Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of recombinant P60 and P80 receptors.

Tumor necrosis factor (TNF) is one of the mediators of inflammatory responses. Recently, the cDNA for two distinct receptors of TNF with predicted molecular masses of 60 kDa and 80 kDa, respectively, were isolated. In this report, we compare the inhibitory effects of these two forms of recombinant soluble TNF receptors (extracellular domains) on the ligand binding and on the antiproliferative effects of TNF and lymphotoxin (LT) in a human histiocytic lymphoma cell line (U-937). Our results show that the soluble form of the p60 receptor is approximately 100-fold more potent than the soluble form of the p80 receptor in inhibiting both the antiproliferative effects of TNF as well as in blocking TNF binding to U-937 cells. In contrast, the antiproliferative effects of LT and its binding to cells is inhibited equally by both the p60 and p80 forms of the soluble receptor. Thus, overall our results indicate that the two soluble receptors differ in their ability to inhibit TNF and LT. The impotance of these soluble receptors in blocking the harmful effects of TNF and LT is discussed.     

Signalling by CD95 and TNF receptors: not only life and death

Members of the TNF family of receptors play important roles in normal physiology and in defence. The recent rapid progress in the understanding of the mechanisms of apoptosis has been accompanied by assumptions that TNF family receptors such as CD95(Fas/APO-1) only have a role in regulating cell survival. While regulation of cell death is one important function of TNF family receptors, they are capable of activating signal transduction pathways that have many other effects. The present review will focus on signalling of some TNF family receptors in the immune system, not only for apoptosis, but also for survival or activation.     

Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.

Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF. These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study of the TNF-like cytotoxicity of a panel of monoclonal antibodies against TBPI. The induction of TNF-like effects by antibodies to a TNF receptor suggests that TNF is not directly involved in intracellular signalling. Rather, it is the receptors to this cytokine which, when properly triggered in a process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior.     

Involvement of TNF in limiting liver pathology and promoting parasite survival during schistosome infection

CD4(+) T cell responses and macrophage activation are essential components of schistosome egg-induced granuloma formation. Previous studies implicated tumour necrosis factor (TNF) as a potential mediator of macrophage recruitment and activation during schistosome infection. Here we demonstrate that signalling by TNF and its receptors can influence granuloma formation, but is ultimately dispensable for granuloma formation in this system. However, we identify a previously unrecognised role for TNF in limiting hepatocellular damage in response to schistosome eggs. Further, we show that this activity of TNF is independent of TNF receptors (TNFR1 and TNFR2). Taken together, these data suggest that additional, as yet unrecognised receptors exist for TNF and that these receptors are capable of mediating important pathological effects in the liver. Finally, we provide evidence that TNF plays an unexpected role in maintaining adult schistosome viability in the portal system.     

肿瘤坏死因子与其受体相互作用的计算机模拟研究

利用同源模建方法,以ＴＮＦＲ５５受体胞外区的晶体结构为参考模板,预测了ＴＮＦＲ７５受体胞外区Ｃｙｓ１８～Ｐｈｅ１４７片段的三维结构．根据Ｒ５５受体胞外区与ＬＴ相结合的复合物的晶体结构,预测了ＴＮＦ与Ｒ５５及Ｒ７５胞外区的复合物的三维结构,模拟了ＴＮＦ与受体之间的相互作用．由于ＴＮＦ与受体的作用形式是三聚体对三聚体,因此在模拟ＴＮＦ与受体相互作用时选择了包括一个非对称的ＴＮＦ三聚体和一个受体（Ｒ５５或Ｒ７５）单体的模拟系统．结合已有的突变体实验结果,利用计算机模拟分析手段,发现了一些ＴＮＦ突变体之所以具有受体选择性的三维结构基础和发挥了关键作用的氨基酸残基以及这些残基之间的主要作用形式．研究深化了对已有的突变体实验结果的认识,建立了不同的实验结果之间的内在关联,为以后有目的的新型突变体设计和实验研究打下了基础．     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-alpha: induction of TNF receptors on human T cells and TNF-alpha-mediated enhancement of T cell responses

The expression of specific tumor necrosis factor (TNF) membrane receptors and biological effects of recombinant TNF (rTNF)-alpha on normal human T lymphocytes were studied. Although resting T cells lacked specific binding capacity for rTNF-alpha, high affinity (Kd 70 pM) TNF receptors were de novo induced upon primary activation of T cells. Comparison of TNF receptor expression with that of high affinity interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) receptors, respectively, revealed similarities to IL 2-receptor expression with respect to kinetics of induction. However, maximum expression of TNF receptors (approximately equal to 5000/cell at day 6) and subsequent decline occurred approximately 3 days after the peak of IL 2-receptor expression. In contrast, no change in the expression of IFN-gamma receptors (Kd 10 pM, 300 to 400 receptors/cell) was found in the course of T cell activation. On activated TNF receptor positive T cells, TNF-alpha exerted multiple stimulatory activities. Thus TNF increased the expression of HLA-DR antigens and high affinity IL 2 receptors. As a consequence, TNF-treated T cells showed an enhanced proliferative response to IL 2. Moreover, TNF-alpha was effective as a co-stimulator of IL 2-dependent IFN-gamma production. These data indicate that TNF-alpha may regulate growth and functional activities of normal T cells.     

Signal transduction by tumor necrosis factor receptors

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.     

Interferon-gamma induces cell surface expression for both types of tumor necrosis factor receptors.

Interferons are known to potentiate various biological effects of tumor necrosis factor (TNF). Recently, two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80), primarily expressed by epithelial cells and myeloid cells, respectively, have been identified. In the present report, we examined the effect of interferon-gamma (IFN-gamma) on each type of TNF receptor. Our results indicate that IFN-gamma induces TNF receptors on both myeloid (e.g. HL-60) and epithelial cells (e.g. HeLa). Furthermore, by using antibodies specific to each type of receptor, we demonstrate that both TNF receptors are equally inducible by IFN-alpha, IFN-beta and IFN-gamma. Thus, the increase in TNF receptors by interferons may play a role in their synergistic cellular response.     

Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.     

TNF induces c-fos via a novel pathway requiring conversion of arachidonic acid to a lipoxygenase metabolite.

Tumour necrosis factor (TNF), a lymphokine released by activated macrophages, has diverse effects on a wide variety of cell types. TNF exerts these effects via specific cell surface receptors; however little is known of the biochemical events that ensue. We have shown that TNF rapidly induces the proto-oncogenes c-fos and c-jun in the adipogenic TA1 cell line and have used these responses to characterize the intracellular mediators of TNF action. We find that arachidonic acid, which is released in response to TNF, induces c-fos, but not c-jun mRNA in quiescent TA1 cells. Pretreatment of the cells with lipoxygenase inhibitors abolishes the induction of c-fos by TNF, while the induction of c-jun is unaffected; in contrast, a cyclooxygenase inhibitor has no effect on either response. Finally, we have demonstrated that TNF stimulates production of lipoxygenase metabolites in TA1 cells and that one of these, 5-HPETE, induces c-fos, but not c-jun. These data suggest that TNF activates two second messenger pathways, one of which is dependent on release of arachidonic acid and its subsequent conversion to a lipoxygenase metabolite.     

Two tumor necrosis factor-binding proteins purified from human urine. Evidence for immunological cross-reactivity with cell surface tumor necrosis factor receptors

Two proteins which specifically bind tumor necrosis factor (TNF) were isolated from human urine by ligand (TNF)-affinity purification, followed by reversed phase high performance liquid chromatography. The molecular weights of the two proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were similar (about 30,000). Both proteins provided protection against the cytocidal effect of TNF in vitro and both bound TNF-alpha more effectively than TNF-beta. Antibodies raised against each of the proteins had an inhibitory effect on the binding of TNF to cells, suggesting that both proteins are structurally related to the TNF receptors. However, the two proteins differed in NH2-terminal amino acid sequences: Asp-Ser-Val-Cys-Pro- in one and Val-Ala-Phe-Thr-Pro- in the other. The NH2-terminal sequence of the former protein was invariable, while that of the latter was truncated to varying degrees. The two proteins were also immunologically distinct. The relative efficacy of anti-sera against the two proteins in inhibiting the binding of TNF to cells varied markedly from one line of cells to another. Evidence has been presented recently for the existence of two distinct molecular species of cell surface receptors for TNF and for differential expression of those two receptors by cells of different lines. The findings presented in this study are consistent with the notion that the urinary TNF-binding proteins constitute soluble forms of the two molecular species of the cell surface TNF receptors.     

Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

Induction of tumor necrosis factor-alpha and its receptors during differentiation in myeloid leukemic cells along the monocytic pathway. A possible regulatory mechanism for TNF-alpha production

We examined the characteristics of tumor necrosis factor (TNF) receptors expressed on immature mouse myeloid leukemic cells (M1), M1 cells induced to differentiate into macrophages, and macrophage cells (Mm1 cells) by binding studies with radioiodinated TNF. Scatchard analysis of TNF binding revealed that a single class of high affinity receptor was present and that 750-1,100 receptors were expressed on each immature M1 cell. The number of TNF receptors was increased 1.5-2-fold on differentiated M1 cells and 4-5-fold on Mm1 cells with no change in affinity. The addition of interferon-gamma (IFN-gamma) up-regulated the expression of TNF receptors in differentiated M1 cells and Mm1 cells, while immature M1 cells were insensitive to IFN-gamma. The number of TNF receptors on the differentiated cells was increased 4-5-fold by the treatment with IFN-gamma with no change in the binding constant. The affinity of TNF receptors to human TNF-alpha (Kd = 1.7-2.8 nM) was lower than that to murine TNF-alpha (Kd = 0.2-0.7 nM). The assays for cell growth and [3H]thymidine incorporation suggested that no relation exists between the sensitivity of the cells to TNF-alpha and the number of TNF receptors. Enhancement of TNF-mediated cytotoxicity by the treatment with IFN-gamma did not correlate with increases in the number of TNF receptors. Cytolytic assays using L929 cells demonstrated that the amount of constitutive and lipopolysaccharide (LPS)-induced secretion of TNF-alpha was markedly increased during differentiation. Both the constitutive expression and IFN-gamma-mediated superinduction of TNF receptors, and the constitutive and LPS-induced secretion of TNF-alpha were closely related to the extent of cellular differentiation along the monocytic pathway. The time course of LPS-induced TNF-alpha activity showed a rise-and-decline profile with a peak at 2 h. On the other hand, the time course of the number of cell surface TNF receptors showed a decline-and-rise profile, a mirror image of the TNF-alpha activity time course profile in the supernatant. Anti-TNF-alpha antibody treatment blocked the LPS-induced down-regulation of TNF receptors and increased TNF-alpha mRNA accumulation. We discussed "an autoinhibitory system" in which an internalization of secreted TNF-alpha mediated by its own receptors is involved not only in decreasing TNF-alpha activity in the supernatant but also in reducing TNF-alpha mRNA expression.     

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.     

The TNF-TNF receptor system

Different forms of tumor necrosis factor (TNF) interact with two specific receptors for TNF (TNFR) on the cell membrane to induce a variety of effects. While sharing structural similarities in their extracellular domains, the two TNFRs differ in their intracellular domain, their signal transduction, and consequently their function. In addition, one of the two TNFRs can be expressed in two differently located isoforms. This makes the TNF-TNFR system very complex. The dual TNF function for either cell death or survival upon interaction of members of the TNF ligand family with members of the TNF receptor family will be discussed.     

Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain of tumor necrosis factor receptors

Tumor necrosis factor (TNF)-alpha has an important role in the pathogenesis of autoimmune and inflammatory diseases such as rheumatoid and septic arthritis. The biological effects of TNF-alpha are mediated by binding to TNF receptors TNFR1 (also known as P60) or TNFR2 (also known as P80). The pre-ligand assembly domain (PLAD) is a portion of the extracellular region of TNFRs that mediates receptor-chain association essential for signaling. We found that soluble versions of PLAD, especially those derived from P60, block the biochemical effects of TNF-alpha in vitro and potently inhibit arthritis in animal models. Thus, targeting the PLAD may have clinical value in the treatment of human arthritis and other disorders involving receptors of the TNFR superfamily.     

Down regulation of the receptors for tumor necrosis factor by interleukin 1 and 4 beta-phorbol-12-myristate-13-acetate

Binding of radiolabeled tumor necrosis factor (TNF) to cell surface receptors was markedly reduced in human foreskin fibroblasts and cells from SV-80 and HeLa cell lines subsequent to treatment with interleukin 1 (IL-1) or 4 beta-phorbol-12-myristate-13-acetate (PMA). The decrease in TNF binding was initiated within minutes of application of IL-1 or PMA and could not be blocked by cycloheximide, suggesting that it is independent of protein synthesis. Scatchard plot analysis of TNF binding to the SV-80 cells indicated that its decrease in response to IL-1 and PMA reflects a reduced amount of TNF receptors, with no change in their affinity. IL-1 and PMA together had an additive effect on TNF binding. Treatment with TNF did not result in decreased binding of IL-1 to its receptors nor did TNF and IL-1 compete directly for their respective receptors. Human U937 cells on which receptors for IL-1 were below detectable levels exhibited no decrease in TNF binding when treated with IL-1, but did so in response to PMA. In addition to a decrease in TNF receptors, cells treated with IL-1 or PMA exhibited a lesser vulnerability to the cytolytic effect of TNF. The two kinds of changes were not completely correlated. A particularly notable dissimilarity was evident when comparing the rate of their reversal: the TNF receptor level was fully recovered within a few hours of removal of IL-1 or of the water-soluble analogue of PMA, 4 beta-phorbol-12,13-dibutyrate, from pretreated SV-80 cells; yet at that time resistance to the cytotoxicity of TNF was still prominent. These findings indicate that IL-1 as well as tumor-promoting phorbol diesters can down regulate cellular response to TNF by inducing a decrease in the number of receptors for TNF, and apparently through some other effect(s) as well.