The role of cytokine gene polymorphism in the formation of arterial hypertension associated with metabolic syndrome.

We investigated the association of polymorphisms of genes tumor necrosis factors and their receptors (-308G/A TNFa, +250A/G Lta, +36 A/G TNFR1, +1663 A/G TNFR2) with the predisposition to the development of essential hypertension (EH) and the features of its clinical course in patients with metabolic syndrome. It has been demonstrated that the molecular genetic marker +36G TNFR1 (OR=1,25) is involved in the formation EH in individuals with metabolic syndrome. The risk of stage III EH in patients with metabolic syndrome is enhanced by genetic variants -308GA TNFa (OR=2,72), -308A TNFa (OR=2,72), +250G Lta (OR=1,80), and combinations thereof -308A TNFa with +1663G TNFR2 (OR=3,85), +250G Lta with +36G TNFR1 (OR=3,85), +250G Lta with +1663G TNFR2 (OR=3,85) while protective properties are inherent in -308GG TNFa (OR=0,32), +250AA Lta (OR=0,45), -308G TNFa (OR=0,37), +250A Lta (OR=0,56) and a combination of genetic markers -308GG TNFa with +250A Lta (OR=0,31), -308G TNFa with +250AA Lta (OR=0,39), -308G TNFa with +250A Lta (OR=0,31).

Association between genetic variations in tumor necrosis factor receptor genes and survival of patients with T-cell lymphoma.

The prognosis of T-cell lymphoma (TCL) has been shown to be associated with the clinical characteristics of patients. However, there is little knowledge of whether genetic variations also affect the prognosis of TCL. This study investigated the associations between single nucleotide polymorphisms(SNPs) in tumor necrosis factor receptor superfamily(TNFRSF) genes and the survival of patients with TCL. A total of 38 tag SNPs in 18 TNFRSF genes were genotyped using Sequenom platform in 150 patients with TCL. Kaplan-Meier survival estimates were plotted and significance was assessed using log-rank tests. Cox proportional hazard models were used to analyze each of these 38 SNPs with adjustment for covariates that might influence patient survival, including sex and international prognostic Index score. Hazard ratios (HRs) and their 95% confidence intervals(CIs) were calculated. Among the 38 SNPs tested, 3 were significantly associated with the survival of patients with TCL. These SNPs were located at LTßR (rs3759333C>T) and TNFRSF17(rs2017662C>T and rs2071336C>T). The 5-year survival rates were significantly different among patients carrying different genotypes and the HRs for death between the different genotypes ranged from 0.45 to 2.46. These findings suggest that the SNPs in TNFRSF genes might be important determinants for the survival of TCL patients.

The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis.

Tumor necrosis factor receptor superfamily (TNFRSF) members were initially identified as immunological mediators, and are still commonly perceived as immunological molecules. However, our understanding of the diversity of TNFRSF members' roles in mammalian physiology has grown significantly since the first discovery of TNFRp55 (TNFRSF1) in 1975. In particular, the last decade has provided evidence for important roles in brain development, function and the emergent field of neuronal homeostasis. Recent evidence suggests that TNFRSF members are expressed in an overlapping regulated pattern during neuronal development, participating in the regulation of neuronal expansion, growth, differentiation and regional pattern development. This review examines evidence for non-immunological roles of TNFRSF members in brain development, function and maintenance under normal physiological conditions. In addition, several aspects of brain function during inflammation will also be described, when illuminating and relevant to the non-immunological role of TNFRSF members. Finally, key questions in the field will be outlined.

T-RMSD: a fine-grained, structure-based classification method and its application to the functional characterization of TNF receptors.

This study addresses the relation between structural and functional similarity in proteins. We introduce a novel method named tree based on root mean square deviation (T-RMSD), which uses distance RMSD (dRMSD) variations to build fine-grained structure-based classifications of proteins. The main improvement of the T-RMSD over similar methods, such as Dali, is its capacity to produce the equivalent of a bootstrap value for each cluster node. We validated our approach on two domain families studied extensively for their role in many biological and pathological pathways: the small GTPase RAS superfamily and the cysteine-rich domains (CRDs) associated with the tumor necrosis factor receptors (TNFRs) family. Our analysis showed that T-RMSD is able to automatically recover and refine existing classifications. In the case of the small GTPase ARF subfamily, T-RMSD can distinguish GTP- from GDP-bound states, while in the case of CRDs it can identify two new subgroups associated with well defined functional features (ligand binding and formation of ligand pre-assembly complex). We show how hidden Markov models (HMMs) can be built on these new groups and propose a methodology to use these models simultaneously in order to do fine-grained functional genomic annotation without known 3D structures. T-RMSD, an open source freeware incorporated in the T-Coffee package, is available online.

A novel role for Glucocorticoid-Induced TNF Receptor Ligand (Gitrl) in early embryonic zebrafish development.

Tumour necrosis factor ligand and receptor superfamily (TNFSF and TNFRSF) members have diverse and well-studied functions in the immune system. Additional, non-immunological roles, such as in the morphogenesis of bone, tooth, hair and skin have also been described for some members. GITRL and its receptor GITR are well-described as co-regulators of the mammalian immune response. Here, we describe the identification and cloning of their zebrafish homologues and demonstrate a novel role for the ligand, but not the receptor, in early vertebrate development. The assignment of zebrafish Gitrl and Gitr was supported by homology and phylogenetic analysis. The ligand exhibited an oscillating pattern of mRNA expression during the first 36 hours post fertilization, during which time gitr mRNA was not detected, and morpholino oligonucleotide-mediated knock-down of gitrl, but not of gitr, resulted in disruption of early embryogenesis, most clearly revealed during gastrulation, which corresponded to the earliest peak in gitrl mRNA expression (5.25-10 hpf). We found Stat3 signalling to be altered in the gitrl-morphants, suggesting that one possible role for Gitrl during embryogenesis may be modulation of Jak/Stat signalling.

Selective stimulation of either tumor necrosis factor receptor differentially induces pain behavior in vivo and ectopic activity in sensory neurons in vitro.

Recent studies suggest that tumor necrosis factor-alpha (TNF) sensitizes primary afferent neurons, and thus facilitates neuropathic pain. Here, we separately examined the roles of tumor necrosis factor receptor (TNFR) 1 and 2 by parallel in vivo and in vitro paradigms using proteins that selectively activate TNFR1 or TNFR2 (R1 and R2). In vivo, intrathecally injected R1, but not R2 slightly reduced mechanical and thermal withdrawal thresholds in rats, whereas co-injection resulted in robust, at least additive pain-associated behavior. In vitro, the electrophysiological responses of dorsal root ganglia (DRG) from rats with spinal nerve ligation were measured utilizing single-fiber recordings of teased dorsal root filaments. In naïve DRG, only R1 (10-1000 pg/ml) induced firing in Ass- and Adelta-fibers, whereas R2 had no effect. In injured DRG, both R1 and R2 at significantly lower concentrations (1 pg/ml) increased discharge rates of Adelta-fibers. Most interesting, in adjacent uninjured DRG, R2 and not R1, increased ectopic activity in both Ass- and Adelta-fibers. We conclude that TNFR1 may be predominantly involved in the excitation of sensory neurons and induction of pain behavior in the absence of nerve injury, TNFR2 may contribute in the presence of TNFR1 activation. Importantly, the effects of individually applied R1 and R2 on injured and adjacent uninjured fibers imply that the role of TNFR2 in the excitation of sensory neurons increases after injury.

Balancing co-stimulation and inhibition with BTLA and HVEM.

The interaction between B- and T-lymphocyte attenuator (BTLA), an inhibitory receptor whose extracellular domain belongs to the immunoglobulin superfamily, and herpesvirus-entry mediator (HVEM), a co-stimulatory tumour-necrosis factor receptor, is unique in that it is the only receptor-ligand interaction that directly bridges these two families of receptors. This interaction has raised many questions about how receptors from two different families could interact and what downstream signalling events might occur as a result of receptor ligation. As we discuss, recent studies show that engagement of HVEM with its endogenous ligand (LIGHT) from the tumour-necrosis factor family induces a powerful immune response, whereas HVEM interactions with BTLA negatively regulate T-cell responses.

Endocrine regulation of suppressor lymphocytes: role of the glucocorticoid-induced TNF-like receptor.

Mechanisms responsible for peripheral immune tolerance are currently under investigation in several laboratories, in order to define the role of immune homeostasis in physiological processes and pathologic conditions, such as autoimmunity and cancer. In this context, recent studies attributed a relevant role to the glucocorticoid-induced TNFR-related gene (GITR). GITR is expressed at high levels on CD4(+)CD25(+). T regulatory (Treg) cells, but only at low levels on resting responder T lymphocytes, and is upregulated after activation. GITR triggering induces both pro- and anti-apoptotic effects through different intracellular pathways, abrogates the suppressive activity of Treg cells, and co-stimulates responder T cells. These data hint that GITR triggering overstimulates the immune system. Indeed, in vivo studies demonstrated that GITR stimulation may both induce autoimmune diseases and strengthen anti-virus and anti-tumor immune responses. Therefore, the GITR-GITRL system appears crucial in regulating immunity. Currently, the majority of studies about GITR's role on regulatory cells are focused on CD4(+)CD25(+) Treg cells, while very little is known about the importance of this molecule in other Treg subtypes. We have recently characterized a subpopulation of CD8+ T suppressor lymphocytes able to inhibit both T cell proliferation and cytotoxicity. Preliminary data show that GITR is expressed on such CD8+ T suppressor cells and that its activation by a specific antibody inhibits generation, but not function, of these cells. These early results suggest the importance of GITR in human T suppressor lymphocytes other than CD4(+)CD25(+) Treg cells.

Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF.

We recently reported that mast cells stimulated via FcepsilonRI aggregation can enhance T cell activation by a TNF-dependent mechanism. However, the molecular mechanisms responsible for such IgE-, Ag- (Ag-), and mast cell-dependent enhancement of T cell activation remain unknown. In this study we showed that mouse bone marrow-derived cultured mast cells express various costimulatory molecules, including members of the B7 family (ICOS ligand (ICOSL), PD-L1, and PD-L2) and the TNF/TNFR families (OX40 ligand (OX40L), CD153, Fas, 4-1BB, and glucocorticoid-induced TNFR). ICOSL, PD-L1, PD-L2, and OX40L also are expressed on APCs such as dendritic cells and can modulate T cell function. We found that IgE- and Ag-dependent mast cell enhancement of T cell activation required secreted TNF; that TNF can increase the surface expression of OX40, ICOS, PD-1, and other costimulatory molecules on CD3(+) T cells; and that a neutralizing Ab to OX40L, but not neutralizing Abs to ICOSL or PD-L1, significantly reduced IgE/Ag-dependent mast cell-mediated enhancement of T cell activation. These results indicate that the secretion of soluble TNF and direct cell-cell interactions between mast cell OX40L and T cell OX40 contribute to the ability of IgE- and Ag-stimulated mouse mast cells to enhance T cell activation.

Development of a chimaeric receptor approach to study signalling by tumour necrosis factor receptor family members.

Members of the tumour necrosis factor receptor family play a pivotal role in cell differentiation, function and apoptosis. However, signalling by many members of the family remains to be elucidated. In the present study, we developed a chimaeric receptor approach for studying signalling by receptors belonging to this family. The chimaeric receptor comprises the human Fas external domain linked to the transmembrane and cytoplasmic domains of a tumour necrosis factor receptor family member of interest. When the chimaera is expressed in mouse cells, the clustering of the chimaera induced by a human Fas-activating antibody activates the intracellular domain of the chimaera without affecting its endogenous counterpart. Since the antibody recognizes only human Fas, this approach can be used to dissect signalling by any tumour necrosis factor family member using any type of mouse cell including those endogenously expressing Fas. Moreover, we also showed that the chimaeric receptor approach can be used to study signalling at any stage of cell differentiation or function.

TNF-related weak inducer of apoptosis receptor, a TNF receptor superfamily member, activates NF-kappa B through TNF receptor-associated factors.

TNF-related weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family that induces angiogenesis in vivo. The TWEAK receptor (TweakR) is a recently identified member of the TNF receptor (TNFR) superfamily and is expressed on smooth muscle cells (SMCs) and endothelial cells (ECs). In this report we identify the TNF receptor-associated factor (TRAF) family of signal transducers as important components of TweakR-mediated NF-kappa B activation. Coimmunoprecipitation experiments suggested potential interactions between the cytoplasmic tail of TweakR with TRAFs 1, 2, 3, and 5. Dominant negative forms of TRAF2 and TRAF5 substantially inhibited TweakR-mediated NF-kappa B activation, suggesting a role of TRAFs in regulating smooth muscle and endothelial cell function. Using alanine-scanning analysis, we defined a TRAF-binding motif, PIEET, in TweakR that mediates TRAF binding and NF-kappa B activation. Furthermore, TweakR mutations within the TRAF-binding motif abolished TweakR-stimulated SMC migration, revealing a role for TRAFs in TweakR-induced activation events.

All in the family: evolutionary and functional relationships among death receptors.

Over the last decade, significant progress has been made towards identifying the signaling pathways within mammalian cells that lead to apoptosis mediated by death receptors. The simultaneous expression of more than one death receptor in many, if not all, cell types suggests that functional innovation has driven the divergence of these receptors and their cognate ligands. To better understand the physiological divergence of the death receptors, a phylogenetic analysis of vertebrate death receptors was conducted based upon amino-acid sequences encoding the death domain regions of currently known and newly identified members of the family. Evidence is presented to indicate an ancient radiation of death receptors that predates the emergence of vertebrates, as well as ongoing divergence of additional receptors both within several receptor lineages as well as modern taxonomic lineages. We speculate that divergence among death receptors has led to their functional specialization. For instance, some receptors appear to be primarily involved in mediating the immune response, while others play critical roles during development and tissue differentiation. The following represents an evolutionary approach towards an understanding of the complex relationship among death receptors and their proposed physiological functions in vertebrate species.

Roles of tumor necrosis factor-alpha receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome.

Tristetraprolin (TTP) is a member of the CCCH tandem zinc-finger class of proteins. It can bind to and destabilize mRNAs encoding tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Conversely, mice deficient in TTP develop a complex syndrome characterized by cachexia, myeloid hyperplasia, and joint and skin inflammation. Studies using anti-TNF-alpha neutralizing antibodies demonstrated that this syndrome, at least in part, is a consequence of the excess production of TNF-alpha in the absence of TTP. To evaluate the role played by each TNF-alpha receptor in the pathogenesis of this syndrome, mice were generated that were deficient in TTP and either or both of the known TNF-alpha receptors (TNFRs), type 1 (TNFR1) and type 2 (TNFR2). Mice deficient in TTP and TNFR1, or in TTP and both receptors, were protected from developing the TNF-alpha-induced cachexia and inflammation. In contrast, mice deficient in TNFR2 were more severely affected than mice deficient in TTP alone, suggesting that TNFR2 might play a protective role in the development of the syndrome. In cultured cells derived from these mice, apparent cooperation between the TNFRs was required to achieve normal TNF-alpha-induced expression of TTP, TNF-alpha, and GM-CSF mRNAs. Finally, the results showed that TNFR1 plays an important role in mediating TNF-alpha-induced changes in TNF-alpha and GM-CSF mRNA stability.

Identification of a novel receptor for B lymphocyte stimulator that is mutated in a mouse strain with severe B cell deficiency.

BLyS (also called BAFF, TALL-1, THANK, and zTNF4), a TNF superfamily member, binds two receptors, TACI and BCMA, and regulates humoral immune responses [1-7]. These two receptors also bind APRIL [7-10], another TNF superfamily member. The results from TACI(-/-) and BCMA(-/-) mice suggest the existence of additional receptor(s) for BLyS. The TACI knockout gives the paradoxical result of B cells being hyperresponsive, suggesting an inhibitory role for this receptor [11, 12], while BCMA null mice have no discernable phenotype [13]. Here we report the identification of a third BLyS receptor (BR3; BLyS receptor 3). This receptor is unique in that, in contrast to TACI and BCMA, BR3 only binds BLyS. Treatment of antigen-challenged mice with BR3-Fc inhibited antibody production, indicating an essential role for BLyS, but not APRIL, in this response. A critical role for BR3 in B cell ontogeny is underscored by our data showing that the BR3 gene had been inactivated by a discrete, approximately 4.7 kb gene insertion event that disrupted the 3' end of the BR3 gene in A/WySnJ mice, which lack peripheral B cells.

New single nucleotide polymorphisms in the coding region of human TNFR2: association with systemic lupus erythematosus.

We recently reported the association of the allele coding for Arg at the position 196 (196R: nucleotide [nt] 587G) of tumor necrosis factor receptor 2 (TNFR2, TNF-R75) with systemic lupus erythematosus (SLE) in Japanese. In the present study, we completed the variation screening of the entire coding region of TNFR2. Three new single nucleotide polymorphisms within the coding sequence (cSNPs), as well as several variations within the promoter, introns and 3'-untranslated region (3' UTR), were identified. Among the new SNPs, nt168G, a synonymous substitution (K56K), was in tight linkage disequilibrium with nt587G. Two other cSNPs, nt543 (C-->T) (P181P) and nt694 (G-->A) (E232K), were not significantly associated with SLE. Thus, among the non-synonymous cSNPs, only nt587 (T-->G) (M196R) was found to be significantly associated with SLE in Japanese.

Role for tumor necrosis factor alpha receptor 1 and interleukin-1 receptor in the suppression of mouse hepatocyte apoptosis by the peroxisome proliferator nafenopin.

Peroxisome proliferators (PPs) cause rodent liver enlargement and tumors. In vitro, PPs induce rat and mouse hepatocyte DNA synthesis and suppress apoptosis, a response mimicked by exogenous tumor necrosis factor alpha (TNFalpha). Here, we determine the role of TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), and nuclear factor kappa beta (NFkappaB) in the response of mouse hepatocytes to the PP, nafenopin. Nafenopin (50 micromol/L) induced DNA synthesis as measured by bromodeoxyuridine (BrdU) incorporation, suppressed cell death as measured by Hoechst 33258 staining, induced peroxisomal beta-oxidation as measured by cyanide insensitive palmitoyl CoA oxidation (PCO) and caused activation of nuclear factor kappa beta (NFkappaB) as determined by electrophoretic mobility gel shift assay (EMSA). The induction of DNA synthesis and the suppression of apoptosis in response to nafenopin was abrogated completely by blocking antibodies to TNFR1 but not to TNFR2. In contrast, the induction of peroxisomal beta-oxidation by nafenopin was not blocked by the anti-TNFR1 antibody. Next, we evaluated the response of hepatocytes to interleukin-1 (IL-1), another proinflammatory cytokine. IL-1alpha (2.5 ng/mL) and, to a lesser extent, IL-1beta (5 ng/mL), shared the ability of TNFalpha to induce DNA synthesis and suppress apoptosis. In addition, anti-IL-1 receptor, type 1/p80 (IL-1R) antibodies were able to abrogate the response to nafenopin. IL-1alpha was still able to perturb hepatocyte growth in the presence of the anti-TNFR1 antibody suggesting that IL-1alpha acts independently rather than by elaborating TNFalpha. In summary, these data provide additional evidence for a role for hepatic cytokines in the perturbation of hepatocyte growth by PPs such as nafenopin.

Differential expression of tumor necrosis factor receptor subtypes on leukocytes in systemic inflammatory response syndrome.

OBJECTIVE: To determine the expression of tumor necrosis factor (TNF) receptor in patients with systemic inflammatory response syndrome (SIRS). DESIGN: Prospective study. SETTING: Intensive care unit and central laboratory. PATIENTS: Blood specimens from 18 healthy volunteers (controls) and 16 patients with SIRS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Using monoclonal antibodies, fluorescence labeling, and high sensitivity flow cytometry, we measured the expression of membrane TNF receptor subtypes TNF-R55 and TNF-R75 on peripheral blood leukocytes. Receptor expression is expressed as mean fluorescence intensity +/- SD (units: detection channel number). In controls, TNF-R55 was only weakly expressed (monocytes: 2.5+/-1.8; neutrophils: 0.7+/-0.8), whereas expression of TNF-R75 was higher (monocytes: 28.6+/-9.0; neutrophils: 4.8+/-1.0) and was also found on lymphocytes (on CD8+ lymphocytes: 5.7+/-1.8; CD16+: 5.5+/-1.2; CD4+: 9.7+/-3.7). In SIRS, we observed increased expression of TNF-R55 on monocytes (6.9+/-3.4, p<.001) and neutrophils (2.2+/-1.9, p<.01), as well as decreased expression of TNF-R75 on monocytes (17.3+/-13.2; p<.001). The extent of TNF-R55 up-regulation did not correlate with that of TNF-R75 down-regulation. TNF-R55 on monocytes and neutrophils strongly correlated with body temperature but not with survival, whereas monocyte TNF-R75 was considerably lower in nonsurvivors, albeit not significantly (12.3+/-7.1 vs. 23.9+/-16.7; p = .07). CONCLUSIONS: These data indicate that leukocyte TNF-R55 and TNF-R75 react differentially and probably serve different functions in SIRS, which prompts the investigation of receptor subtype-specific therapeutic approaches.

Heterogeneous expression of tumor necrosis factor-alpha receptors I and II on human peripheral eosinophils.

BACKGROUND: Expression of the tumor necrosis factor-alpha receptor by human eosinophils has not been determined. We examined the surface expression and mRNA for tumor necrosis factor-alpha receptors I and II (TNF-alphaRI and TNF-alphaRII) on peripheral eosinophils. METHODS: Eosinophils were obtained from 17 asthma patients and 3 healthy volunteers. Flow cytometry was used to examine receptor expression, and the reverse transcriptase-polymerase chain reaction was used to examine receptor mRNA expression. RESULTS: Flow cytometry revealed that 16 out of 20 subjects had eosinophils expressing TNF-alphaRI and 18 subjects had eosinophils expressing TNF-alphaRII. All eosinophils expressed at least one receptor. The mRNA for TNF-alphaRII was detected in all eosinophils examined, but the signals for TNF-alphaRI mRNA were only found in cells expressing this receptor. CONCLUSIONS: Human peripheral eosinophils show heterogeneous expression of TNF-alphaRI and TNF-alphaRII.