and several virally transformed T or B cell lines also show CD30 expression. The interaction of CD30 with its ligand induces cell death or proliferation, depending on the cell type. In this report we characterize the signals mediated by the intracellular domain of CD30 and show that, in combination with signal(s) transduced by the T cell receptor, the multimerization of CD30 cytoplasmic domain induces Fas(CD95)-independent cell death in T cell hybridomas. Deletion analysis shows that the COOH-terminal 66 amino acids of CD30 are required to induce cell death. Using the yeast two-hybrid system, we have identified that the same region of CD30 interacts with tumor necrosis factor receptor-associated factor (TRAF)1 and TRAF2. These results indicate that TRAF1 and/or TRAF2 play an important role in cell death in addition to their previously identified roles in cell proliferation.     

Impaired preneoplastic changes and liver tumor formation in tumor necrosis factor receptor type 1 knockout mice

Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.     

Level of soluble tumor necrosis factor receptor type I in patients with systemic scleroderma

AIM: To study content and clinical correlations of sTNF-RI in patients with systemic sclerosis (SS). MATERIAL AND METHODS: Thirty nine SS patients were examined with enzyme immunoassay for serum levels of sTNF-RI and soluble adhesion molecules (sSAM) sVSAM-1, sISAM-1 and sR-selectine using R&D System kits (USA). The control group consisted of 14 healthy subjects (donors). Content of sTNF-RI and sSAM was considered as elevated if it exceeded relevant mean values by 1SD. RESULTS: sTNF-RI content was significantly higher in the patients than in the controls (p = 0.0001) and was similar in patients with diffuse and limited forms of the disease. A rise in sTNF-RI correlated with a rise in pulmonary artery pressure. In patients with pulmonary hypertension or restrictive pulmonary affection sTNF-RI was higher than in patients free of pulmonary hypertension or impaired external respiration function. A marked correlation was found between sTNF-RI and sVSAM-1. Patients with high CRP had significantly higher sTNF-RI level than patients with normal CRP. CONCLUSION: SS is characterized by elevated content of sTNF-RI. This content may serve a diagnostic marker of the disease progression.     

Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis

The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.     

Glutaihione protects a human insulinoma cell line from tumor necrosis factors-α-mediated cytotoxicity

It is postulated that glutathione acting as a free oxygen radical scavenger may protect beta-cells from cytokine-mediated cytotoxicity in insulin-dependent diabetes. In this study the effect of glutathione in preventing the cytotoxic damage mediated by tumor necrosis factor-a in vitro towards a human beta-cell line (CM insulinoma) was investigated. CM cells were exposed in vitro to tumor necrosis factor-α, tumor necrosis factor-α plus glutathione or glutathione alone at different concentrations. The resulting cytotoxicity was measured using a colorimetric assay. Glutathione significantly reduced the cytotoxicity mediated by tumor necrosis factor-α in a dose-dependent fashion (P<0.001). These results suggest a protective effect of glutathione on beta-cell cytotoxicity induced by tumor necrosis factor-α and encourage the use of glutathione in trials aimed at reducing the beta-cell damage occurring in insulin-dependent diabetes.     

Synergistic enhancement of TRAIL- and tumor necrosis factor alpha-induced cell death by a phenoxazine derivative

2-Amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1) has been developed as a novel phenoxazine derivative having an anticancer activity on a variety of cancer cell lines as well as transplanted tumors in mice with minimal toxicity to normal cells. We examined the effects of Phx-1 on Jurkat cells, a human T cell line. Phx-1 inhibited proliferation of the cells in a dose-dependent manner but hardly induced cell death, suggesting that Phx-1 acts primarily as an antiproliferative reagent but not as a cytocidal drug. Phx-1 enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic cell death about 100-fold. Tumor necrosis factor alpha, which alone does not induce cell death of Jurkat cells, caused apoptosis in combination with Phx-1. These enhancements of cell death were not due to up-regulation of the death receptors. Phx-1 decreased serum-induced phosphorylation of Akt, a kinase involved in cell proliferation and survival, and inhibited complex III of mitochondrial respiratory chain. Considering that both TRAIL and Phx-1 have only marginal cytotoxicity to most normal cells, Phx-1 may provide an ideal combination for cancer therapy with TRAIL.     

Interleukin-2, soluble interleukin-2 receptor and tumor necrosis factor in sera from patients with rheumatoid arthritis

Summary Interleukin-2 (IL-2), soluble interleukin-2 receptor (IL-2R) and tumor necrosis factor (TNF) have been measured in sera from 47 patients affected by classic rheumatoid arthritis (RA) using an enzyme-linked immunosorbent assay. The patients were divided into 4 groups as follows: group A, 18 patients with inactive disease; group B, 19 patients with active disease under treatment with non-steroidal antiinflammatory drugs (NSAID) and second-line drugs; group C, 5 patients with active disease under treatment with NSAID and cyclosporine A (CSA) for at least 4 months; group D, 5 patients in the same condition as patients of group C, but treated with azathioprine (AZA) instead of CSA. IL-2 was undetectable in all patients except two, both characterized by active disease. Soluble IL-2R levels were above the upper limit of the normal range in most of the patients studied, but the mean value (±1 SD) was significantly higher in patients of group B (1,288±421 U/ml) than in patients of group A (686±205 U/ml) and group C (842±414 U/ml). In two patients affected by active RA treated with pulse methylprednisolone therapy (1 g/day for 3 alternate days) the values of soluble IL-2R dropped from 948 to 662 U/ml and from 660 to 518 U/ml, respectively. No statistically significant correlation was observed between the serum level of IL-2R and the RF titre or percentage of C1q-binding activity, respectively. TNF was found within the normal range in all patients except one, who was characterized by active arthritis, high number of rheumatoid skin nodules and extremely high RF titre. Since this patient was not responsive to second- and third-line drugs, an immunomodulatory therapy with high-dose intravenous immunoglobulins was established. Following such treatment a transient clinical improvement as well as a drop in TNF level and RF titre were observed.     

肿瘤坏死因子诱导肿瘤细胞凋亡及其调节机制的研究

背景肿瘤坏死因子(TNF)在体外能诱导某些正常细胞和大多数肿瘤细胞株(包括Hela细胞株)凋亡,其凋亡发生机制与细胞周期的关系中尚有许多问题需要探讨.目的研究细胞周期时相对TNF诱导Hela细胞凋亡的影响.设计非随机非对照实验研究.地点和对象实验在广东医学院生物化学与分子生物学研究所完成,对象为重组人肿瘤坏死因子-α(rhTNF-α,Sigma产品),Hela细胞株引自中国科学院上海细胞生物研究所.干预通过胸腺嘧啶核苷酸(TdR)阻断法阻滞Hela细胞的细胞周期,采用MTT法、流式细胞术和荧光染色分析TdR阻滞细胞和周期化的Hela细胞对TNF诱导凋亡的敏感性.主要观察指标TNF诱导TdR阻滞和周期化Hela细胞凋亡数.结果TdR阻滞细胞周期较周期化的Hela细胞对TNF诱导凋亡的敏感性降低.结论TNF诱导Hela细胞凋亡与细胞周期有关.     

TNFR2基因多态性与系统性红斑狼疮的相关性研究

目的 探讨TNFR2基因587位点的多态性是否与中国南方系统性红斑狼疮（SLE）人群的易感性有关,并进一步分析其与SLE实验室指标的相关性。方法 采用序列特异引物聚合酶链反应（PCR—SSP）法检测128例SLE患者和135例健康人群中TNFR2基因nt587的多态性频率。收集SLE患者的临床资料,利用x^2检验或t检验分析nt587的基因分型与ANA、抗dsDNA抗体、C3、C4等实验室指标的相关性。结果 128例SLE中,nt587G的等位基因频率为54个（21．1％）;而135例健康人群中nt587G的频率为35个（13．0％）;SLE组明显高于健康人群（P〈0．05）,携带nt587G的个体SLE发病危险性大。128例SLE患者中有77例为TT基因型,48例为TG基因型,3例为GG基因型,统计结果表明基因分型与ANA、抗dsDNA抗体相关,与C3、C4不相关。结论 TNFR2基因nt587（T—G）的多态性与中国南方SLE人群相关。     

2型糖尿病发病机制研究:肿瘤坏死因子α与胰岛素抵抗的关系

目的:回顾肿瘤坏死因子α的生物学效应以及与胰岛素抵抗之间的关系,为糖尿病一级康复预防提供理论依据。资料来源:应用计算机检索ScienceDirect1996-01/2004-05期间的相关文章,检索词“tumornecrosisfactor-α,insulinresistance”,并限定文章语言种类为英文。同时计算机检索“中国期刊网”2003-01/2004-06期间的相关文章,限定文章语言种类为中文,检索词为“肿瘤坏死因子α,胰岛素抵抗”等。资料选择:对资料进行初审,选择肿瘤坏死因子α与胰岛素抵抗之间的相互作用的研究,治疗糖尿病的方法方面的实验研究。资料提炼:共收集到15篇关于肿瘤坏死因子α与胰岛素抵抗之间的作用以及减重与肥胖方面的文献。资料综合:肿瘤坏死因子α在肥胖动物模型及肥胖症患者的脂肪组织中过度表达,在与肥胖相关的胰岛素抵抗中起核心作用。实验研究也显示出减重治疗由肿瘤坏死因子α导致的糖尿病有一定疗效。结论:肿瘤坏死因子α与胰岛素抵抗之间分子水平联系的研究越来越细致化,糖尿病的治疗向分子水平及基因水平发展,为研究糖尿病发病机制,早期预防及运动干预和开发胰岛素抵抗药物奠定分子生物学基础。     

肿瘤坏死因子受体2基因多态性与汉族人群冠心病的相关性研究

目的 探讨肿瘤坏死因子受体2(tumor necrosis factor receptor 2,TNFR2)第6外显子196位点基因多态性与冠心病的相关性.方法 采用病例对照研究的方法,用ShineRoar探针技术检测142例冠心病患者、215例冠状动脉粥样硬化患者和631名健康体检者的TNFR2第6外显子196位点的基因型.结果 TNFR2第6外显子196位点GG型基因携带者发牛冠心病的风险(OR=2.556,95%CI:1.051～6.218,χ2=4.57,P=0.033)及冠状动脉粥样硬化的风险(OR=2.547,95%CI:1.162～5.579,χ2=5.81,P=0.016)明显高于TT型基因携带者;但两分类Logistic回归分析结果显示,在控制性别和年龄不变的情况下,TNFR2第6外显子GG型基因与冠心病(OR=0.614,95%CI:0.166～2.279,χ2=0.53,P=0.466)及冠状动脉粥样硬化(OR=0.644,95%CI:0.200～2.069,χ2=0.55,P=0.459)的关联无统计学意义.结论 汉族人群TNFR2第6外显子196位点基因多态性不是汉族人发生冠心病及冠状动脉粥样硬化的独立风险因素.     

The tumor necrosis factor receptor and human neutrophil function. Deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation.

Despite numerous reports, the role of tumor necrosis factor (TNF) in polymorphonuclear leukocyte (PMN) function remains controversial. We found TNF to be a potent, pertussis toxin-independent stimulator of PMN adhesion (ED50 2.6 pM). TNF-stimulated PMN under adherent conditions released up to 65% of their transcobalamine content (ED50 3.9 pM) and increased their burst activity 10-fold (ED50 3.2 pM) as measured by the hexose monophosphate shunt, whereas PMN held in suspension hardly degranulated at all and only little burst activity was demonstrable. However, preincubation of PMN with TNF in suspension led to a decrease in cellular adhesiveness, degranulation, and burst activity in response to a secondary stimulus of TNF under adherent conditions, although cells remained fully responsive toward phorbol myristate acetate. A concomitant dose-dependent decline of TNF receptor numbers that correlated well with the inhibition of PMN function (r = 0.91) suggests receptor down-regulation as the mechanism of functional PMN deactivation. Remarkably, preincubation with other PMN stimuli such as N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor, leukotriene B4, complement component fragment 5a (C5a)/C5a (desarginated), and endotoxin also led to a reduction of TNF-specific PMN responses (cross-deactivation) from 35% (LTB4) to 90% (endotoxin), corresponding with the down-regulation of TNF receptors. Deactivation and receptor down-regulation are independent of pertussis toxin-sensitive G proteins and protein kinase C but seemed to depend on changes in calcium metabolism. Granulocyte hyporesponsiveness towards TNF in sepsis (with elevated blood levels of endotoxin and TNF) might be a mechanism of self-protection or, to the contrary, might impair a possibly central mode of host defense.     

AMP-activated protein kinase protects against anti-epidermal growth factor receptor-Pseudomonas exotoxin A immunotoxin-induced MA11 breast cancer cell death

We have shown previously that our 425.3PE immunotoxin inhibits protein synthesis and induces apoptosis in human breast cancer cells. In attempts to further elucidate the intracellular pathways implicated in its cellular effects, we found that the immunotoxin induced an initial stress response, which rapidly caused an imbalance in the cellular energy status with an increase in reactive oxygen species. The AMP-activated protein kinase (AMPK), a sensor of increased cellular AMP/ATP ratio, was activated by 425.3PE. An immunotoxin-induced activation of c-Jun NH2-terminal kinase (JNK) preceded and overlapped caspase-mediated cleavage of the alpha-subunit of AMPK in a time- and dose-dependent manner. The JNK activation occurred already at a dose level too low to induce any detectable changes in the apoptotic machinery or protein synthesis. In contrast, cycloheximide, even at a concentration causing a 90% inhibition of protein synthesis, did neither affect the ATP level nor activate JNK and AMPK. Pretreatment of the cells with the specific AMPK inhibitor compound C and JNK inhibitor SP600125 blocked activation of AMPK and JNK, respectively, and subsequently sensitized the cells to 425.3PE-induced cell death. Whereas the antioxidant N-acetyl-l-cysteine blocked the generation of reactive oxygen species and activation of JNK and AMPK, it did not block immunotoxin-induced apoptosis. Together, the results show that 425.3PE induces several parallel signaling events, observed initially as an early activation of survival pathways, protecting the cells against the toxic effects of the immunotoxin, followed by subsequent apoptosis induction and protein synthesis inhibition. Conceivably, therapeutic manipulation of the signaling intermediates AMPK and JNK might provide a means to maximize the anticancer effects of the 425.3 immunotoxin.