Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNFalpha-blockers in rheumatoid arthritis and spondyloarthritis patients

OBJECTIVE: To examine whether the G-to-A polymorphism at position -308 in the promoter of the tumour necrosis factor-alpha (TNFalpha) gene influences the therapeutic response to TNFalpha-blockers in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS). METHODS: A total of 54 patients with RA, 10 with PsA and 22 with AS were genotyped by polymerase chain reaction for the -308 TNFalpha promoter polymorphism. They were treated with infliximab (n = 63), adalimumab (n = 10) or etanercept (n = 13). Clinical response was assessed after 24 weeks by the Disease Activity Score in 28 joints (DAS28) for RA and PsA, and the Bath Ankylosing Spondylitis Activity Index (BASDAI) for AS patients. RESULTS: All patients with the A/A genotype (n = 3, all RA) and two patients with the A/G genotype (AS) failed to respond to anti-TNF treatment. Irrespective of the underlying disease, moderate response (n = 44) was predominantly associated with the A/G genotype (A/G 18/22, G/G 4/22), whereas good response (n = 59) was exclusively seen in patients with the G/G genotype. The average improvement in the DAS28 score was 0.83 in the A/A, 1.50 in the A/G and 2.64 in the G/G group of RA and PsA patients (P < 0.0001). The BASDAI score in AS improved on average by 1.21 in the A/G and by 3.30 in the G/G group (P < 0.005). CONCLUSIONS: The data suggest that humans with a TNFalpha -308 G/G genotype are better responders to anti-TNFalpha treatment than those with A/A or A/G genotypes independent of the treated rheumatic disease (RA, PsA or AS).

Increased lipopolysaccharide-induced tumour necrosis factor-alpha, interferon-gamma and interleukin-10 production in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is based on a genetic predisposition, but environmental factors may trigger skin inflammation. According to the hygiene hypothesis, decreased exposure to microbial products in early childhood does not allow sufficient maturation of the immune system that is associated with an increased risk of atopic sensitization. OBJECTIVES: The effect of lipopolysaccharide (LPS) on the cytokine production of peripheral blood mononuclear cells (PBMC) of AD patients and nonatopic controls was studied. PATIENTS AND METHODS: PBMC were isolated from heparinized blood of 10 patients with AD and 10 nonatopic individuals, suspended in culture medium and stimulated with LPS. Cytokine levels in the supernatants were measured by immunoassays. Results Upon stimulation with LPS, PBMC from AD patients produced significantly higher amounts of tumour necrosis factor-alpha, interferon-gamma and interleukin (IL)-10 compared with control PBMC. LPS stimulation blocked the increased spontaneous production of IL-4 and IL-5 by PBMC from AD patients, but had no effect on IL-13 production. CONCLUSIONS: These results demonstrate that the effects of LPS stimulation depend on both the type of cytokine and the origin of PBMC. Endotoxin exposure is suggested to modulate the disease course of AD.

Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis

Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) converting enzyme (TACE) contribute synergistically to the pathophysiology of bacterial meningitis. TACE proteolytically releases several cell-surface proteins, including the proinflammatory cytokine TNF-alpha and its receptors. TNF-alpha in turn stimulates cells to produce active MMPs, which facilitate leucocyte extravasation and brain oedema by degradation of extracellular matrix components. In the present time-course studies of pneumococcal meningitis in infant rats, MMP-8 and -9 were 100- to 1000-fold transcriptionally upregulated, both in CSF cells and in brain tissue. Concentrations of TNF-alpha and MMP-9 in CSF peaked 12 h after infection and were closely correlated. Treatment with BB-1101 (15 mg/kg subcutaneously, twice daily), a hydroxamic acid-based inhibitor of MMP and TACE, downregulated the CSF concentration of TNF-alpha and decreased the incidences of seizures and mortality. Therapy with BB-1101, together with antibiotics, attenuated neuronal necrosis in the cortex and apoptosis in the hippocampus when given as a pretreatment at the time of infection and also when administration was started 18 h after infection. Functionally, the neuroprotective effect of BB-1101 preserved learning performance of rats assessed 3 weeks after the disease had been cured. Thus, combined inhibition of MMP and TACE offers a novel therapeutic strategy to prevent brain injury and neurological sequelae in bacterial meningitis.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer

The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Comparison of drug retention rates and causes of drug discontinuation between anti-tumor necrosis factor agents in rheumatoid arthritis

OBJECTIVE: Tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of severe rheumatoid arthritis (RA), yet drug discontinuation is common. The aim of this study was to compare treatment retention rates and specific causes of anti-TNF discontinuation in a population-based RA cohort. METHODS: All patients treated with etanercept, infliximab, or adalimumab within the Swiss Clinical Quality Management RA cohort between 1997 and 2006 were included in the study. Causes of treatment discontinuation were broadly categorized as adverse events (AEs) or nontoxic causes, and further subdivided into specific categories. Specific causes of treatment interruption were analyzed using a Cox proportional hazards model and adjusted for potential confounders. RESULTS: A total of 2,364 anti-TNF treatment courses met the inclusion criteria. Treatment discontinuation was reported 803 times: 309 with etanercept, 249 with infliximab, and 245 with adalimumab. Drug inefficacy represented the largest single cause of treatment discontinuation (55.8% of cases). The median time of receiving anti-TNF therapy was 37 months, but discontinuation rates differed between the 3 anti-TNF agents (P < 0.001), with shorter retention rates for infliximab (hazard ratio [HR] 1.24, 99% confidence interval [99% CI] 1.01-1.51). The specific causes of treatment discontinuation revealed an increased risk of AEs with infliximab (HR 1.4, 99% CI 1.003-1.96), mostly due to an increased risk of infusion or allergic reactions (HR 2.11, 99% CI 1.23-3.62). Other discontinuation causes were equally distributed between the anti-TNF agents. CONCLUSION: In this population, infliximab was associated with higher overall discontinuation rates compared with etanercept and adalimumab, which is mainly due to an increased risk of infusion or allergic reactions.

Hypoxia inducible factor-1 alpha induction by tumour necrosis factor-alpha, but not by toll-like receptor agonists, modulates cellular respiration in cultured human hepatocytes

BACKGROUND/AIMS: Genes encoding for some of the mitochondrial proteins are under the control of the transcriptional factor hypoxia inducible factor-1 alpha (HIF-1 alpha), which can accumulate under normoxic conditions in inflammatory states. The aim of this study was to evaluate the effects of cobalt chloride (CoCl(2), a hypoxia mimicking agent), tumour necrosis factor-alpha (TNF-alpha) and toll-like receptor (TLR) -2, -3 and -4 agonists on HIF-1 alpha accumulation, and further on HIF-1 alpha-mediated modulation of mitochondrial respiration in cultured human hepatocytes. METHODS: The human hepatoma cell line HepG2 was used in this study. Cells were treated with CoCl(2), TNF-alpha and TLR-2, -3 and -4 agonists. HIF-1 alpha was determined by Western blotting and mitochondrial respiration in stimulated cells by high-resolution respirometry. RESULTS: CoCl(2), TNF-alpha and TLR agonists induced the expression of HIF-1 alpha in a time-dependent fashion. TNF-alpha and CoCl(2), but not TLR agonists, induced a reduction in complex I-, II- and IV-dependent mitochondrial oxygen consumption. TNF-alpha-associated reduction of cellular oxygen consumption was abolished through inhibition of HIF-1 alpha activity by chetomin (CTM). Pretreatment with cyclosporine A prevented CoCl(2)-induced reduction of complex I- and II-dependent mitochondrial oxygen consumption and TNF-alpha-induced reduction of complex-I-dependent respiration, implicating the involvement of the mitochondrial permeability transition pore openings. TNF-alpha and TLR-2, -3 and -4 agonists induced the expression of vascular endothelial growth factor, which was partially abolished by the blockage of HIF-1 alpha with CTM. CONCLUSIONS: The data suggest that HIF-1 alpha modulates mitochondrial respiration during CoCl(2) and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.

Transmembrane tumor necrosis factor is a potent inducer of colitis even in the absence of its secreted form

Tumor necrosis factor (TNF) is cleaved proteolytically from a 26-kilodalton transmembrane precursor protein into secreted 17-kilodalton monomers. Transmembrane (tm) and secreted trimeric TNF are biologically active and may mediate distinct activities. We assessed the consequences of a complete inhibition of TNF processing on the course of colitis in recombination activating gene (RAG)2 -/- mice on transfer of CD4 CD45RB hi T cells.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

Ras signaling in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is important in immunologic cytotoxicity, autoimmunity, sepsis, normal embryonic development, and wound healing. TNF exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. We found that enforced expression of an activated H-ras oncogene converted the non-tumorigenic TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells (10TEJ) that also became very sensitive to TNF-induced apoptosis. This finding suggested that the oncogenic form of H-Ras, in which the p21 is locked in the GTP-bound form, could play a role in TNF-induced apoptosis of these cells. To investigate whether Ras activation is an obligatory step in TNF-induced apoptosis, we introduced two different molecular antagonists of Ras, namely the Rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras transformed 10TEJ cells. Expression of either Rap1A or RasN17 in 10TEJ cells resulted in abrogation of TNF-induced apoptosis. Similar results were obtained by expression of either Ras antagonist in L929 cells, a fibroblast cell line that is sensitive to TNF-induced apoptosis but does not have a ras mutation. The effects of Rap-1A and RasN17 appear to be specific to TNF, since cytotoxicity induced by doxorubicin and thapsigargin are unaffected. Additionally, constitutive apoptosis sensitivity in isolated nuclei, as measured by activation of Ca$\sp{2+}$-dependent endogenous endonuclease, is not affected by Rap-1A or RasN17. Moreover, TNF treatment of L929 cells increased Ras-bound GTP, indicating that Ras activation is triggered by TNF. Thus, Ras activation is required for TNF-induced apoptosis in mouse cells. ^

Cloning, sequencing and partial functional characterization of the 5' region of the human p75 tumor necrosis factor receptor-encoding gene (TNF-R)

A 1887-bp region at the 5' flank of the human p75 tumor necrosis factor receptor (p75 TNF-R)-encoding gene was found to be active in driving expression of the luc (luciferase-encoding) reporter gene, suggesting that it contains the promoter for the receptor. Rather unexpectedly, a 1827-bp region at the 3' end of the first intron of the p75 TNF-R gene also displayed promoter activity. This activity may be artefactual, reflecting only the presence of an enhancer in this region; yet it also raises the possibility that p75 TNF-R is controlled by more than one promoter and that it encodes various forms of the receptor, or even other proteins. We present here the nucleotide sequences of the 5' flanking and intron regions. Possible implications for the transcriptional regulation of the p75 TNF-R gene are discussed.