TNF-α Ab Research Articles

Anti-Inflammatory Effects of Sphingosine Kinase Modulation in Inflammatory Arthritis

Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S1P(1) receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling via S1P(1) enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E(2) production. Here, we examined the role of SphK/S1P in RA using a potent SphK inhibitor, N,N-dimethylsphingosine (DMS), and a molecular approach against one of its isoenzymes, SphK1. We observed that levels of S1P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-alpha, IL-6, IL-1beta, MCP-1, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-alpha, IFN-gamma, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK1 knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflammatory cytokines.

Trichostatin a (TSA) sensitizes the human prostatic cancer cell line DU145 to death receptor ligands treatment

The human prostatic carcinoma cell line DU145 has previously been found to be resistant to treatment with TNF-family ligands. However, TRAIL, TNF-α, and anti-Fas antibodies (Ab) treatment in combination with the histone deacetylase inhibitor Trichostatin A (TSA) converted the phenotype of DU145 from resistant to sensitive. TSA induced 15% cell death but simultaneous treatment with TRAIL, TNF-α, and anti-Fas Ab resulted in 55%, 70%, and 40% cell death, respectively. Simultaneous treatment did not increase the level of TSA-induced histone acetylation, but induced the release of acetylated histones from chromatin into the cytosol. This release was caspase dependent since it was abrogated by Z-VAD-fmk. In addition, treatment with TSA induced caspase-9 activation and resulted in the release of cytochrome c and Smac/DIABLO from mitochondria. To further investigate the role of caspase-9 in TSA-mediated apoptosis we used two different approaches: 1) cells were pretreated with the caspase-9 inhibitor Z-LEHD-fmk, and 2) cells were transfected with a dominant-negative form of caspase-9. Both approaches gave similar results: cells became resistant to treatment with TSA. These data indicate that TSA mediates its effect via the mitochondrial pathway. This was confirmed by examining DU145 overexpressing Bcl-2. These transfectants were resistant to TSA treatment. Taken together, our data shows that only simultaneous treatment with TNF-family ligands and TSA in DU145 resulted in caspase activity sufficient to induce apoptosis. The combination of TSA and TNF-family ligands could potentially be the basis for the treatment of prostate cancer.

Requirements of NK cells and proinflammatory cytokines in T cell-dependent neonatal autoimmune ovarian disease triggered by immune complex.

A model of neonatal autoimmune disease has been described recently in which an epitope-specific autoantibody to murine zona pellucida 3 induces severe ovarian disease in neonatal, but not adult, mice (neonatal AOD). The autoantibody forms immune complex with endogenous ovarian zona pellucida 3, and a pathogenic CD4(+) T cell response is triggered. The basis for the predominant neonatal susceptibility has not been clarified. In this study innate immunity, including neonatal NK cells, in neonatal AOD was investigated. Neonatal spleen contained readily detectable NK1.1(+)TCRVbeta(-), but not NK1.1(+)TCRVbeta(+), cells. Ab depletion of NK1.1(+)TCRVbeta(-) cells inhibited neonatal AOD development. Moreover, in adoptive transfer of neonatal AOD, recipient disease was ameliorated when either donor or recipient NK cells were depleted. Thus, NK cells operate in both induction and effector phases of the disease. IFN-gamma was produced by neonatal NK cells in vivo, and it may be important in neonatal AOD. Indeed, ovaries with neonatal AOD expressed high levels of IFN-gamma and TNF-alpha which correlated with disease severity, and the disease was inhibited by IFN-gamma or TNF-alpha Ab. Importantly, disease was enhanced by recombinant IFN-gamma, and treatment of T cell donors with IFN-gamma Ab also significantly reduced adoptive transfer of neonatal AOD. Finally, neonatal AOD was ameliorated in mice deficient in FcgammaRIII and was enhanced in FcgammaRIIB-deficient mice. We conclude that neonatal NK cells promote pathogenic T cell response at multiple stages during neonatal autoimmune disease pathogenesis. Also operative in neonatal AOD are other mediators of the innate system, including proinflammatory cytokines and FcgammaRIII signaling.

Effects of Tumor Necrosis Factor-.ALPHA. on the Synthesis of DNA, the Secretion of Matrix Metalloproteinases/Tissue Inhibitors of Metalloproteinases, and the Activity of Invasive Migration in Cultured Vascular Smooth Muscle Cells.

To address the question of whether or not tumor necrosis factor-α (TNF-α) regulates the functions of vascular smooth muscle cells (SMCs), dense or sparse cultures of the cells derived from human aorta were treated with TNF-α or TNF-αneutralizing antibody (TNF-α Ab). The incorporation of [3H]thymidine into the acid-insoluble fraction of SMCs was significantly inhibited by TNF-α, but stimulated by TNF-α Ab only when the cells had a high cell density. TNF-αsignificantly increased the accumulation of matrix metalloproteinase-1 and -3 (MMT-1 and -3, respectively) in the conditioned medium of dense SMCs, but does not affect that of tissue inhibitors of metalloproteinases-1 (TIMP-1); MMP-9 and TIMP-2 were undetectable. The invasive migration of SMCs determined by a Transwell system was stimulated by neither TNF-α nor TNF-α Ab. Taking these results together, it is suggested that TNF-α regulates DNA and MMP synthesis in dense SMCs but does not affect their invasive migration.

Cerebral Endothelial Cells Release TNF-α After Stimulation with Cell Walls of Streptococcus pneumoniae and Regulate Inducible Nitric Oxide Synthase and ICAM-1 Expression Via Autocrine Loops

Abstract TNF-α, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-α, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-α mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-α protein peaked at 4 h (9.4 ± 3.6 vs 0.1 ± 0.1 pg/μg protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 ± 4 vs 5.8 ± 1.8 at 12 h; 18.1 ± 4 vs 5.8 ± 1.8 pmol/μg protein at 72 h). The addition of TNF-α Ab significantly reduced nitrite production to 62.2 ± 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-α Ab (142 ± 18.6 vs 97.5 ± 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-α mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-α, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.

Interferon-γ Modulates a p53-independent Apoptotic Pathway and Apoptosis-related Gene Expression

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Membrane Form of TNFα Induces both Cell Lysis and Apoptosis in Susceptible Target Cells

Tumor necrosis factor α, in the secreted as well as membrane-associated (mTNFα) form, represents a cytotoxic effector mechanism of activated macrophages; in contrast, direct evidence of the mTNFα involvement in cytotoxic T lymphocyte (CTL)-mediated lysis has not yet been obtained. We observed that following activation with anti-CD3 monoclonal antibody (mAb), both cloned CTL and peritoneal exudate lymphocytes rapidly upregulated mTNFα; a similar effect was observed in the macrophage cell line J774 after stimulation with lipopolysaccharide endotoxin. Activated effector cells, which were fixed with paraformaldehyde before testing, exerted lytic activity against the TNF-sensitive WEHI 164 tumor cell line, but not against the TNF-resistant P-815 mastocytoma. This effect was completely inhibited in the presence of anti-mouse TNFα Ab. Moreover, both mTNFα-expressing macrophages and CTL induced nuclear DNA fragmentation in WEHI 164 cells, which was also blocked by anti-TNFα Ab and was accompanied by a morphologic degeneration characteristic of the apoptotic form of cell death. These data on the whole indicate a common mode of action for mTNFα expressed on different cell populations endowed with cytotoxic capability and also imply a role for this molecule in T-cell-mediated cytotoxicity.