Etanercept: recombinant human soluble tumor necrosis factor receptor fusion protein

TNF is a central cytokine in the pathogenesis of rheumatoid arthritis(RA), which induces synovitis as well as joint damage. Etanercept is a recombinant human soluble TNF receptor that binds specifically to TNF receptor, and inhibits TNF receptor-mediated signaling cascade. Recent investigations have revealed successful clinical efficacy of biologics in RA including etanercept. We reviewed here the structure and efficacy of etanercept in RA according to the published evidence.     

Modulation of endothelial cell hemostatic properties by tumor necrosis factor

Tumor necrosis factor/cachectin (TNF) is a mediator of the septic state, which involves diffuse abnormalities of coagulation throughout the vasculature. Since previous studies have shown that endothelial cells can play an active role in coagulation, we wished to determine whether TNF could modulate endothelial cell hemostatic properties. Incubation of purified recombinant TNF with cultured endothelial cells resulted in a time- and dose-dependent acquisition of tissue factor procoagulant activity. Concomitant with enhanced procoagulant activity, TNF also suppressed endothelial cell cofactor activity for the anticoagulant protein C pathway; both thrombin-mediated protein C activation and formation of functional activated protein C-protein S complex on the cell surface were considerably attenuated. Comparable concentrations of TNF (half-maximal affect at approximately 50 pM) and incubation times (half-maximal affect by 4 h after addition to cultures) were required for each of these changes in endothelial cell coagulant properties. This unidirectional shift in cell surface hemostatic properties favoring promotion of clot formation indicates that, in addition to leukocyte procoagulants, endothelium can potentially be instrumental in the pathogenesis of the thrombotic state associated with inflammatory and malignant disorders.     

Pharmacokinetics of recombinant human tumor necrosis factor alpha in rhesus monkeys after intravenous administration

Recombinant human tumor necrosis factor alpha (TNF-alpha) was administered to rhesus monkeys by i.v. short-term infusions (0.5 hr) of 10, 20, 30 and 120 micrograms/kg and long-term infusions (6.5 hr) of 22, 54, 135 and 325 micrograms/kg. At high plasma levels of TNF-alpha (doses of 120 micrograms/kg of short-term infusions and greater than or equal to 54 micrograms/kg of long-term infusion) the pharmacokinetics of TNF-alpha were practically first order. A plasma T1/2 of 1.2 to 2.1 hr was calculated. However, at low plasma levels (doses of 10-30 micrograms/kg of short-term infusion and 22 micrograms/kg of long-term infusion) the elimination rate increased steadily in dependence on concentration and time. We conclude that at low concentrations of TNF-alpha the pharmacokinetics were not first order. Simultaneous long-term infusion of recombinant human TNF-beta (200 micrograms/kg) in addition to 22 micrograms/kg of TNF-alpha reduces the elimination rate of TNF-alpha, which can be concluded from the elevation of the TNF-alpha plasma levels. Furthermore, there was no time-dependent increase of the elimination rate that was detected without infusion of TNF-beta. Based on these results two different elimination mechanisms of TNF-alpha in rhesus monkeys are postulated: an unspecific, nonsaturable process as well as a specific, saturable mechanism.     

Nonhematopoietic cells selected for resistance to tumor necrosis factor produce tumor necrosis factor

TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse TNF: it is a protein of 43 kD, composed of 16 kD subunits, that competes with TNF for receptor binding, induces hemorrhagic necrosis of the TNF-sensitive mouse sarcoma Meth A, has synergistic cytotoxic action with interferon, and its activity is neutralized by antibody to TNF. The two conclusions of this study are that cells selected for TNF resistance spontaneously produce a molecule resembling macrophage TNF, and that cells of nonhematopoietic origin are capable of producing TNF.     

Adaptation to bacterial lipopolysaccharide controls lipopolysaccharide-induced tumor necrosis factor production in rabbit macrophages.

These experiments provide an explanation for the observation that two intravenous injections of lipopolysaccharide (LPS) spaced 5 h apart in rabbits cause tumor necrosis factor/cachectin (TNF) levels to rise in the blood only after the first LPS injection. Herein we show that treatment of elicited peritoneal exudate rabbit macrophages (PEM) with two doses of LPS given 9 h apart results in a marked reduction in TNF production by the second LPS exposure. This state of hyporesponsiveness is a result of adaptation to LPS, is induced by LPS concentrations that are 1,000-fold less than required to induce TNF production (picograms vs. nanograms), is characterized by a decrease in LPS-induced TNF mRNA without any change in TNF mRNA half-life, is not changed by including indomethacin in cultures, and is specific for LPS since LPS-adapted cells display a TNF response to heat-killed Staphylococcus aureus that is at least as good as that observed in control PEM.     

TRANCE, a tumor necrosis factor family member, enhances the longevity and adjuvant properties of dendritic cells in vivo

Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo.     

肿瘤坏死因子α诱导人皮肤角质形成细胞β-防御素的体外表达

背景:人β-防御素在皮肤的固有免疫中占据重要地位,同时由于其广谱的抗菌活性,从而成为抗感染临床研究中的热点。目的:观察原代培养人皮肤角质形成细胞在浓度梯度肿瘤坏死因子α的刺激下,其免疫分子β-防御素及Toll样受体的表达情况。方法:取8例30岁以下患者包皮组织,分别原代培养获取角质形成细胞,并采用浓度梯度肿瘤坏死因子α(10,50,100,150,200μg/L)进行刺激,实时荧光定量PCR检测β-防御素1,2,3及Toll样受体2,4的基因表达水平。结果与结论:肿瘤坏死因子α对β-防御素1,2,3基因表达均有明显诱导作用,且在150μg/L时达到最大效应(P<0.05),相对于对照组分别出现明显的升高,而在200μg/L时又出现明显降低,呈现出浓度依赖性趋势。肿瘤坏死因子α诱导角质形成细胞后,Toll样受体2,4的表达规律类似于β-防御素。结果可见肿瘤坏死因子α对体外培养的皮肤角质形成细胞有显著的免疫诱导效应。     

Radioimmunoassay of tumor necrosis factor in serum

We present a double-antibody radioimmunoassay for determination of the concentration of tumor necrosis factor (TNF) in serum. TNF in serum competes with a fixed amount of 125I-labeled TNF for the binding sites of specific rabbit antibodies. The bound TNF is precipitated with Sepharose-bound anti-rabbit IgG, then centrifuged, and the radioactivity of the pellets is counted. The detection limit of the assay is 7 ng/L (B0-3 SD). Bound radioactivity in the range of 10% to 90% of the B0 counts corresponds to TNF concentrations of 26 to 10,000 ng/L. Of 40 sera from healthy subjects, 21 (53%) contained TNF concentrations greater than 7 ng/L (range 8-40 ng/L). Some patients with parasitic or neoplastic disease and patients with septic shock had highly increased TNF values. Three of the 14 sera (21%) from patients with rheumatoid arthritis had TNF concentrations greater than 40 ng/L.     

Evaluation of recombinant human tumor necrosis factor by scheduled intratumoral administration in mice bearing transplantable tumors

The antitumor effect of recombinant human tumor necrosis factor (rTNF) was examined against Meth A fibrosarcoma in BALB/c mice and Sarcoma-180 in ddY mice. Significant hemorrhagic necrosis in tumor tissues occurred within 24 hr when optimal rTNF (1,000 to 5,000 units per mouse) was injected intratumorally on day 5 after intradermal inoculation of 5 x 10(5) tumor cells. Complete tumor regression resulted when two repeated courses of administration a week, each for 3 consecutive days, were given. For this marked effect to occur, however, initial tumor weight should not be greater than 1 g. When the initial tumor was greater than 1 g the surgical removal of tumor tissues was conducted and followed by rTNF administration. This caused hemorrhagic necrosis and the regression was the case with smaller tumors. When the cured mice were rechallenged with same tumors, more than 60% of mice rejected the tumors in a specific manner. In spite of such demonstration of specific immunity, well-known immunological effector mechanisms such as augmentation of natural killer cell activity, activation of antibody dependent cellular cytotoxicity or induction of interferon activity by rTNF were not detected in normal and tumor-bearing mice, suggesting that the activation of immunoregulatory cells by TNF itself may not involve at least in an early stage of TNF treatment. These results suggest that rTNF is a potent therapeutic agent for a certain solid tumor when the protocol of administration is optimized.     

Interleukins and tumor necrosis factor in inflammation

Intense research efforts have been directed toward characterizing mediators that control the inflammatory response and regulate the growth, differentiation, and function of cells involved in inflammation. Tumor necrosis factor, or cachectin, and members of a heterogeneous group of peptides called interleukins exhibit a wide spectrum of activities, some of which appear to influence the evolution of inflammatory processes. This review outlines the observations that have led to our current understanding of the biology of tumor necrosis factor and the interleukins. Particular attention is directed toward the evidence suggesting that these cytokines function as mediators of inflammatory responses.     

Augmentation of murine tumor necrosis factor production by amphotericin B in vitro and in vivo.

Murine peritoneal macrophages were preincubated with amphotericin B (AMPH) and were then stimulated with bacterial lipopolysaccharide or streptococcal preparation (OK432). These macrophages produced a large amount of tumor necrosis factor. When administered to mice, the priming activity of amphotericin B for tumor necrosis factor production in vivo was also observed.     

Novel method for detection of ex vivo tumor necrosis factor alpha production by monocytes

Tumor necrosis factor alpha (TNF-alpha) has come into recent focus as a proinflammatory cytokine derived from monocytes/macrophages. We developed a novel system (the SEK-5001 system) for measurement of ex vivo production of TNF-alpha stimulated by lipopolysaccharide (LPS) as a marker of immune function. In the present study, we evaluated the performance of this system as a diagnostic tool. Furthermore, we compared TNF-alpha levels with data from other immune function tests, including lymphocyte blast formation test and differential leukocyte counts. Incubation of whole blood with a stimulation of low-dose LPS (100 EU/mL blood) for 4 hr at 37 degrees C gave acceptable results. After incubation, plasma TNF-alpha levels were determined by enzyme-linked immunosorbent assay (ELISA). The specificity, reproducibility, and recovery of the SEK-5001 system were excellent. No correlation between TNF-alpha levels and total leukocyte counts was found. Lymphocyte blast formation test and monocyte counts, however, were correlated with TNF-alpha levels in blood from patients with hematological malignancy and aplastic anemia before treatment. This assay system may potentially be clinically applicable to assess in vivo immune function.     

Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis

The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.     

Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha

The immunoregulatory effects of transforming growth factor beta (TGF-beta) and recombinant murine tumor necrosis factor alpha (rMuTNF-alpha) on CTL generation and activity were examined. The results demonstrate that TGF-beta, in a dose-dependent manner, inhibited CTL generation but not CTL activity. The inhibitory effects were detected only when TGF-beta was added within the first 48 h of the MLC. Little activity was seen when it was added thereafter, including the addition of TGF-beta to the cytotoxicity assay. The production of TNF-alpha, which occurs during early phases of the MLC and which is inhibited in the presence of TGF-beta, appears to have an important regulatory role, as altering the levels of TNF-alpha in an MLC can significantly influence CTL development. The inhibitory effects of TGF-beta on the MLC can be significantly reversed by the addition of rMuTNF-alpha to the cultures. These results demonstrate that TGF-beta can inhibit MLC and subsequent CTL generation at early stages of the reaction, and such inhibition may involve the suppression of TNF-alpha production.     

急性中小量脑出血患者血浆TNF-α、IL-2的变化及羚蝎胶囊干预的疗效观察

目的：观察急性中小量脑出血患者血浆肿瘤坏死因子－α（TNF－α）、白介素－2（IL－2）的变化及羚蝎胶囊干预的临床疗效。方法：治疗组31例、西医对照组32例均为急性中小量脑出血患者,2组基础疗法相同,治疗组加羚蝎胶囊,比较2组治疗后血浆TNF－α、IL－2水平的变化。结果：治疗组治疗后第2周、第3周血浆TNF－α均低于对照组（P均＜0．01）,IL－2均高于对照组（P均＜0．05）。结论：羚蝎胶囊通过调节脑出血患者血浆TNF－α、IL－2水平来改善预后。     

Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor.

We have developed a rat model of ischemic bowel necrosis associated with shock by injection of platelet-activating factor (PAF) or a combination of PAF and endotoxin. Recent investigations have shown that tumor necrosis factor (TNF) also induces shock and necrosis of the gastrointestinal tract. The morphological changes of TNF-induced bowel lesions are indistinguishable from those caused by PAF. The mechanism of TNF-induced bowel necrosis is unclear. In the present study, we have shown that (a) TNF caused PAF production in bowel tissue; (b) the effects of TNF and LPS on PAF production in the intestine are additive; (c) TNF and LPS are synergistic in inducing bowel necrosis; and (d) TNF-induced bowel necrosis is due to PAF release and can be prevented by pretreatment with PAF antagonists.     

肿瘤坏死因子诱导肿瘤细胞凋亡及其调节机制的研究

背景肿瘤坏死因子(TNF)在体外能诱导某些正常细胞和大多数肿瘤细胞株(包括Hela细胞株)凋亡,其凋亡发生机制与细胞周期的关系中尚有许多问题需要探讨.目的研究细胞周期时相对TNF诱导Hela细胞凋亡的影响.设计非随机非对照实验研究.地点和对象实验在广东医学院生物化学与分子生物学研究所完成,对象为重组人肿瘤坏死因子-α(rhTNF-α,Sigma产品),Hela细胞株引自中国科学院上海细胞生物研究所.干预通过胸腺嘧啶核苷酸(TdR)阻断法阻滞Hela细胞的细胞周期,采用MTT法、流式细胞术和荧光染色分析TdR阻滞细胞和周期化的Hela细胞对TNF诱导凋亡的敏感性.主要观察指标TNF诱导TdR阻滞和周期化Hela细胞凋亡数.结果TdR阻滞细胞周期较周期化的Hela细胞对TNF诱导凋亡的敏感性降低.结论TNF诱导Hela细胞凋亡与细胞周期有关.     

Role of lipid A in the production of tumor necrosis factor and differences in antitumor activity between tumor necrosis factor and lipopolysaccharide

The role of lipopolysaccharide (LPS) in the production of tumor necrosis factor (TNF) was examined. Alkaline treatment of LPS greatly reduced the TNF-producing activity of LPS, but TNF was produced when a large amount was injected. Free lipid A and lipid A-mouse serum albumin complex, which were prepared from the acid hydrolyzate, effectively induced TNF. However, the polysaccharide-rich fraction of the acid hydrolyzate was not capable of inducing TNF. Preincubation of LPS with polymixin B largely abrogated the TNF-producing activity of LPS. The differences in antitumor activity between TNF and LPS were also tested. TNF has a direct cytotoxicity against cancer cells in vitro but LPS does not. The activity of TNF was not inhibited by preincubation with polymixin B. Tumor necrosis in vivo was inhibited by preincubation of LPS with polymixin B but not by that of TNF. Galactosamine was found to induce susceptibility to the lethal effects of LPS, but did not influence the action of TNF. Lipid A is largely responsible for the TNF-inducing activity of LPS, but is not essential for the antitumor activity of TNF.     

Glucocorticoid-mediated inhibition of endotoxin-induced intratumor tumor necrosis factor production and tumor hemorrhagic necrosis and regression

Intravenous injection of 25 micrograms of bacterial endotoxin on day 9 of growth of the SA1 sarcoma results in extensive necrosis of the core of this tumor and in its subsequent complete regression. Tumor hemorrhagic necrosis and regression failed to occur in mice that were given a subcutaneous injection of cortisone acetate or dexamethasone 12 h before being giving endotoxin. Inhibition of tumor hemorrhagic necrosis and regression by glucocorticoids was associated with inhibition of endotoxin-induced intratumor TNF production that normally takes place several h after endotoxin is given. In contrast, glucocorticoids had no effect on the ability of intravenously injected rTNF to cause tumor hemorrhagic necrosis and regression. The results lend further support to the belief that TNF is the predominant mediator of endotoxin-induced hemorrhagic necrosis of established murine tumors, and that hemorrhagic necrosis is a prerequisite for the immunologically mediated regression that follows.