Identification and characterization of a novel spliced variant that encodes human soluble tumor necrosis factor receptor 2

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation and cell death induction in several cell types. The biological effects of TNF-alpha are mediated via the cell-surface TNF receptors TNFR1 and TNFR2. Soluble forms of these two receptors, which contain the extracellular ectodomains, are proteolytically cleaved from the membrane. High levels of soluble (s) TNFR2 in serum have been documented in multiple inflammatory pathologies. We describe here a new differential spliced isoform of human TNFR2 missing exons 7 and 8, DS-TNFR2(Delta7,8). This novel isoform lacks the transmembrane and cytoplasmic domains. Expression studies with DS-TNFR2(Delta7,8) cDNA transiently transfected COS cells showed that it encodes a sTNFR2 receptor of approximately 42 kDa. Soluble DS-TNFR2(Delta7,8) blocked TNF-alpha-induced apoptosis, which suggests that it regulates TNF-alpha function by antagonizing its biological activity. An ELISA was developed that quantifies sTNFR2 generated by alternative splicing. Our data show that sTNFR2 generated by alternative splicing can be found in sera of healthy individuals, at increased levels in patients with sepsis and at high concentrations in rheumatoid arthritis patients.     

类风湿关节炎患者血清sTNF—R、TNF—α的变化

目的为探讨类风湿关节炎 (RA)与可溶性肿瘤坏死因子受体 (s TNF- R)、TNF- α、TNF- α/ s TNF- R比值的关系。方法应用双抗体夹心 EL ISA法检测了活动期 RA(2 8例 ) ,稳定期 RA (12例 )及健康人 (30例 )血清中 s TNF- R 、s TNF- R 、TNF-α的水平。结果活动期 RA患者血清 s TNF- R 、s TNF- R 、TNF-α水平明显高于健康人及稳定期 RA组 ,P均 <0 .0 1。稳定期 RA患者血清 s TNF- R 、s TNF- R 、TNF-α水平亦明显高于健康人 ,P<0 .0 1。在 RA患者中 ,血清 s TNF- R 、s TNF- R 水平与 ESR、CRP、Ritchie index呈显著正相关 ,与类风湿因子 (RF)水平无相关性。RA患者治疗 3个月后 s TNF- R 、s TNF- R 及 TNF- α/ s TNF R比值显著下降。结论 RA患者血清 s TNF- R 、s TNFR- 水平显著增高 ,且与疾病活动度呈正相关。测定血清 s TNF- R 、s TNF- R 、TNF- α/ s TNF- R水平可作为 RA诊断 ,监测疾病活动、治疗及判断预后的一项有意义的实验室指标     

Two inhibitors of pro-inflammatory cytokine release,interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes

The biological activity of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α depends on the level of TNF-α itself, the expression of the p55 and p75 cell surface receptors for TNF on target cells and the concentrations of the natural inhibitors of TNF-α, the soluble p55 and p75 TNF receptors (TNF-R). Interleukin (IL)-10 and IL-4 are known to inhibit TNF-α production by monocytes. We, therefore, investigated the effects of IL-10 and IL-4 on the cell surface expression and release of TNF-R by human monocytes to determine whether these cytokines also indirectly modulated the biological activity of TNF-α. Exposure to IL-10 (1-10 U/ml) for 24 or 48 h increased soluble p75 TNF-R expression and concomitantly reduced surface expression of p75 TNF-R. Further, IL-l α-stimulated production of TNF-α was diminished by IL-10 and only a small proportion of this TNF-α was bioactive, consistent with increased production of inhibitory soluble TNF-R. IL-10 also induced down-regulation of surface p55 TNF-R on monocytes, and increased release of soluble p55 TNF-R. However, the expression of soluble p55 TNF-R was much lower than soluble p75 TNF-R, indicating that it contributed less importantly to neutralization of TNF-α under these conditions. Like IL-10, IL-4 supressed the release of TNF-α by monocytes. In contrast to IL-10, however, IL-4 (0.1-10 ng/ml) supressed the release of soluble p75 TNF-R from monocytes in a dose-dependent manner. Release of soluble p55 TNF-R was also supressed by IL-4. IL-10, therefore, reduces the pro-inflammatory potential of TNF in three ways: by down-regulating surface TNF-R expression whilst increasing production of soluble TNF-R and inhibiting the release of TNF-α itself. This suggests that IL-10 may be useful in the treatment of diseases where overexpression of TNF-α occurs.     

Polymorphic analysis of the high-affinity tumor necrosis factor receptor 2

The tumor necrosis factor receptor 2 (TNF-RII, CD120b, TNF-R p75/80) gene has recently been characterised. It is located on chromosome 1p362 and consists of 10 exons and 9 introns A number of biallelic polymorphisms have been found in exons 4, 6, 9 and 10 based on differences between published sequences. In this study we have used polymerase chain reaction methodology in association with sequence-specific primers (PCR-SSP) incorporating mismatches at the 3' end to identify these polymorphisms. We were able to confirm the presence of a single biallelic polymorphism in exon 6 corresponding to a (T/G) at nucleotide 676 of TNF-RII mRNA (gb:M32315) which results in an amino acid change and three biallelic polymorphisms in exon 10 (in the3'UTR) corresponding to (A/G) at nucleotide 1663, (T/G) at nucleotide 1668 and a (C/T) at nucleotide 1690 of gb:M32315, whereas no polymorphisms were observed in exons 4 and 9. Here we report that in 192 unrelated UK Caucasian individuals the allele frequencies determined by direct counting were: 676-T (0.77), 1663-G (0.51), 1668-T (0.95), and 1690-T (0.64) and the calculated gene frequencies were; 676-T (0.52), 676-G (0.12); 1663-G (0.30), 1663-A (0.28); 1668-T (0.77), 1668-G (0.025); and 1690-T (0.40), 1690-C (0.20). Furthermore, the presence of an A allele at nucleotide position 1663 was found to be strongly associated with the presence of a C allele at nucleotide position 1690 and a G allele at nucleotide position 1668 whereas the presence of a G allele at position 1663 was associated with the absence of a C allele at nucleotide position 1690.     

Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions

A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)-associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR-superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T-cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3-mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post-translational modifications involving ubiquitin that are important in various signaling pathways.     

Elevated soluble tumor necrosis factor receptor levels in seasonal allergic rhinitis patients

In this study, we examined the symptom scores and tumor necrosis factor-alpha (TNF-alpha), p55 soluble tumor necrosis factor receptor (sTNFR1), and p75 soluble tumor necrosis factor receptor (sTNFR2) levels in the sera and nasal epithelial lining fluids (ELF) of 20 patients with Japanese cedar pollinosis from the pre- to the postseason period, and compared the results with those of 10 nonallergic control subjects. The symptom scores of the allergic subjects were significantly (P<0.01) higher than those of the nonallergic subjects during the early stage and mid-stage of the season. There were no statistical differences between the allergic and nonallergic subjects in the TNF-alpha levels in sera and ELF from the pre- to the postseason. In the allergic subjects, however, the levels of sTNFR1 and sTNFR2 in ELF were significantly elevated during the early stage (P<0.05) and mid-stage (P<0.01) of the season, whereas those in sera did not change from the pre- to the post-season period. The levels of TNF-alpha in ELF were more than 10 times higher than those in sera, whereas the levels of sTNFR1 and sTNFR2 in ELF were less than half of those in sera in the allergic and nonallergic subjects. These results suggest that sTNFR1 and sTNFR2 may play a role in the pathogenesis of nasal allergic reaction.     

Tumor necrosis factor receptor p55 mediates deletion of peripheral cytotoxic T lymphocytes in vivo

Cellular death of activated lymphocytes down-regulates immune responses and is involved in maintaining self tolerance. Signals associated with ligation of the membrane molecule Fas lead to lymphocyte apoptosis, but additional, Fasindependent mechanisms have been postulated. Here, we show a marked expansion and prolonged persistence of functional activated cytotoxic T cells in mice lacking the tumor necrosis factor (TNF) receptor p55. In the absence of this receptor, peripheral lymphocyte apoptosis was significantly reduced in vivo. The prolonged thymocyte survival was associated with functional anergy, since the T cells no longer proliferated in vitro when stimulated with peptide antigen. However, specific cytotoxic effector function was easily detected in vitro. We conclude that the TNF receptor p55 is involved in peripheral T cell deletion in vivo.     

SLE患者血清可溶性肿瘤坏死因子受体水平及意义

目的探讨系统性红斑狼疮 (SLE)与可溶性肿瘤坏死因子受体 (sTNF R)的关系。方法利用双抗体夹心ELISA法检测了活动期SLE患者35例,稳定期患者25例及健康对照40例血清中sTNF RI和sTNF RII的水平。结果患者血清sTNF RI和sTNF RII的水平分别为(2.34±0.76)μg/L和(4.33±1.15)μg/L ;健康对照组分别为(1.09±0.11)μg/L和(2.05±0.29)μg/L ,患者明显高于健康对照组(P<0.01)。活动期SLE患者血清sTNF RI和sTNF RII水平分别为(2.93±0.32)μg/L和(5.19±0.53)μg/L ,明显高于稳定期SLE组分别为(1.46±0.15)μg/L和(3.04±0.28)μg/L(P∨0.01)。稳定期也明显高于健康对照组(P<0.01)。在SLE患者组中 ,血清sTNF RI和sTNF RII的水平与疾病活动积分呈显著的正相关(相关系数分别为0.76和0.69)(P<0.01) ;与抗ds DNA抗体水平亦呈显著的正相关(相关系数分别为0.67和0.58)(P<0.01) ;与补体C3的水平呈显著的负相关(相关系数分别为 -0.62和 -0.84)(P<0.01)。结论SLE患者血清sTNF RI和sTNFRII的水平明显增高 ,且与疾病的活动度呈显著正相关 ,这对于SLE的诊断及监测疾病的活动性 ,以及患者的判断预后可能是一种有用的实验室指标。     

Crucial role of tumor necrosis factor (TNF) receptor 2 and membrane-bound TNF in experimental cerebral malaria

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of experimental cerebral malaria (CM), but the respective role of its two types of receptors has not been established. A significant increase in the expression of TNF-receptor 2 (TNFR2, p75), but not of TNFR1 (p55), was found on brain microvessels at the time of CM in susceptible animals. Moreover, mice genetically deficient for TNFR2 (Tnfr2°) were significantly protected from experimental CM, in contrast to TNFR1-deficient (Tnfr1°) mice, which were as susceptible as wild-type mice. To identify the factors involved in the protection from CM conferred by the lack of TNFR2, we assessed in both knockout and control mice the serum concentrations of mediators that are critical for the development of CM, as well as the up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the brain microvessels. No significant difference in serum levels of TNF and interferon-γ was found between infected wild-type and Tnfr1° or Tnfr2° mice. Interestingly, the pronounced ICAM-1 up-regulation and leukocyte sequestration, typically occurring in brain microvessels of CM-susceptible animals, was detected in infected control and Tnfr1° mice – both of which developed CM – whereas no such ICAM-1 up-regulation or leukocyte sequestration was observed in Tnfr2° mice, which were protected from CM. Making use of microvascular endothelium cells (MVEC) isolated from wild-type, Tnfr1° or Tnfr2° mice, we show that soluble TNF requires the presence of both TNF receptors, whereas membrane-bound TNF only needs TNFR2 for TNF-mediated ICAM-1 up-regulation in brain MVEC. Thus, only in MVEC lacking TNFR2, neither membrane-bound nor soluble TNF cause the up-regulation of ICAM-1 in vitro. In conclusion, these results indicate that the interaction between membrane TNF and TNFR2 is crucial in the development of this neurological syndrome.     

Induction of soluble tumor necrosis factor receptor (sTNF-R75) release by HIV adsorption on cultured human monocytes

Soluble tumor necrosis factor (TNF) receptors were recently detected in the circulation of patients with early HIV-induced disease, at significantly higher levels than in control subjects. They were proposed as markers of disease progression and of the degree of immunodeficiency. We report that adsorption of heat-inactivated HIV-1 LAI to isolated human monocytes triggers the release of both TNF-α and its natural specific inhibitor, the soluble TNF receptor (sTNF-R)75, but not that of sTNF-R55. Only limited inhibition of sTNF-R release was obtained in the presence of a fully neutralizing anti-TNF-α monoclonal antibody, suggesting that stimulation by TNF-α was only partially responsible for sTNF-R release. HIV-1 LAI induced a higher sTNF-R/TNF ratio than lipopolysaccharide, a well-known monocyte activator. Monocytes thus represent a cellular source of sTNF-R that can be detected in the circulation of HIV-infected patients from seroconversion onwards. The release of sTNF-R could be of great significance in the control of HIV infection via the cytokine network and especially TNF-α.     

Association of tumor necrosis factor receptor 2 (TNFR2) polymorphism with susceptibility to systemic lupus erythematosus

Multiple genetic as well as environmental factors are considered to be involved in the development of systemic lupus erythematosus (SLE). A number of previous studies have suggested a possible role for tumor necrosis factor (TNF) in the pathogenesis of SLE. In addition, one of the candidate loci suggested by the genome-wide linkage analysis corresponds to the chromosomal position encompassing the TNF receptor 2 gene (TNFR2). The purpose of this study was to analyze the polymorphism of TNFR2 and its possible association with the susceptibility to SLE, using the case-control association analysis. Polymorphism screening of the exons containing previously reported nonsynonymous base substitutions was carried out by the polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method, using genomic DNA from 81 Japanese patients with SLE and 207 healthy individuals. Two alleles were present in exon 6, coding for methionine (196M) and arginine (196R) at position 196. 30 of 81 patients (37.0%) with SLE were positive for the 196R allele, which was significantly more frequent compared with 39 of 207 healthy individuals (18.8%) (chi2=10.6, df=l, P=0.001, odds ratio=2.53, 95% CI: 1.45-4.43). Genotype analysis revealed that the presence of one 196R allele was sufficient for rendering susceptibility. The association of 196R allele with SLE was independent from that of HLA-DRB1*1501. In conclusion, the TNFR2 196R allele was found to be significantly associated with the susceptibility to SLE in the Japanese population. Further population and functional studies will be of particular importance to establish TNFR2 as one of the susceptibility genes to SLE.     

Tumor necrosis factor, tumor necrosis factor receptors type 1 and 2, lymphotoxin-α, and HLA-DRB1 gene polymorphisms in human T-Cell lymphotropic virus type I associated myelopathy

We studied tumor necrosis factor (TNF), lymphotoxin-α (LT-α), and TNF receptors type 1 (TNFR-1) and type 2 (TNFR-2) gene polymorphisms as well as HLA class II DRB1 alleles in Japanese patients with human T-cell lymphotropic virus type I (HTLV-I) associated myelopathy (HAM) (n = 51), patients with adult T-cell leukemia/lymphoma (ATL) (n = 48), asymptomatic HTLV-I carriers (n = 50), and HTLV-I seronegative, normal controls (n = 112). There were significant differences between HAM patients and normal controls in the distributions of TNF promoter region polymophism at position −857, the LT-α gene NcoI polymorphism, and the T-G substitution in exon 6 of the TNFR-2 gene. The distribution of the NcoI polymorphism of the LT-α gene was also significantly different between HAM patients and asymptomatic HTLV-I carriers. In contrast, we failed to detect any difference in the frequency of DRB1, TNF promoter at position −1031, −863, or the TNFR-1 promoter −383 polymorphism. The results suggest that the TNF/LT-α gene region within the HLA class III of chromosome 6 and the TNFR-2 gene region located on chromosome 1p36 might contribute to susceptibility to HAM, and that aberrant expression or function of these cytokines and the receptor could be involved in the development of HAM.     

肿瘤坏死因子抑制剂治疗类风湿关节炎

肿瘤坏死因子-α(TNF-α)是类风湿关节炎(RA)发病和维持关节慢性滑膜炎症反应的最重要的致炎性细胞因子之一。TNF-α可作用于RA关节组织中的滑膜细胞、巨噬细胞、软骨细胞和破骨细胞,使这些细胞活化,产生金属蛋白酶、胶原酶、基膜溶解酶等,导致局部炎症反应和血管翳形成,进一步引起软骨破坏和骨侵蚀。以TNF抑制剂为代表的生物制剂可有效改善RA病情,成为近年来RA治疗的新里程碑。诸多临床试验已证实,TNF抑制剂可改善关节炎症和关节功能,减少RA临床活动性,并延缓关节的放射学进展。目前共有3种TNF-α抑制剂:Etaner-cept,Inflixim ab和Adalimum ab。Etanercept是重组人II型肿瘤坏死因子受体-抗体融合蛋白,由II型TNF受体(p75)与IgG1的Fc部分组成二聚体;Inflixim ab是针对TNF的特异性IgG1单克隆抗体(由人Ig稳定区和鼠Ig可变区组成的嵌合体);Adalimum ab也是特异性针对TNF的IgG1单克隆抗体(Ig的稳定区和可变区均为人源的)。TNF抑制剂的主要不良反应有注射部位反应、感染、肿瘤、淋巴增殖性疾病、神经脱髓鞘病变以及狼疮样综合征。     

头帕肿瘤综合征蛋白与肿瘤坏死因子受体介导的坏死样凋亡

头帕肿瘤综合征蛋白(cylindromatosis,CYLD)是一种去泛素化酶,其C-末端USP结构域具有催化功能,可移除受体相互作用蛋白激酶1(receptor interacting protein kinase 1,RIPK1)的K63连接泛素链,调节RIPK1的泛素化水平,从而参与调节肿瘤坏死因子受体1(tumor necrosis factor receptor 1, TNFR1)介导的RIPK依赖的细胞坏死样凋亡等病理生理过程。阐明CYLD对RIPK1去泛素化调节的详细机制,寻找针对CYLD的特异性抑制剂,可为与坏死样凋亡相关的损伤与疾病提供治疗的新策略。     

胰岛素样生长因子-I受体与肿瘤

胰岛素样生长因子I受体(insulin-like growth factor I receptor,IGF-IR)为跨膜蛋白，是酪氨酸蛋白激酶类受体家族的主要成员之一。IGF-IR在多种恶性肿瘤中均有表达，它可促进肿瘤细胞的增殖、抑制肿瘤细胞的凋亡，在肿瘤的发生发展中起重要作用。现已在多种肿瘤细胞中证实，用抗IGF-IR的抗体或相关药物及以反义IGF-IR RNA封闭IGF-IR来阻断IGF-IR信号转导，可以抑制肿瘤细胞的生长和增殖，促进其凋亡。因此针对IGF-IR靶向治疗将是恶性肿瘤生物治疗值得关注的一个研究方向。     

胰岛素样生长因子-Ⅰ受体与肿瘤

胰岛素样生长因子Ⅰ受体(insulin-like growth factor Ⅰ receptor,IGF-IR)为跨膜蛋白,是酪氨酸蛋白激酶类受体家族的主要成员之一.IGF-IR在多种恶性肿瘤中均有表达,它可促进肿瘤细胞的增殖、抑制肿瘤细胞的凋亡,在肿瘤的发生发展中起重要作用.现已在多种肿瘤细胞中证实,用抗IGF-IR的抗体或相关药物及以反义IGF-IR RNA封闭IGF-IR来阻断IGF-IR信号转导,可以抑制肿瘤细胞的生长和增殖,促进其凋亡.因此针对IGF-IR靶向治疗将是恶性肿瘤生物治疗值得关注的一个研究方向.     

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4⁺ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.     

Serum levels of the soluble receptor for tumor necrosis factor in patients with renal disease

Tumor necrosis factor- (TNF-) has been found to be elevated in patients during hemodialysis and is thought to mediate some of the immune and metabolic dysfunctions in these patients. It has been speculated that infusions of soluble TNF receptor (sTNF-R) may prevent some of the cytotoxic effects of TNF. However, little is still known about preexisting serum TNF-R levels in patients with chronic renal failure, with or without hemodialysis. Therefore we analyzed serum samples of sTNF-R in 26 patients with chronic renal failure (group I), 6I hemodialysis patients (group II), 9 renal transplant recipients with acute renal failure requiring posttransplant dialysis (group III), 13 renal transplant patients with rejection and moderate kidney dysfunction (group IV), and 21 renal transplant recipients with borderline kidney dysfunction and diverse infectious complications (group V). Control groups consisted of 34 blood donors and diseased controls (11 renal transplant recipients with normal kidney function without complications). All patient groups showed significantly higher sTNF-R levels compared to the control groups. In groups I, IV, and V comparable levels were observed. In group I there was a clear correlation between sTNF-R levels and serum creatinine. The highest sTNF-R serum levels were seen in groups II and III, but there was no correlation with creatinine. In the posttransplant cases (group III and diseased controls) there was a decrease in sTNF-R with improvement of kidney function. These data strongly suggest that sTNF-R serum levels are dependent on kidney function.Abbreviations TNF tumor necrosis factor - TNF-R tumor necrosis factor receptor - sTNF-R soluble tumor necrosis factor receptor - RTX renal transplantation - ELISA enzyme-linked immunosorbent assay Correspondence to: G. Halwachs