Recombinant Soluble Tumor Necrosis Factor Research Articles

A Case of Etanercept Induced Palmoplantar Pustular Psoriasis

Observations: Etanercept is a human recombinant soluble tumor necrosis factor  (TNF-α) receptor fusion protein. Etanercept has been used successfully to treat a wide range of inflammatory disorders including psoriasis and psoriatic arthritis resistant to classical disease-modifying treatments. The widespread use of TNF-α antagonists led to the recognition of adverse effects. Herein, we report a case of palmoplantar pustular psoriasis in a 37-year-old man during the etanercept therapy.

Blocking of tumor necrosis factor activity promotes natural repair of osteochondral defects in rabbit knee

Background and purpose Osteochondral defects have a limited capacity for repair. We therefore investigated the effects of tumor necrosis factor (TNF) signal blockade by etanercept (human recombinant soluble TNF receptor) on the repair of osteochondral defects in rabbit knees.Material and methods Osteochondral defects (5 mm in diameter) were created in the femoral patellar groove in rabbits. Soon after the procedure, a first subcutaneous injection of etanercept was performed. This single injection or, alternatively, 4 injections in total (twice a week for 2 weeks) were given. Each of these 2 groups was divided further into 3 subgroups: a low-dose group (0.05 μg/kg), an intermediate-dose group (0.4 μ g/kg), and a high-dose group (1.6 μ g /kg) with 19 rabbits in each. As a control, 19 rabbits were injected with water alone. The rabbits in each subgroup were killed 4 weeks (6 rabbits), 8 weeks (6 rabbits), or 24 weeks (7 rabbits) after surgery and repair was assessed histologically.Results Histological examination revealed that the natural process of repair of the osteochondral defects was promoted by 4 subcutaneous injections of intermediate-dose etanercept and by 1 or 4 injections of high-dose etanercept at the various time points examined postoperatively (4, 8, and 24 weeks). Western blot showed that rabbit TNFα had a high affinity for etanercept.Interpretation Blocking of TNF by etanercept enabled repair of osteochondral defects in rabbit knee. Anti-TNF therapy could be a strategy for the use of tissue engineering for bone and cartilage repair.

The Treatment of Ankylosing Spondylitis and Psoriatic Arthritis with Etanercept: A Comprehensive Review

Etanercept is a dimeric recombinant soluble tumor necrosis factor (TNF) receptor protein utilized in the treatment of various inflammatory diseases, including ankylosing spondylitis and psoriatic arthritis. Etanercept binds the proinflammatory cytokine TNF and blocks its interaction with soluble TNF receptors, thus decreasing the inflammatory response. Several randomized controlled clinical trials have demonstrated the efficacy, safety and tolerability of etanercept in the treatment of both ankylosing spondylitis and psoriatic arthritis. These trials have also shown significant reductions in markers of systemic inflammation and improvements in patient-reported quality of life measures. Inhibition of radiographic progression has been established in etanercept-treated psoriatic arthritis patients, and ankylosing spondylitis patients have demonstrated decreases in bony inflammation; however, current data does not support a reduction in bone proliferation in ankylosing spondylitis patients. Concerns regarding long-term toxicity, efficacy, and cost-effectiveness considerations persist and guidelines have been published to assist the clinician with appropriate patient selection for this biologic therapy. Current data indicates etanercept therapy has been a very successful and well-tolerated therapy for numerous ankylosing spondylitis and psoriatic arthritis patients and will likely continue to be a cornerstone therapy for treatment of these challenging diseases in the foreseeable future.

Molecular Determinants of Kinase Pathway Activation by Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) mainly activates programmed cell death through caspases. By contrast, TNF primarily induces gene transcription through the inhibitor of kappaB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. Apo2L/TRAIL also can stimulate these kinases, albeit less strongly; however, the underlying mechanisms of this stimulation and its relation to apoptosis are not well understood. Here we show that Apo2L/TRAIL activates kinase pathways by promoting the association of a secondary signaling complex, subsequent to assembly of a primary, death-inducing signaling complex (DISC). The secondary complex retained the DISC components FADD and caspase-8, but recruited several factors involved in kinase activation by TNF, namely, RIP1, TRAF2, and NEMO/IKKgamma. Secondary complex formation required Fas-associated death domain (FADD), as well as caspase-8 activity. Apo2L/TRAIL stimulation of JNK and p38 further depended on RIP1 and TRAF2, whereas IKK activation required NEMO. Apo2L/TRAIL induced secretion of interleukin-8 and monocyte chemoattractant protein-1, augmenting macrophage migration. Thus, Apo2L/TRAIL and TNF organize common molecular determinants in distinct signaling complexes to stimulate similar kinase pathways. One function of kinase stimulation by Apo2L/TRAIL may be to promote phagocytic engulfment of apoptotic cells.

Etanercept or intravenous immunoglobulin attenuates expression of genes involved in post-myocardial infarction remodeling

Persistently elevated levels of inflammatory cytokines such as tumor necrosis factor (TNF)alpha after acute myocardial infarction (MI) may contribute to maladaptive ventricular remodeling. The aim of the present study was to examine the effects of immunomodulatory therapy with recombinant soluble TNF receptor (TNFR:Fc) or intravenous immunoglobulin (IVIg) on left and right ventricular post-MI remodeling in rats. Adult male Sprague-Dawley rats were subjected to MI by left coronary artery ligation and randomized to treatment with vehicle, TNFR:Fc, or IVIg and sacrificed after 7 days. The main findings were that: (i) TNFR:Fc- and IVIg-treated rats developed less right ventricular (RV) hypertrophy compared to vehicle-treated controls. (ii) LV and arterial pressures in post-MI rats were not affected by the TNFR:Fc or IVIg treatment. (iii) As determined by real-time RT-PCR, both treatments reduced the expression of the hypertrophy-related genes, atrial natriuretic peptide and the ratio of beta/alpha-myosin heavy chains, and genes related to extracellular matrix remodeling (i.e., collagens I and III, matrix metalloproteinase [MMP]-2 and its tissue inhibitor TIMP-1) in the non-ischemic segment of LV and, in particular, in the RV. (iv) Treatment with IVIg, but not TNFR:Fc, reduced MMP-2 zymographic activity in the RV and the expression of genes for TNFalpha and monocyte chemoattractant protein-1. Therapy targeted directly against TNFalpha (i.e., TNFR:Fc) and a more general immunomodulatory approach (i.e., IVIg) in the acute phase of MI attenuates the cardiac remodeling process and expression of genes that are involved. These findings raise the possibility that initiation of immunomodulatory therapy post-MI could be beneficial in preventing the later development of heart failure.

Tumor Necrosis Factor Increases MUC1 mRNA in Cultured Human Nasal Epithelial Cells

Objective--Mucins are high molecular weight glycoproteins which are normally expressed on the surface of a variety of epithelia. It is possible that shedding of such molecules from the epithelium could play a role in preventing bacterial colonization at the mucosal surface. Immunohistochemical and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of human inferior turbinates have shown the existence of MUC1 mucin in nasal mucosa. However, the regulatory mechanisms of MUC1 mucin are poorly understood. In order to clarify the modulation of mucin gene expression, we developed a real-time semi-quantitative RT-PCR based on TaqMan fluorescence methodology to quantify MUC1 mRNA in primary cultured human nasal epithelial cells (HNECs).Material and Methods--HNECs were stimulated with recombinant human tumor necrosis factor (TNF)-α (20 pg/ml to 20 ng/ml) for specified time periods (0, 12, 24 and 48 h) and MUC1 mRNA was determined by means of semi-quantitative RT-PCR.Results--Significant increases in MUC1 gene expression in HNECs were initially detected at 12 h, peaking at 24 h after stimulation. TNF-mediated MUC1 mRNA expression at 24 h was significantly inhibited by co-incubation with human recombinant soluble TNF receptor.Conclusions--TNF-mediated MUC1 gene expression may contribute to the pathogenesis of human inflammatory upper airway disorders. Also, our mucin mRNA real-time PCR provides a quantitative method for investigating the regulation of mucin gene expression in both healthy and diseased samples.

Juvenile dermatomyositis: recognition and treatment.

Juvenile dermatomyositis (JDM) is a multisystem disease characterized by acute and chronic lymphocytic inflammation of the skeletal muscle and skin. The disease is marked early in its course by the presence of a vasculopathy or vasculitis, and later by the development of calcinosis. Research has focused on the epidemiology, etiology, and pathogenesis of the disease with, until more recently, limited therapeutic interventions. This article highlights treatment regimens, both traditional and more recent interventions. Traditional treatment for JDM includes high dose corticosteroid treatment with additional agents used in resistant disease or children with unwarranted adverse effects. Traditional therapy begins with daily oral corticosteroids, with intravenous corticosteroids utilized in severe disease; however, recent data suggests that short-term use of intravenous corticosteroids will allow a short-term improvement in strength, with no long-term change in outcome. More recent investigations suggest that early intervention with additional immunomodulatory agents will allow for a faster recovery, with less medication and disease sequelae. Use of methotrexate as an agent early in the disease course is becoming common place. Methotrexate, in conjunction with oral corticosteroids, allows a rapid improvement in symptoms, and allows for a more rapid reduction in corticosteroid dose. Methotrexate is considered as a steroid sparing agent, whether oral or intravenous corticosteroids are used. Additional immunomodulatory agents include the use of cyclosporine with or without methotrexate. Intravenous immunoglobulin has been reported to have benefit in resistant disease. There are exciting new agents which have great potential in treating JDM. Many of these agents are termed biologics and are being tested in adult myositis and juvenile arthritis. These include tumor necrosis factor (TNF)-alpha inhibitors, such as a chimeric monoclonal antibody to TNF-alpha, and a recombinant soluble human TNF receptor (p75)-Fc fusion protein. Many other new biological agents are also being tested in myositis.

Human Immunodeficiency Virus Type 1 Nef Mediates Sustained Membrane Expression of Tumor Necrosis Factor and the Related Cytokine LIGHT on Activated T Cells

Human immunodeficiency virus (HIV) Nef downregulates the antigen recognition molecules major histocompatibility complex class I and CD4. Downregulation of surface CD4 by Nef relies on the ability of this viral protein to redirect the endocytic machinery to CD4. However, by redirecting the endocytic machinery, Nef may affect the internalization rates of other proteins. Here we show that Nef simultaneously enhances surface expression of the effector cytokines tumor necrosis factor (TNF) and LIGHT, leading to enhanced cytokine activity. A dileucine motif in Nef, which is essential for CD4 downregulation and is involved in the recruitment of adapter protein complexes by Nef, was required to increase surface levels of both cytokines. The physiological impact of the Nef-mediated interference with endocytosis was demonstrated by the fact that a TNF-responsive T-cell line chronically infected with HIV produced higher levels of p24 viral protein following expression of a Nef-green fluorescent protein (GFP) fusion protein. This enhancement was dependent on the levels of membrane-bound TNF, since it was abrogated by a recombinant soluble TNF receptor. Expression of Nef-GFP in human 293T cells reduced the endocytosis of LIGHT, whereas at the same time CD4 internalization was accelerated. Taken together, these results suggest that in infected cells Nef interferes with the internalization of these effector cytokines. By increasing TNF expression, Nef could accelerate disease progression in infected individuals. These findings may help explain the pleiotropic functions that Nef plays during infection and disease.

Clinical and pharmacological experience with etanercept

Tumour necrosis factor (TNF) is the dominant mediator of the cytokine cascade that causes inflammation and joint destruction in rheumatoid arthritis (RA). A new class of agent under investigation, the biological TNF inhibitors, inhibit the activity of TNF. Recombinant human TNF receptor p75 Fc fusion protein (TNFR:Fc; etanercept) blocks the activity of the cytokine TNF. Etanercept (Enbrel™) was recently approved by the Food and Drug Administration for the treatment of RA. Etanercept is a genetically engineered recombinant soluble TNF receptor fusion protein that binds to TNF body and inhibits its biologic action; it is the first biotechnology product approved for the treatment of RA. Remarkable progress has been achieved in the treatment of RA with the development of TNF antagonists. Recent clinical trials have improved our understanding of the major role played by the cytokine TNF in provoking joint inflammation in RA.

Cloning and expression of porcine recombinant soluble tumor necrosis factor receptor 1

Abstract Objective To clone, sequence, and express porcine recombinant soluble tumor necrosis factor receptor 1 (sTNFR1). Procedure A polymerase chain reaction (PCR)-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a fusion protein. Protein product was purified by immunoaffinity chromatography, using a commercially available affinity gel specific for the marker peptide of the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay. Results A 927-base pair fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains, as well as 75 amino acids of the cytoplasmic domain, was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120 to 150 μg/L of culture. Affinity-purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in dose-dependent manner. Conclusions Porcine recombinant sTNFR1 inhibits TNF bioactivity in vitro. This recombinant protein will be useful for developing TNFR1 antibodies and studying the roles of TNF and TNFR1 in the pathogenesis of infectious diseases in swine. (Am J Vet Res 1998;59:1317–1322)

Mapping the Domain(s) Critical for the Binding of Human Tumor Necrosis Factor-α to Its Two Receptors

The extracellular domains of the two human tumor necrosis factor (TNF) receptors critical for binding TNF-alpha were examined by deletion mapping. The ligand binding capability of full-length and truncated recombinant soluble TNF receptors (TNFRs) was assessed by ligand blot analysis and their binding affinity determined by Scatchard analysis. The results showed that deletion of the fourth cysteine-rich domain of the p55 receptor (TNFR-1) did not alter ligand binding affinity significantly. Deletion of domains 3 and 4 of TNFR-1 resulted in no ligand binding, suggesting that domain 3, but not 4, of TNFR-1 binds directly to ligand. Deletion of domain 4 of TNFR-2 resulted in drastically reduced protein yield and 3-fold reduction in ligand binding affinity, while deletion of both domains 4 and 3 yielded no protein. Thus, the domain 4 of TNFR-2, but not that of TNFR-1, appears to be involved directly in binding TNF, although it is also possible that the domain 4 of TNFR-2 is involved in the correct folding of other domains. These results suggest that the modes of interaction between TNF-alpha and its dual receptors are different, providing opportunity to modulate each receptor specifically for research and therapeutic purposes.

Treatment by human recombinant soluble TNF receptor of pulmonary fibrosis induced by bleomycin or silica in mice.

Intratracheal instillation of bleomycin (0.08 U) or silica (2 mg) to mice leads, after 15 days, to a patchy pulmonary fibrosis associated with a significant increase of the lung hydroxyproline. Since tumour necrosis factor (TNF) seems to be an important mediator in pulmonary fibrosis, we wondered whether this fibrosis might be prevented by a new TNF-alpha antagonist. Infusion of a 55 kD human recombinant soluble TNF receptor rsTNFR-beta, at a rate of 20 micrograms.day-1, prevented the bleomycin/silica induced increase of lung hydroxyproline content, as measured 15 days after instillation. Infusion of rsTNFR-beta was also effective in the treatment of an established fibrosis, i.e. administered 25 or more days after instillation of bleomycin or silica, since it reduced lung collagen content. Recombinant soluble TNFR-beta had no significant influence on the number of cells, mostly macrophages, recovered by bronchoalveolar lavage. The examination of histological sections indicated that the rsTNFR-beta reduced the proportion of areas of damaged lung and, in silicosis, the formation of nodules with a rich collagen content. This study suggests that rsTNFR-beta might be useful in the therapy of pulmonary fibrosis.

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.

Recombinant soluble tumor necrosis factor receptor proteins protect mice from lipopolysaccharide-induced lethality.

The in vivo efficacy of human recombinant soluble tumor necrosis factor (TNF) receptor protein to prevent and to treat lipopolysaccharide (LPS)-induced lethal toxicity in D-galactosamine-treated mice was investigated. Chimeric proteins of the receptor extracellular domains fused to the hinge region of human IgG3 were expressed in myeloma cells (rsTNFR-h gamma 3). The fusion proteins had a disulfide-bonded dimeric structure. Upon intravenous injection, their serum concentration decreased relatively slowly after an initial phase of rapid elimination. D-galactosamine-sensitized mice were fully protected from the toxic effects of LPS, if the animal were pretreated with rsTNFR-h gamma 3 at 20 micrograms/animal. Partial protection was seen at significantly lower doses and when rsTNFR-h gamma 3 was given up to 3 h after LPS.

Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF

Membrane-associated tumor necrosis factor (TNF) and soluble TNF were compared as to their lytic activities, and as to the kinetics of their expression by macrophages activated with LPS and/or IFN-γ in the presence or absence of cycloheximide. EL 4 tumor cells, resistant and sensitive to lysis by recombinant TNF or membrane-associated TNF (paraformaldehyde (PF) -fixed activated macrophages) were used as targets. In the presence of cycloheximide the TNF-resistant S-EL4 cells were lysed by both TNFs. PF-fixed macrophages was cytolytic after 1 hr activation but not after 3 or more hours of activation. Their activity was totally inhibited by anti-TNF antibodies and was a composite of transmembrane (integral) TNF and soluble TNF conjugated to macrophage membrane TNF receptors. Treatment of the macrophages with glycine pH 3.0 buffer dissociated the conjugated TNF without affecting the integral membrane TNF. When macrophages were activated with LPS ± IFN-γ in the presence of cycloheximide or activated just with IFN-γ their activity after fixation with paraformaldehyde was no longer detected. Nonfixed macrophages under these conditions still remained cytotoxic. Tumor cell susceptibility to membrane-associated TNF activity, in contrast to recombinant (soluble) TNF, was greatly reduced in the presence of nicotinamide, an inhibitor of ADP-ribosyltransferase, suggesting that the mechanisms of lysis by these TNFs may be different. The lytic activity of both TNFs was found to be receptor-dependent in that tumor cells, whose TNF binding sites were “down-regulated” by TPA, were rendered resistant to lysis by both membrane-associated and soluble TNFs.

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.