Mechanism of crosstalk inhibition of IL-6 signaling in response to LPS and TNFα

Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli (“crosstalk”). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor α (TNFα), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFα. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFα.     

Dysfunction of microvascular endothelial cells induced by TNFαand its molecular mechanism

Dysfunction of microvascular endothelial cells induced by TNFαand its molecular mechanism     

Constitutively active NF-κB triggers systemic TNFα-dependent inflammation and localized TNFα-independent inflammatory disease

NF-κB is well established as a key component of the inflammatory response. However, the precise mechanisms through which NF-κB activation contributes to inflammatory disease states remain poorly defined. To test the role of NF-κB in inflammation, we created a knock-in mouse that expresses a constitutively active form of NF-κB p65 dimers. These mice are born at normal Mendelian ratios, but display a progressive, systemic hyperinflammatory condition that results in severe runting and, typically, death 8–20 d after birth. Examination of homozygous knock-in mice demonstrates significant increases in proinflammatory cytokines and chemokines. Remarkably, crossing this strain with mice lacking TNF receptor 1 (TNFR1) leads to a complete rescue of the hyperinflammatory phenotype. However, upon aging, these rescued mice begin to display chronic keratitis accompanied by increased corneal expression of TNFα, IL-1β, and MMP-9, similar to that seen in human keratoconjunctivitis sicca (KCS) or “dry eyes.” Therefore, our results show that, while constitutively active NF-κB can trigger systemic inflammation, it does so indirectly, through increased TNF production. However, certain inflammatory disease states, such as keratitis or KCS, a condition that is seen in Sjogren''s syndrome, are dependent on NF-κB, but are independent of TNFR1 signaling.     

Adipokine expression and secretion by canine adipocytes: stimulation of inflammatory adipokine production by LPS and TNFα

Adiposity and obesity are increasing in dogs. We have examined here the endocrine function of canine adipose tissue and the regulation of production of inflammation-related adipokines by dog adipocytes. Adiponectin, leptin, IL-6, MCP-1 and TNFα genes were expressed in the main adipose depots of dogs, but there were no major depot differences in mRNA levels. Each adipokine was expressed in canine adipocytes differentiated in culture and secreted into the medium (leptin undetected). IL-6, MCP-1 and TNFα were also expressed and secreted by preadipocytes; adiponectin and leptin were only expressed after adipocyte differentiation. The inflammatory mediators LPS and TNFα had major stimulatory effects on the expression and secretion of IL-6, MCP-1 and TNFα; there was a >5,000-fold increase in IL-6 mRNA level with LPS. IL-6 release into the medium was increased >50-fold over 24 h with LPS and TNFα, while MCP-1 release was increased 23- and 40-fold by TNFα and LPS, respectively. However, there was no effect, or small reductions, in adiponectin and leptin mRNA levels with the inflammatory mediators. Dexamethasone-stimulated leptin gene expression, had no effect on adiponectin expression, but decreased the expression and secretion of IL-6 and MCP-1. The PPARγ agonist rosiglitazone stimulated both adiponectin and leptin expression and inhibited the expression of IL-6, MCP-1 and TNFα; MCP-1 secretion was reduced. These results demonstrate that canine adipocytes express and secrete key adipokines and show that adipocytes of this species are highly responsive to inflammatory mediators with the induction of major increases in the production of inflammation-related adipokines.     

Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance?

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3' UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.     

TNFα的中枢神经系统来源

ＴＮＦα是一个重要的细胞因子，有着广泛的生物学作用，主要来源于单核／巨噬细胞和Ｔ细胞。ＣＮＳ一直被认为是免疫豁免部位，这是部分因为血脑屏障的存在限制了免疫能细胞，细胞因子和免疫球蛋白的通过，部分因为ＣＮＳ的细胞缺乏免疫活性。     

TNFα的中枢神经系统来源

ＴＮＦα是一个重要的细胞因子,有着广泛的生物学作用,主要来源于单核／巨噬细胞和Ｔ 细胞。ＣＮＳ一直被认为是免疫豁免部位,这是部分因为血脑屏障的存在限制了免疫能细胞、细胞因子和免疫球蛋白的通过,部分因为ＣＮＳ的细胞缺乏免疫活性。目前后者受到越来越多的研究结果的冲击。ＴＮＦα不但可以在周围循环中产生,而且可以在ＣＮＳ内由小胶质细胞、星形细胞、神经细胞和血管内皮细胞产生,在ＣＮＳ免疫炎性反应中发挥着重要作用     

Antioxidants inhibit TNFα-induced motility and invasion of human osteosarcoma cells: Possible involvement of NFκB activation

Osteosarcoma is the most frequent malignant bone tumor in children. It is highly invasive, however, the mechanisms behind osteosarcoma cell invasion are as yet still unknown. In the present study, treatment with TNFα enhanced the invasiveness of two human osteosarcoma cell lines, OST and MNNG. TNFα treatment also induced tumor cell motility, adhesion to laminin, the expression of matrix metalloproteinase 9 (MMP9), and the nuclear translocation of nuclear factor κB (NFκB) in the osteosarcoma cells. Moreover, antioxidants inhibited TNFα-induced osteosarcoma cell invasion, motility and NFκB nuclear translocation, but not adhesion to laminin or MMP9 expression. NFκB decoy, another NFκB inhibitor, also inhibited TNFα-induced osteosarcoma cell invasion and motility. Therefore, motility and NFκB activation were possibly related to TNFα-induced osteosarcoma cell invasion. However, adhesion to laminin or MMP did not demonstrate any correlation with TNFα-induced osteosarcoma cell invasion. Although NFκB is known to regulate TNFα-induced phenotypes, it may influence only motility and invasion, but not the MMP or laminin-mediated adhesion of these osteosarcoma cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mathematical modeling and sensitivity analysis of the integrated TNFα-mediated apoptotic pathway for identifying key regulators

TNFα-mediated apoptosis is one of the complex and tightly regulated cellular processes as it involves the activation of both pro- and anti-apoptotic signaling pathways. Thus, it is important to elucidate the molecular players of this process and their dynamics in order to gain an in-depth understanding of the mechanisms underlying apoptosis. To this end, we proposed an integrated model of TNFα-mediated apoptosis pathway in Type I cells, formulated based on the principles of mass action kinetics. The model includes major apoptotic modules—the extrinsic and intrinsic pathways, the NFκB survival signaling and various regulatory mechanisms. We performed simulations and sensitivity analyses to study the role of NFκB pathway in regulating apoptosis, and identified IAP as one of the more potent regulators of apoptosis.     

Differential regulation of TNFα, IL-1β, IL-6, IL-8, TNFβ, and IL-10 by pentoxifylline

Pentoxifylline (PTX) is a methylxanthine drug known to inhibit the production of tumor necrosis factor-alpha (TNFα), which plays a key role in inflammation. Recent studies also revealed that other cytokines may be inhibited by PTX. We investigated PTX effects on production and mRNA expression of TNFα, IL-1β, IL-6, IL-8, TNFβ and IL-10. Cytokine release was studied in 1/10 diluted whole blood culture (WB) and in peripheral blood mononuclear cell (PBMC) culture. Cytokine production was triggered in both culture systems by endotoxin (LPS) or by phorbol ester (PMA) plus phytohemagglutinin (PHA). Our results showed that expression and production of TNFα and TNFβ were inhibited by PTX in a dose-dependent manner. Moreover, we observed that depending on the way of activating cells, PTX induced an up- or a down-regulation (in PMA+PHA or LPS stimulated cells, respectively) for IL-1 and IL-6 release. We also noted that the effects of PTX on IL-6, IL-8 and IL-10 production were different in WB and in PBMC culture. In conclusion PTX acts on cytokine in a complex manner depending on cellular environment and on the method of activation.     

TNF—α与肝细胞损伤

在内毒素、脂多糖、半乳糖胺、氯仿等引起的肝细胞损伤中，由巨噬细胞分泌的α肿瘤坏死因子起着重要的介导作用。通过抑制巨噬细胞的功能，或使用抗肿瘤坏死因子抗体或肿瘤坏死因子受体来中和肿瘤坏死因子，可以减轻肝损伤     

Up-regulation of Cks1 and Skp2 with TNFα/NF-κB signaling in chronic progressive nephropathy

The cyclin-dependent kinase (CDK) inhibitor p27 level is associated with progression of renal damage. We previously reported that mRNA of Skp2, a component of Skp/Cullin/F-box (SCF)-ubiquitin ligase which targets to p27, was increased in unilateral ureteral obstructive kidneys in mice and that the nephritis was attenuated in Skp2-deficient mice. However, the details have not been fully clarified. Here, we found that not only Skp2 but also cdc kinase subunit 1 (Cks1), an essential cofactor for the SCF-Skp2 ubiquitin ligase in targeting p27, was increased in another chronic progressive model, anti-thymocyte serum (ATS) rat nephropathy. After induction of ATS nephropathy, Skp2(+) /Cks1(+) /Ki67(+) tubular epithelial cell numbers increased, and p27(+) tubular epithelial cells decreased transiently. Moreover, we found that TNFα was involved in expression of both Skp2 and Cks1 in NRK cell line as well as the in ATS nephropathy. Nuclear accumulations of NF-κB subunits RelB and p52 were increased in the tubular epithelial cells of the nephritic kidney. Both Skp2 and Cks1 were colocalized with RelB in these cells. These data suggest that both Skp2 and Cks1 are up-regulated by the TNFα-RelB/p52 pathway in the early stages of renal damage and are collaboratively involved in down-regulation of p27 in proliferative tubular dilation and the progression of chronic nephropathy.     

Effects of Fas,NF-κB and caspases on rat microvascular endothelial cell apoptosis induced by TNFα

Effects of Fas,NF-κB and caspases on rat microvascular endothelial cell apoptosis induced by TNFα     

TNFα Inhibitors in the Treatment of Psoriasis and Psoriatic Arthritis

Psoriasis is a chronic inflammatory skin disease that can lead to significant physical and psychologic distress for patients. Psoriatic arthritis (PsA), originally thought to be quite a mild disorder, is now recognized as a progressive and destructive arthritis. To date, therapies for both these conditions have been non-specific and unable to maintain long-lasting remission. In addition, many of the current therapies have significant adverse effects, limiting their usefulness. However, elucidation of the pathogenesis of psoriasis and PsA at a molecular level and the development of selective biologic agents have led to an enormous expansion of the armamentarium available to psoriasis patients. Two agents (infliximab and etanercept) selectively block the role of the cytokine tumor necrosis factor (TNF)-alpha and have proved effective in clinical trials in the treatment of both the skin and the joint manifestations of psoriasis. A third anti-TNF alpha agent (adalimumab Humira) is licensed for the treatment of rheumatoid arthritis; however, no studies have been published to date on its use in PsA or psoriasis. It is known that TNF alpha is elevated in both the skin and synovium of psoriatic patients and the effectiveness of its blockade by these two agents in psoriasis and PsA confirms its role in their pathogenesis. Randomized, double-blind, placebo-controlled trials have been performed with both agents in the treatment of psoriasis and PsA; in the case of etanercept these have been to support US FDA approval for use in psoriatic arthropathy. These studies are supported by smaller cohorts in open-label studies and anecdotal reports in the literature. Anti-TNF alpha therapy has proved to have disease-reducing activity in PsA and psoriasis and appears to be well tolerated. These studies have generally featured small numbers of patients and, until a larger cohort of treated patients is available, vigilance must be exercised. A considerable body of post-marketing safety data exists on the use of infliximab in rheumatoid arthritis and Crohn disease and for etanercept in rheumatoid arthritis and PsA. Certain issues, particularly the risk of infection, have emerged as features of the use of these agents. It remains to be seen whether effects seen in other disease entities may be extrapolated to psoriatic patients. More long-term data and experience are needed to define the role of anti-TNF alpha agents in the management of psoriasis and PsA. In particular, more studies are required to elucidate the finer points of co-medication; in some studies both agents have been used with other medications but there have been no formal trials of various possible combinations.     

Interaction of human TNF and β2-microglobulin with Tanapox virus-encoded TNF inhibitor, TPV-2L

Tanapox virus (TPV) encodes and expresses a secreted TNF-binding protein, TPV-2L or gp38, that displays inhibitory properties against TNF from diverse mammalian species, including human, monkey, canine and rabbit. TPV-2L also has sequence similarity with the MHC-class I heavy chain and interacts differently with human TNF as compared to the known cellular TNF receptors or any of the known virus-encoded TNF receptor homologs derived from many poxviruses. In order to determine the TNF binding region in TPV-2L, various TPV-2L C-terminal truncations and internal deletions were created and the muteins were expressed using recombinant baculovirus vectors. C-terminal deletions from TPV-2L resulted in reduced binding affinity for human TNF and specific mutants of TNF that discriminate between TNF-R1 and TNF-R2. However, deletion of C-terminal 42 amino acid residues totally abolished the binding of human TNF and its mutants. Removal of any of the predicted internal domains resulted in a mutant TPV-2L protein incapable of binding to human TNF. Deletion of C-terminal residues also affected the ability of TPV-2L to block TNF-induced cellular cytotoxicity. In addition to TNF, TPV-2L can also form complexes with human β2-microglobulin to form a novel macromolecular complex. In summary, the TPV-2L protein is a bona fide MHC-1 heavy chain family member that binds and inhibits human TNF in a fashion very distinct from other known poxvirus-encoded TNF inhibitors, and also can form a novel complex with the human MHC-1 light chain, β2-microglobulin.     

乳腺癌患者血清TNFα和TGFα的临床意义

肿瘤坏死因子α(TNFα)和转化因子α(TGFα)均为多肽类细胞因子,前者由巨噬细胞产生,具有多种生物效应;后者粘附在细胞膜上,具有自分泌和旁分泌刺激功能.二者与肿瘤的发生、发展及预后相关联.本文将探讨乳癌患者血清TNFα和TGFα的临床意义.     

shRNA抑制淋巴细胞TNFα的表达

目的观察RNA干扰技术是否有效抑制原代培养的脾淋巴细胞肿瘤坏死因子α(TNFα)的表达。方法体外合成针对TNFα基因序列的特异性发夹状双链RNA(shRNA)的表达载体,与L ipofectam ine2000结合后转染原代培养的由脂多糖(LPS)激活的脾淋巴细胞,用ELISA和RT-PCR法检测TNFα表达的变化。结果ELISA检测结果表明,特异性RNA干扰组TNFα蛋白表达较对照组下降34.2%,而非特异性干扰组TNFα表达与对照组无明显差异;RT-PCR检测结果表明,特异性RNA干扰组TNFαmRNA表达较对照组下降61.3%。结论RNA干扰可以有效抑制原代培养的淋巴细胞TNFα的表达,该过程具有严格的基因序列特异性,为TNFα相关疾病的基因治疗提供了新策略。