Heterotrimers formed by tumor necrosis factors of different species or muteins

Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.     

Characterization of the interaction between murine tumour necrosis factor and monoclonal antibodies

We characterized the interaction between murine TNF (mTNF) and the neutralizing monoclonal antibodies TN3 and 1F3F3. The epitopes were localized by comparing the detection efficiency for a panel of TNF chimaeric proteins and site-specific muteins in ELISA. Mutation of mTNF amino acid Q131 inhibited the interaction with 1F3F3, whereas mutation of D71/Y72 inhibited the binding to TN3. As D71/Y72 are located in an exposed loop near the TNF intersubunit groove, binding of TN3 promoted the dissociation and/or interfered with the reassociation of subunits into mTNF trimers. 1F3F3, on the other hand, prevented the spontaneous dissociation of bound (hetero)trimeric mTNF.     

Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors

Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition.     

3种人肿瘤坏死因子衍生物的研制

本文在详细分析肿瘤坏死因子（ＴＮＦ）结构的基础上应用ＰＣＲ技术和基因工程手段改造人ＴＮＦ分子,构建了３种人ＴＮＦ的衍生物。３种人ＴＮＦ衍生物是;Ｎ端缺失７个氨基酸且８、９、１０位的ＰｒｏＳｅｒＡｓｐ改为ＡｒｇＬｙｓＡｒｇ的ＴＮＦ（简写为ｈＴＮＦＤ１）：Ｃ端１５７位Ｌｅｕ改为Ｐｈｅ的ＴＮＦ（简写为ｈＴＮＦＤ２）;Ｎ端和Ｃ端同时作上述改变的ＴＮＦ（简写为ｈＴＮＦＤ３）。这３种衍生物在大肠杆菌中均获得较高表述,它们对Ｌ９２９细胞的细胞毒活性较重组天然人ＴＮＦ有很大升高,尤其是ｈＴＮＦＤ１和ｈＴＮＦＤ３,升高达３个数量级。文中对３种衍生物活性升高的原因作了分析。     

Expression of genes for orthopoxviral TNF-binding proteins and study resulted recombinant proteins

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.     

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.     

Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-alpha (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MPhi), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MPhi with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MPhi as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.     

Aspartic acid residues 72 and 75 and tyrosine-sulfate 73 of heparin cofactor II promote intramolecular interactions during glycosaminoglycan binding and thrombin inhibition

We used site-directed mutagenesis to investigate the role of Glu(69), Asp(70), Asp(71), Asp(72), Tyr-sulfate(73), and Asp(75) in the second acidic region (AR2) of the serpin heparin cofactor II (HCII) during formation of the thrombin.HCII complex with and without glycosaminoglycans. E69Q/D70N/D71N recombinant (r)HCII, D72N/Y73F/D75N rHCII, and E69Q/D70N/D71N/D72N/Y73F/D75N rHCII were prepared to localize acidic residues important for thrombin inhibition. Interestingly, D72N/Y73F/D75N rHCII had significantly enhanced thrombin inhibition without glycosaminoglycan (4-fold greater) and with heparin (6-fold greater), showing maximal activity at 2 microg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) with maximal activity at 20 microg/ml heparin. The other rHCII mutants had lesser-enhanced activities, but they all eluted from heparin-Sepharose at significantly higher ionic strengths compared with wt-rHCII. Neutralizing and reversing the charge of Asp(72), Tyr-sulfate(73), and Asp(75) were done to characterize their individual contribution to HCII activity. Only Y73K rHCII and D75K rHCII have significantly increased heparin cofactor activity compared with wt-rHCII; however, all of the individual rHCII mutants required substantially less glycosaminoglycan at maximal inhibition than did wt-rHCII. Inhibition of either alpha-thrombin/hirugen or gamma(T)-thrombin (both with an altered anion-binding exosite-1) by the AR2 rHCII mutants was similar to wt-rHCII. D72N/Y73F/D75N rHCII and D75K rHCII were significantly more active than wt-rHCII in a plasma-based thrombin inhibition assay with glycosaminoglycans. These results indicate that improved thrombin inhibition in the AR2 HCII mutants is mediated by enhanced interactions between the acidic domain and anion-binding exosite-1 of thrombin and that AR2 may be a "molecular rheostat" to promote thrombin inhibition in the presence of glycosaminoglycans.     

Interaction between transmembrane TNF and TNFR1/2 mediates the activation of monocytes by contact with T cells

Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-alpha implies that the interaction between transmembrane TNF-alpha (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-alpha-blocking Ab was found to significantly decrease TNF-alpha production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-alpha production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-alpha production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-alpha production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-alpha production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-alpha production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-alpha production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-alpha production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-alpha Abs.     

In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.     

抗人肿瘤坏死因子-α单链抗体与其配体的相互作用

为了在大肠杆菌中表达纯化抗人 TNF- α单链抗体并检测其结合活性与中和活性 .利用GST融合蛋白系统在大肠杆菌中表达抗人 TNF- α单链抗体 E6Sc Fv;分离包含体后进行变性和复性 ,再用亲和层析法进行纯化 ;用 ELISA法和酵母双杂交系统检测 E6Sc Fv与配体的结合 ;用 L92 9细胞检测 E6Sc Fv对人 TNF- α细胞毒作用的中和活性 .经变性 ,复性与亲和层析 ,E6Sc Fv被纯化 ,在 SDS- PAGE上为单一蛋白带 ;体外结合与中和实验表明 ,表达纯化的 E6Sc Fv可与人 TNF-α结合并中和其细胞毒活性 ;进一步用酵母双杂交系统证明当表达于细胞内时 ,E6Sc Fv仍保持了与TNF-α相结合的能力 .     

Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes

Reverse signaling of transmembrane TNF (mTNF) contributes to the versatility of this cytokine superfamily. Previously, we could demonstrate that mTNF acting as receptor confers resistance to bacterial lipopolysaccharide in monocytes and macrophages (MO/MPhi). Reverse signaling can be induced by incubation with the monoclonal anti-TNF antibody 195F and other TNF antagonists, such as the humanized monoclonal antibody infliximab and the humanized soluble TNF receptor construct etanercept, respectively, all in former or present clinical use. Here, we addressed the question whether there are differences in modulating the LPS response in MO/MPhi among these three antagonists. Whereas 195F and infliximab suppress both, the release of an LPS-induced endothelial cell apoptotic factor and proinflammatory cytokines, etanercept only protected against the LPS-triggered apoptosis activity, but left the LPS-induced cytokine release unchanged. These data could have clinical impact with regard to TNF neutralization strategies.     

Tumour necrosis factor induced an early release of superoxide and a late mitochondrial membrane depolarization in L929 cells. Increase in the production of superoxide is not sufficient to mimic the action of TNF

Tumour necrosis factor alpha (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O2-*). In the present study, we attempted to examine the role of O2-* in the regulation of mitochondrial membrane potential (Delta(Psi)m) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O2-*. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O2-* and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the Delta(Psi)m. Reduction in the release of O2-* by rotenone (50 microM) or thenoyltrifluoroacetone (250 microM) suppressed both the TNF-mediated Delta(Psi)m depolarization and cyto c release. However, increase in the production of O2-* by antimycin A (25 microM) only slightly enhanced the TNF effect in altering the Delta(Psi)m and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in Delta(Psi)m nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O2-* was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.     

Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist

Tumor necrosis factor-alpha (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.     

Membrane TNFα: Importance for the Effector Function of Dendritic Cells and Potential Ways of Its Targeted Modulation

Membrane TNFα (mTNFα) is expressed on many immune cell types and performs various biological functions. Dendritic cells (DC) of high-grade glioma patients exhibit impaired cytotoxic activity against TNFα-sensitive HEp-2 tumor cells. The mechanisms leading to the impairment of the TNFα- dependent tumoricidal activity of DC and the possibility of regulating the cytotoxic activity of DC mediated by the TNFα/TNF-R1 signaling pathway have been studied. The study was conducted on healthy donors and patients with newly diagnosed high-grade glioma. DC were generated by culturing the plastic-adherent peripheral blood mononuclear cell fraction in the presence of GM-CSF and interferon-α (IFN-DC). It was shown that the impairment of the cytotoxic activity of patient IFN-DC was associated with a low number of DC expressing mTNFα and a low level of TNFα mRNA expression in DC. IFN-DC of patients exhibited a tendency of high activity of the TNFα-converting enzyme (TACE), which accomplishes shedding of mTNFα from the cell membrane. An increased number of IFN-DC with mTNFα caused by TACE blocking enhanced cytotoxic activity of the patient’s IFN-DC against HEp-2 cells. It was established that exogenous interleukin-2 and extracellular DNA are up-regulators of the mTNFα expression on IFN-DC of the patients, but their effects are mediated by different mechanisms.     

TNF plays an essential role in tumor regression after adoptive transfer of perforin/IFN-gamma double knockout effector T cells

We have recently shown that effector T cells (T(E)) lacking either perforin or IFN-gamma are highly effective mediators of tumor regression. To rule out compensation by either mechanism, T(E) deficient in both perforin and IFN-gamma (perforin knockout (PKO)/IFN-gamma knockout (GKO)) were generated. The adoptive transfer of PKO/GKO T(E) mediated complete tumor regression and cured wild-type animals with established pulmonary metastases of the B16BL6-D5 (D5) melanoma cell line. PKO/GKO T(E) also mediated tumor regression in D5 tumor-bearing PKO, GKO, or PKO/GKO recipients, although in PKO/GKO recipients efficacy was reduced. PKO/GKO T(E) exhibited tumor-specific TNF-alpha production and cytotoxicity in a 24-h assay, which was blocked by the soluble TNFRII-human IgG fusion protein (TNFRII:Fc). Blocking TNF in vivo by administering soluble TNFR II fusion protein (TNFRII:Fc) significantly reduced the therapeutic efficacy of PKO/GKO, but not wild-type T(E). This study identifies perforin, IFN-gamma, and TNF as a critical triad of effector molecules that characterize therapeutic antitumor T cells. These insights could be used to monitor and potentially tune the immune response to cancer vaccines.     

Differences in binding and effector functions between classes of TNF antagonists

There are currently two Food and Drug Administration-approved classes of biologic agents that target tumor necrosis factor-α (TNF-α): anti-TNF monoclonal antibodies (mAbs) (adalimumab and infliximab), and soluble TNF receptors (etanercept). This study examined the ability of the TNF antagonists to: (1) bind various polymorphic variants of cell surface-expressed Fc receptors (FcγRs) and the complement component C1q, and (2) mediate Ab-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) killing of cells expressing membrane-bound TNF (mTNF) in vitro. Both mAbs and the soluble TNF receptor demonstrated low-level binding to the activating receptors FcγRI, FcγRIIa, and FcγRIIIa, and the inhibitory receptor FcγRIIb, in the absence of exogenous TNF. However, upon addition of TNF, the mAbs, but not etanercept, showed significantly increased binding, in particular to the FcγRII and FcγRIII receptors. Infliximab and adalimumab induced ADCC much more potently than etanercept. In the presence of TNF, both mAbs bound C1q in in vitro assays, but etanercept did not bind C1q under any conditions. Infliximab and adalimumab also induced CDC in cells expressing mTNF more potently than etanercept. Differences in the ability to bind ligand and mediate cell death may account for the differences in efficacy and safety of TNF antagonists.     

A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in 'reverse signalling'

We have identified a putative signalling feature of the cytoplasmic domains of the tumour necrosis factor (TNF) family members based on available amino acid sequence data. A casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of six of 15 members of the type II integral membrane TNF ligand family. We examined the phosphorylation state of transmembrane tumour necrosis factor-alpha (mTNF) with [32P]orthophosphate labelling and in vitro kinase assays, in lipopolysaccharide-stimulated RAW264.7 cells. A dimeric form of the type I soluble TNF receptor (sTNFR) was found to dephosphorylate mTNF. This effect could be prevented by treatment with phosphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that had been dephosphorylated by sTNFR ligation in vivo, and this was less effective if phosphatase inhibitors had been used to prevent mTNF dephosphorylation. A mutated form of mTNF, lacking the CKI recognition site, cannot be phosphorylated by the enzyme. Binding of sTNFR to mTNF induced an increase in intracellular calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phosphorylated in other TNF ligand members, and that it represents a new insight into the mechanism of 'reverse signalling' in this cytokine family.     

Increase of MnSOD expression and decrease of JNK activity determine the TNF sensitivity in bcl2-transfected L929 cells.

To investigate the protection mechanism of Bcl-2 against tumour necrosis factor (TNF)-mediated cell death, the bcl2 gene was transfected into the L929 cells and stably expressed. Two clones having different sensitivity among bcl2-transfected L929 clones had been isolated, and termed clone R1 and R2. It was observed that activation of manganese superoxide dismutase (MnSOD) and suppression of Jun kinase of clone R1 and R2 were correlated with protection from TNF cytotoxicity. Upon treatment with TNF, clone R1 and R2 were more resistant than control L929 cells against TNF cytotoxicity and the protective effect of clone R1 was stronger than clone R2. However, in case of TNF plus actinomycin D treatment, clone R1 was still resistant against TNF cytotoxicity, whereas clone R2 became more sensitive than control L929 cells. The JNK activities of clone R1 and R2 were suppressed upon TNF treatment and in case of TNF plus actinomycin D treatment, clone R2 showed a marked increase in JNK activities and had higher activity than control L929 cells. The specific activities of MnSOD of clone R1 and R2 upon TNF treatment were 70 U/ml and 33 U/ml, respectively, while the MnSOD activity was not detectable in control L929 cells. When TNF and actinomycin D were treated simultaneously, MnSOD activity was not detectable in control L929 cells and bcl2 -transfected L929 cells (clone R1, R2). Consistent with these results, both clone R1 and R2 showed higher levels of MnSOD mRNA expression than control L929 cells after TNF treatment. These data suggest that suppression of Jun kinase and increase of MnSOD may be involved in inhibitory action of Bcl-2 against TNF, and the balance between MnSOD and JNK signalling pathway may be an important factor for the protection of bcl2-transfected L929 cells from TNF cytotoxicity.