Tumor necrosis factor as a pharmacological target

As indicated by its name, tumor necrosis factor (TNF), cloned in 1985, was originally described as a macrophage-derived endogenous mediator that can induce hemorrhagic necrosis of solid tumors and kill some tumor cell lines in vitro. Unfortunately, its promising use as an anticancer agent was biased by its toxicity, which was clear soon from the first clinical trials with TNF in cancer. Almost at the same time TNF was being developed as an anticancer drug, it became clear that TNF was identical to a mediator responsible for cachexia associated with sepsis, which was termed cachectin. This research led to the finding that TNF is, in fact, the main lethal mediator of sepsis and to the publication of a huge number of articles showing that TNF inhibits the toxic effects of bacterial endotoxins, which are now described as systemic inflammatory response. Although the clinical trials with anti-TNF in sepsis have not been successful thus far, undoubtedly as a result of the complexity of this clinical setting, these studies ultimately led to the identification of TNF as a key inflammatory mediator and to the development of anti-TNF molecules (soluble receptors and antibodies) for important diseases including rheumatoid arthritis and Crohn’s disease. On the other side, the mechanisms by which TNF and related molecules induce cell death have been studied in depth, and their knowledge might, in the future, suggest means of improve the therapeutic index of TNF in cancer.     

Tumor necrosis factor is markedly synergistic with interleukin 1 and interferon-γ in stimulating the production of nerve growth factor in fibroblasts

A possible interaction between tumor necrosis factor- (TNF) and other cytokines/growth factors in stimulating the production of nerve growth factor (NGF) in Swiss 3T3 cells was studied. TNF's stimulatory activity on fibroblast NGF production was synergized by interleukin-1 (IL-1), IL-1β and interferon-γ (IFN-γ), but was antagonized by transforming growth factor-β (TGF-β). The most remarkable synergistic effect was observed between TNF and IL-1/β; as little as 0.003 ng/ml of IL-1β markedly enhanced TNF's stimulatory activity on NGF production in the cells. These findings reinforce the idea that TNF, in concert with IL-1/β, plays an essential role in regulating the regeneration of peripheral nerves following injury through an indirect mechanism by which it stimulates NGF production in fibroblasts.     

NT FRZ 基因多态性与SEL 的相关性研究

本研究通过调查中国南方SLE人群和健康对照人群中TNFR2基因两个位点（nt587,nt694）的多态性频率,探讨TNFR2基因多态性是否与中国汉族SLE人群的易感性相关。结果发现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组明显高于健康对照人群（P〈0．05）,携带nt587G的个体SLE发病危险性大。同时128例SLE中,舶94A的等位基因频率为41个（16．0％）;而健康人群中舶94A的频率为32个（11．9％）;两组比较无显著差异（P=0．149）。提示TNFR2基因nt587的多态性与中国南方SLE人群相关,可能通过影响TNFR2的表达而参与SLE的发病,而nt694（G—A）的多态性与中国南方SLE人群不相关。     

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.     

Interleukin-6 is a better marker of lethality than tumor necrosis factor in endotoxin treated mice

Abstract We established a mouse model to differentiate between a lethal and non-lethal presentation of endotoxic shock. The model involved injecting different amounts of Escherichia coli LPS into C3H/HeN mice which had been 'primed' with BCG. We found that the mice receiving non-lethal and lethal doses of LPS could not be differentiated in terms of their physical symptoms for the first 8 h post-injection. Tumour necrosis factor (TNF) was detected at concentrations 2–9-fold greater in mice receiving lethal doses of LPA when compared with non-lethally injected mice. However, given that (i) the successful detection of this differential was dependent on the time of sampling and (ii) that TNF was only detected in the first 3–4 h post LPS challenge, we suggest that TNF may not be very useful as a prognostic marker in endotoxic shock. In contrast, circulating IL-6 appeared to mirror the symptoms of the endotoxic mice. The relative disappearance of IL-6 after 10 h in the non-lethally injected mice corresponded with their symptomatic recovery, while IL-6 continued to circulate up to the time of death in the lethally injected mice. Furthermore, there appeared to be a good correlation between the levels of injected LPS and the levels of IL-6 induced into the circulation. Our results suggest that IL-6, rather than TNF, may serve as a prognostic marker for endotoxic shock.     

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.     

Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.     

Analysis of human tumour necrosis factor receptor 1 dominant-negative mutants reveals a major region controlling cell surface expression

Tumour necrosis factor receptor 1 (TNFR1) plays a critical role in host defence and inflammation. We have identified a membrane proximal region (aa 218–324) of TNFR1 that restricts surface expression. This was prompted by comparing the dominant-negative properties of a C-terminal truncation of TNFR1 with a point mutant that prevents signalling. C-terminal truncation (aa 218–426) generates a better dominant-negative TNFR1 mutant than inactivation of the death domain by point mutation. The increased dominant-negative activity correlates with increased cell surface expression. The membrane proximal region is the most important region of the receptor for restricting expression.     

Tumor necrosis factor-α induces substance P in sympathetic ganglia through sequential induction of interleukin-1 and leukemia inhibitory factor

The present study establishes that tumor necrosis factor-α (TNF-α) induction of sympathetic substance P (SP) requires sequential induction of both interleukin (IL-1) and leukemia inhibitory factor (LIF). TNF-α dose-dependently induces SP, an induction that is secondory to an increase in the SP precursor, preprotachykinin (PPT), mRNA. Since TNF-α conditioned medium (CM) mimics the effect of TNF-α by raising SP, actions that are not antagonized by a neutralizing TNF-α antibody, TNF-α induction of SP is mediated by a soluble intermediate or intermediates. The blockade of TNF-α action by a specific IL-1 receptor antagonist and the induction of IL-1 mRNA by TNF-α suggest that IL-1 is one of the intermediates. Moreover, because immunoprecipitation with LIF antibodies decreases SP-inducing activity of TNF-α CM, and because LIF mRNA is also induced by TNF-α, LIF is a second intermediate. Furthermore, TNF-α-induced LIF mRNA is blocked by the IL1 receptor antagonist, whereas IL-1-induced LIF mRNA is not affected by TNF-α antibodies, suggesting that TNF-α first induces IL-1, and IL-1 subsequently induces LIF. These data suggest that TNF-α induces SP in sympathetic ganglia through the sequential inductions of IL1 and LIF. © 1995 John Wiley & Sons, Inc.     

Fumonisin B(1) alters sphingolipid metabolism and tumor necrosis factor alpha expression in heart and lung of mice

Fumonisin B(1) (FB(1)), produced by Fusarium verticillioides, is a common contaminant in foods and feeds. Increase in tissue free sphingoid bases resulting from the inhibition of ceramide synthase is a biomarker of fumonisin exposure. Tumor necrosis factor alpha (TNFalpha) is induced in liver in response to FB(1) treatment. This study determined whether fumonisin B(1) caused increases in free sphingoid bases and altered the expression of TNFalpha in heart and lung, organs that are not targets of FB(1) toxicity, of male and female mice treated with 5-daily subcutaneous injection of 2.25 mg/kg FB(1). A significant increase in free sphingoid bases was observed in both heart and lung of FB(1)-exposed mice. The magnitude of increases in free sphingoid bases in both organs of female mice was much higher than that in males. The expression of TNFalpha was increased by FB(1) treatment in the lung of male mice and in the heart of female mice, whereas the expression of interferon gamma was unaltered. Results suggest that both sphingolipid accumulation and TNFalpha induction are observed in the tissues of mice that are not associated with FB(1) toxicity.     

Tumor necrosis factor receptor-associated protein 1 improves hypoxia-impaired energy production in cardiomyocytes through increasing activity of cytochrome c oxidase subunit II

Tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes against hypoxia, but the underlying mechanisms are not completely understood. In the present study, we used gain- and loss-of-function approaches to explore the effects of tumor necrosis factor receptor-associated protein 1 and cytochrome c oxidase subunit II on energy production in hypoxic cardiomyocytes. Hypoxia repressed ATP production in cultured cardiomyocytes, whereas overexpression of tumor necrosis factor receptor-associated protein 1 significantly improved ATP production. Conversely, knockdown of tumor necrosis factor receptor-associated protein 1 facilitated the hypoxia-induced decrease in ATP synthesis. Further investigation revealed that tumor necrosis factor receptor-associated protein 1 induced the expression and activity of cytochrome c oxidase subunit II, a component of cytochrome c oxidase that is important in mitochondrial respiratory chain function. Moreover, lentiviral-mediated overexpression of cytochrome c oxidase subunit II antagonized the decrease in ATP synthesis caused by knockdown of tumor necrosis factor receptor-associated protein 1, whereas knockdown of cytochrome c oxidase subunit II attenuated the increase in ATP synthesis caused by overexpression of tumor necrosis factor receptor-associated protein 1. In addition, inhibition of cytochrome c oxidase subunit II by a specific inhibitor sodium azide suppressed the ATP sy nthesis induced by overexpressed tumor necrosis factor receptor-associated protein 1. Hence, tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxia at least partly via potentiation of energy generation, and cytochrome c oxidase subunit II is one of the downstream effectors that mediates the tumor necrosis factor receptor-associated protein 1-mediated energy generation program.     

Recruitment of T lymphocytes and induction of tumor necrosis factor in thyroid cancer by a local immunotherapy

Summary To elucidate the mechanism of action for intratumoral injection of immunopotentiators, infiltrating mononuclear cells and tumor necrosis factor (TNF) were assayed by immunostaining tissue samples of differentiated thyroid cancer resected with or without presurgical local application of OK-432, a streptococcal preparation. Frozen sections of resected specimens were stained with monoclonal antibodies using either a conventional or a modified immunoperoxidase method. The tumors injected with OK-432 showed increased T lymphocyte infiltration and HLA-DR expression on cancer cells as compared to the non-injected controls. Among these T cells, the CD4⁺ subset was more numerous than the CD8⁺ population. In four out of the seven cases constituting the injected group, numerous TNF-positive cells were seen in clusters or lines as well as scattered, while none of the seven cases in the control group was associated with a considerable amount of these cells. In their morphology and distribution pattern, these TNF-positive cells appeared to be of macrophage lineage. Thus local injection of OK-432 in thyroid cancer was shown to recruit T lymphocytes of predominantly the CD4⁺ subset and to induce in situ production of TNF, a known potent tumoricidal cytokine. The present data warrant further studies in this direction besides wider clinical intratumoral application of the reagent.     

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102     

The potential biological and clinical significance of the soluble tumor necrosis factor receptors

The role of TNF receptors (TNF-Rs) is not limited to signal transduction but includes extracellular regulatory functions affecting systemic TNF bioavailability. This review summarizes the regulation of TNF-R shedding and its kinetics, the complex interaction between the soluble receptors and their ligand in vitro and in vivo, and the potential diagnostic, prognostic and therapeutic value of the soluble receptors in malignant, inflammatory, infectious and autoimmune disorders.