P55 TNF Research Articles

The p55 tumor necrosis factor receptor (CD120a) induces endothelin-1 synthesis in endothelial and epithelial cells

Synthesis of the vasoconstrictor peptide endothelin-1 by endothelial and epithelial cells is strongly induced by tumor necrosis factor α (TNF-α). The actions of TNF-α are mediated by two transmembrane receptors of approximately 55 (p55, CD120a) and 75 kDa (p75, CD120b). Reagents activating selectively these receptor subtypes have been used to identify which TNF receptor mediates the induction of endothelin-1 synthesis. Stimulation of bovine aortic endothelial cells or human HEp-2 epithelial cells with a p55-selective mutant of human TNF-α (R32W-S86T) induced significant and concentration-dependent increases in endothelin-1 release. A p75 receptor-selective TNF-α mutant (D143N-A145R) was ineffective alone or in combination with the p55-selective mutant. Competitive binding experiments with [ 125I]TNF-α showed the p55-selective mutant, but not the p75-selective mutant, to inhibit the binding of [ 125I]TNF-α to endothelial and HEp-2 cells. Similar results were obtained with the p55 agonist antibody htr1 in both cell lines. These results establish the p55 TNF receptor as the main receptor involved in the induction of endothelin-1 synthesis by TNF-α.

Regulated on activation normal T expressed and secreted chemokine is induced by tumor necrosis factor-alpha in granulosa cells from human preovulatory follicle.

We examined the production of regulated on activation normal T expressed and secreted (RANTES) chemokine, which may contribute to the recruitment and local accumulation of leukocytes in human preovulatory follicles. Cells were obtained from follicular aspirates collected from in vitro fertilization patients, then cultured. RANTES production in culture was measured by immunoenzymatic assay, RANTES-producing cells were measured by flow cytometry, and messenger ribonucleic acid as measured by RT-PCR and in situ hybridization. RANTES was detected in follicular fluids and culture supernatants; RANTES protein and messenger ribonucleic acid were expressed in granulosa cells. RANTES production was stimulated by tumor necrosis factor-alpha (TNFalpha) and was inhibited in cultures containing a neutralizing anti-TNFa antibody. p55 TNF receptors were detected by RT-PCR and visualized on granulosa cells by flow cytometry. RANTES production was increased by phorbol 12-myristate 13-acetate, but not by 8-bromo-cAMP. RANTES was produced by granulosa cells from human preovulatory follicles. This production was activated by TNFalpha, probably through TNF receptor p55. This suggests that RANTES may play an active role in ovarian processes involving the local accumulation of immune cells.

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

Cytokine cascade induced by endotoxin in TNF double receptor knockout mice: evidence supporting a role for IL-10 in mediating antipyretic action of TNF

Tumor necrosis factor double receptor-knockout (TNFR KO) mice lacking functional genes for the TNF p55 and p75 receptors show exacerbated febrile responses to endotoxin. TNFR KO mice and wild-type (WT) control animals were intraperitoneally treated with 2.5 mg/kg of lipopolysaccharide (LPS), and the effect of this treatment on plasma levels of TNF, interleukin (IL)-6, IL-10 and IL-1 type II (decoy) receptor was studied. TNFR KO mice showed markedly higher levels of TNF 1.5 and 8 h after LPS compared to WT controls. There was no difference in circulatory levels of IL-6 and IL-1 type II receptor between TNFR KO and WT mice 1.5 and 8 h after LPS challenge. There was no difference in plasma IL-10 levels 1.5 h after LPS treatment between TNFR KO and WT mice. However, 8 h after LPS injection TNFR KO mice showed significantly lower levels of IL-10 in plasma than control animals (13.2±2.7 pg/ml vs. 47.6±11.4 pg/ml, P=0.001). These results indicate that under certain conditions the antipyretic action of TNF may be mediated via IL-10 which is considered a major antiinflammatory and antipyretic cytokine. The data obtained also suggest that the exacerbated fever in TNFR KO mice is probably not due to alterations of IL-6 or IL-1 type II (decoy) receptor concentrations in the plasma.

Clinical experience with soluble TNF p75 receptor in rheumatoid arthritis

Objectives: Managing rheumatoid arthritis (RA) can be difficult: the disease may follow an unpredictable course, and therapies are often ineffective or toxic. Etanercept, a bioengineered fusion protein derived from the human soluble tumor necrosis factor p75 receptor, recently has been approved for use in patients with refractory RA. Methods: Published data on clinical experience with etanercept in conjunctionwith case illustrations are presented. Results: Reports from clinical trials of patients with refractory RA indicate that etanercept significantly improves measures of disease activity, including swollen and tender joint counts, morning stiffness, pain, and erythrocyte sedimentation rate, compared with placebo. In the phase 3 trial, swollen joint counts improved by 47% in patients receiving etanercept 25 mg compared with a 7% worsening in patients receiving placebo. The drug is well tolerated; injection site reaction, the most frequent adverse event, was minor and manageable. In long-term studies, etanercept remains well tolerated and effective. Our clinical experience indicates that patients with refractory RA experience dramatic symptomatic relief, along with reduced fatigue and improved quality of life. One of 18 patients discontinued treatment; the rest have remained on therapy for up to 18 months. Conclusions: Etanercept diminishes disease activity in patients with refractoryRA. Its favorable safety profile provides symptom control without major toxicity. Etanercept is an important addition to RA therapeutic agents.

PEGylated recombinant human soluble tumour necrosis factor receptor type I (r-Hu-sTNF-RI): novel high affinity TNF receptor designed for chronic inflammatory diseases

The proinflammatory cytokine, tumour necrosis factor α (TNFα) has been shown to play a pivotal part in mediating acute and chronic inflammation. The activities of TNFα are modulated by the...

A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor alpha expressed from an adenovirus vector induces an antitumor response with reduced toxicity.

The toxic effects of tumor necrosis factor alpha (TNFalpha) have greatly limited its use in tumor therapy. Recently, clear evidence has been obtained linking the p55 TNF receptor (TNFR) to the induction of systemic toxicity. We have generated a p75 murine TNFR (mTNFR)-specific mutant of mTNFalpha (D142N-A144R), cloned this gene into a recombinant adenovirus vector (Ad-75), and studied its efficacy for tumor immunotherapy of a murine transgenic breast cancer model. Cell culture supernatants from Ad-75-transduced cells showed no cytotoxic activity on L929 cells, but retained the ability to induce proliferation of a murine T-cell line (CT6); this activity was not blocked by soluble p55 mTNFR. Furthermore, it was shown that the mutant form of mTNFalpha was able to coimmunoprecipitate only with the p75 mTNFR and not with the p55 mTNFR. Tumors injected with Ad-75 became necrotic, and mice injected with < or =1 x 10(9) plaque-forming units showed no mortality, whereas both wild-type murine and human TNF vectors induced lethality at doses of 1 and 5 x 10(8) plaque-forming units. All Ad-TNF vectors induced partial or permanent tumor regressions, with cured mice showing immune memory against the tumor. These results demonstrate that a p75 mTNFR agonist expressed from a recombinant adenovirus vector does not induce mortality at doses that cause tumor regression.

Susceptibility to FAS-induced apoptosis in human nontumoral enterocytes: role of costimulatory factors.

FAS-FAS ligand interaction has been implicated in increased enterocyte apoptosis seen in immune-mediated bowel injury. However, scant information exists on the role of FAS in physiological enterocyte turnover. In the present study, the regulation of enterocyte FAS and FAS ligand expression by cytokines and its functional role in human intestinal epithelial cell apoptosis and proliferation were analyzed with two different models: a nontransformed human intestinal epithelial cell line (HIEC) and normal colonic explant cultures. HIEC constitutively expressed FAS, as analyzed by flow cytometry. However, stimulation with agonistic anti-FAS antibody (1-500 ng/ml) did not induce HIEC apoptosis. In contrast, in the presence of tumor necrosis factor alpha (TNFalpha) and/or interferon gamma (IFNgamma), HIEC became highly susceptible to FAS-induced apoptosis. The sensitizing effect to FAS-induced apoptosis was mediated via TNFalpha- and IFNgamma-induced upregulation of FAS expression (maximally 348%). Receptor studies showed that the effect of TNFalpha on FAS was mediated via the p55 TNF receptor. In colonic organ cultures, IFNgamma and TNFalpha also enhanced colonocyte FAS expression, resulting in a markedly increased apoptotic response to stimulation of this receptor, as shown by in situ terminal deosyuridine triphosphate nick-end staining. Neither FAS ligand expression nor its induction by cytokines was observed in HIEC or colonic explants. Proliferation studies showed that FAS is not implicated in regulating HIEC growth. These findings suggest that, despite the fact that normal human enterocytes express FAS, costimulatory factors, such as TNFalpha or IFNgamma, abundantly secreted under inflammatory conditions, are necessary to sensitize intestinal epithelial cells to FAS-induced apoptosis by upregulating this receptor.

TNF-Induced Haptoglobin Release from Human Neutrophils: Pivotal Role of the TNF p55 Receptor

Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-alpha induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-alpha was not due to nonspecific lysis of the cells. TNF-alpha is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-alpha mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-alpha for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-alpha-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-alpha may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.

Oligodendrocyte Apoptosis Mediated by Caspase Activation

Treatment with NGF causes long-term cultures of oligodendrocytes to die via a yet undefined mechanism mediated by the p75 neurotrophin receptor. The p75 receptor belongs to the TNF receptor superfamily of molecules, which includes Fas and p55 TNF receptors. The Fas and TNF receptors use adaptor molecules to recruit and activate caspase-8 to the receptor. Using a combination of immunohistochemical and Western blotting assays, we have examined caspase activity during NGF-induced apoptosis. Interestingly, although caspase-1 [interleukin-1beta-converting enzyme (ICE)], caspase-2, caspase-3, and caspase-8 were expressed in oligodendrocytes, only caspase-1, -2, and -3 were activated after NGF treatment, whereas caspase-8 was not. These data suggest that the mechanism of apoptosis by NGF through the p75 receptor is different from TNF and Fas-mediated killing. gamma Radiation of oligodendrocytes also activated a similar subset of caspases as NGF, indicating that NGF-induced oligodendrocyte apoptosis uses a similar cell death execution mechanism as injury models. This consolidates a potential role of the p75 neurotrophin receptor during stress and inflammatory conditions.

TNF Receptor p55 Plays a Pivotal Role in Murine Keratinocyte Apoptosis Induced by Ultraviolet B Irradiation

Excess exposure of skin to ultraviolet B (UVB) results in the appearance of so-called sunburn cells. Although it has been demonstrated that sunburn cells represent apoptotic keratinocytes, the molecular mechanisms for UVB-induced apoptosis in keratinocytes have not been fully elucidated. The cytokine, TNF-alpha, has been shown to induce apoptosis in a variety of cell types. Since UVB induces keratinocytes to release TNF-alpha, we hypothesized that TNF-alpha is involved in UVB-induced apoptosis in keratinocytes. In order to confirm this hypothesis and to further delineate which type of TNF receptor signaling mediates the apoptosis pathway, we performed both in vivo and in vitro experiments using gene-targeted knockout mice lacking either the TNF p55 receptor or the TNF p75 receptor. In the in vivo study, wild-type and mutant mice were exposed to UVB, and apoptotic keratinocytes were detected by examining DNA fragmentation using in situ nick-end labeling. For the in vitro experiments, keratinocytes derived from the wild-type and mutant mice were irradiated with UVB, and the degree of apoptosis was determined by flow cytometry, nick-end labeling of DNA, and a DNA ladder assay. Both in vivo and in vitro studies demonstrated that the deletion of TNF receptor p55 could suppress UVB-induced apoptosis in keratinocytes. Our observations support the notion that TNF-alpha is involved in UVB-induced keratinocyte apoptosis, and demonstrate that p55 receptor signaling plays a pivotal role in this event.

A 63 kDa tumor necrosis factor inhibitor released from a human monocytic leukemia cell line, THP-1.

A human monocytic cell line, THP-1-S, was cultured in a serum-free medium. The effect of the culture supernatant of THP-1-S on the cytotoxicity of rTNF-alpha to three kinds of cell lines and the binding of rTNF to its receptor were tested. The supernatant inhibited the cytotoxicity of rTNF-alpha when tested by the neutral red uptake method. In addition, the supernatant blocked the binding of 125I-rTNF-alpha to its receptor. Furthermore, following precipitation with PEG we detected complexes between rTNF-alpha and the inhibitory factor which formed during incubation with the culture supernatant from THP-1-S cells. However, the supernatant did not bind to or down-regulate the receptor for TNF-alpha on the cell surface of L-M-2d6 cells. This factor eluted with an apparent molecular mass of 63,000 Da by gel filtration and did not react with antibodies against p55 and p75 TNF receptors. These data suggest that human monocytic cells are capable of releasing an inhibitory factor against rTNF-alpha in serum-free culture conditions.

BRE: a modulator of TNF-alpha action.

A stress-responsive gene highly expressed in brain and reproductive organs (BRE) is down-regulated after UV irradiation, DNA damaging agents, or retinoic acid treatment. The human BRE gene encodes a mRNA of 1.9 kb, which gives rise to a protein of 383 amino acids with a molecular size of 44 kilodaltons. BRE is not homologous to any known gene and its function has not been defined. Here we report that BRE was identified multiple times in a yeast two-hybrid screen of a murine cerebellar cDNA library, using the juxtamembrane domain of the p55 tumor necrosis factor alpha (TNF) receptor. The interaction between the p55 receptor and BRE was verified by an in vitro biochemical assay by using recombinant fusion proteins and by co-immunoprecipitation of transfected mammalian cells. In the yeast two-hybrid assay, BRE specifically interacted with p55 TNF receptor but not with other TNF family members such as the Fas receptor, the p75 TNF receptor, and p75 neurotrophin receptor. Overexpression of BRE inhibited TNF-induced NFkappaB activation, indicating that the interaction of BRE protein with the cytoplasmic region of p55 TNF receptor may modulate signal transduction by TNF-alpha.

Complementation of lymphotoxin alpha knockout mice with tumor necrosis factor-expressing transgenes rectifies defective splenic structure and function.

Lymphotoxin (LT)alpha knockout mice, as well as double LTalpha/tumor necrosis factor (TNF) knockout mice, show a severe splenic disorganization with nonsegregating T/B cell zones and complete absence of primary B cell follicles, follicular dendritic cell (FDC) networks, and germinal centers. In contrast, as shown previously and confirmed in this study, LTbeta-deficient mice show much more conserved T/B cell areas and a reduced but preserved capacity to form germinal centers and FDC networks. We show here that similar to the splenic phenotype of LTbeta-deficient mice, complementation of LTalpha knockout mice with TNF-expressing transgenes leads to a p55 TNF receptor-dependent restoration of B/T cell zone segregation and a partial preservation of primary B cell follicles, FDC networks, and germinal centers. Notably, upon lipopolysaccharide challenge, LTalpha knockout mice fail to produce physiological levels of TNF both in peritoneal macrophage supernatants and in their serum, indicating a coinciding deficiency in TNF expression. These findings suggest that defective TNF expression contributes to the complex phenotype of the LTalpha knockout mice, and uncover a predominant role for TNF and its p55 TNF receptor in supporting, even in the absence of LTalpha, the development and maintenance of splenic B cell follicles, FDC networks, and germinal centers.

A p55 TNF Receptor Immunoadhesin Prevents T Cell-Mediated Intestinal Injury by Inhibiting Matrix Metalloproteinase Production

Anti-TNF-alpha Ab therapy has been shown to be of benefit in the treatment of active Crohn's disease, but the tissue-injuring processes in the gut mediated by TNF-alpha that might be inhibited by neutralizing Ab are unknown. In this work, we have used a p55 TNF receptor-human IgG fusion protein (TNFR-IgG) to prevent the severe mucosal injury that ensues when lamina propria T cells in explant cultures of human fetal small intestine are directly activated with the lectin PWM. Following T cell activation and associated with mucosal injury, there is a marked elevation of soluble TNF-alpha in organ culture supernatants and a large increase in TNF-alpha mRNA transcripts. The addition of TNFR-IgG at the onset of cultures greatly reduced PWM-induced tissue injury, without inhibiting the increase in TNF-alpha and IFN-gamma transcripts seen following T cell activation. Mucosal injury in this model is mediated by endogenously-produced matrix metalloproteinases (MMPs). When TNFR-IgG was added to PWM-stimulated explants, there was a reduction in MMPs in the explant culture supernatants, especially stromelysin-1. Recombinant TNF-alpha and IL-1beta added directly to mucosal mesenchymal cell lines also caused an increase in MMP production, but only the former was inhibited by the TNFR-IgG. These results suggest that one of the ways in which TNF-alpha causes tissue injury in the gut is by stimulating mucosal mesenchymal cell to secrete matrix-degrading metalloproteinases. Neutralization of this activity should help maintain tissue integrity.

Cloning and expression of murine IFN beta and a TNF antagonist for gene therapy of experimental allergic encephalomyelitis.

Immunomodulation of an ongoing autoimmune disease can be achieved by inhibitory cytokines or cytokine inhibitors such as TNF antagonists, delivery by gene therapy. The aim of this study was to design and test plasmid and retrovirus vectors expressing the mouse IFN beta gene and a chimeric protein containing the extracellular domain of human p55 TNF receptor linked to a murine Ig. These vectors were transiently expressed in COS-7 cells and permanently in amphotropic packaging cell lines or ABH mouse immortalized fibroblasts. Expression levels were assessed by ELISA. Western blotting and biological activity. In order to achieve tissue-specific expression in the CNS, the IFN beta gene was cloned and expressed under the control of the rat NSE promoter. We evaluated these constructs by direct intracranial injections of DNA-liposome complexes during the induction phase of experimental allergic encephalomyelitis, a murine model of multiple sclerosis, with therapeutic benefit.

Influence of fatty acid ethanolamides and Δ9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells

The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and Δ 9-tetrahydrocannabinol on the production of tumor necrosis factor- α (TNF- α), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon- γ, p55 and p75 TNF- α soluble receptors by stimulated human peripheral blood mononuclear cells as well as [ 3 H ]arachidonic acid release by non-stimulated and N-formyl-Met–Leu–Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3–30 nM) but inhibited the production of TNF- α, interferon- γ, interleukin-4 and p75 TNF- α soluble receptors at higher concentrations (0.3–3 μM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF- α soluble receptors at concentrations similar to those of anandamide but failed to influence TNF- α and interferon- γ production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. Δ 9-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF- α, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM Δ 9-tetrahydrocannabinol but stimulated by 3 μM Δ 9-tetrahydrocannabinol, as was interleukin-8 and interferon- γ synthesis. The level of interleukin-4, interleukin-10 and p75 TNF- α soluble receptors was diminished by 3 μM Δ 9-tetrahydrocannabinol. [ 3 H ]Arachidonate release was stimulated only by high Δ 9-tetrahydrocannabinol and anandamide concentrations (30 μM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and Δ 9-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.

Modulation of soluble CD40 ligand bioactivity with anti-CD40 antibodies.

The B cell surface molecule CD40 may be activated either by its ligand CD40L or by anti-CD40 antibodies. In this study, five new anti-CD40 monoclonal antibodies (MAb) were characterized. Bioactivity of the MAb was assessed using a receptor hybrid consisting of the extracellular domain of CD40 and the intracellular domain of the p55 TNF receptor as a model for CD40 activation. Two agonistic MAb were able to enhance the activation of this CD40 hybrid CD40L. These MAb bound to an epitope that was not located within the CD40L-binding region indicating that activation of CD40 occurs epitope-independent. A second pair of ligand mimetic anti-CD40 MAb which appeared to bind to the CD40L binding site decreased CD40L bioactivity. With regard to ligand mimetic effects binding of the CD40L epitope was not of advantage. Combining anti-CD40 MAb with different epitope specificities or cross linking anti-CD40 MAB with secondary antibodies enhanced ligand mimetic effects. These data clearly show that ligand or antibody-mediated receptor aggregation is the major mechanism by which CD40 is activated. Furthermore, our data support that an aggregate of activated receptors is favorable in regard to CD40 activation.

Constitutive shedding of both p55 and p75 murine TNF receptors in vivo.

Recently, we reported the generation and characterization of hamster mAbs specific for the murine p55 and p75 TNF receptors. Upon characterizing these mAbs in vivo, we discovered that administration of TNF receptor-specific mAb to normal mice resulted in the linear accumulation of the appropriate class of soluble TNF receptor in the circulation. The mechanism underlying soluble receptor accumulation was found to be due to an abrogation of the clearance of constitutively shed soluble receptor by receptor-specific mAb. Levels of p55 or p75 accumulated in the presence of nonblocking, nonagonistic TNF receptor mAb were capable of inhibiting murine TNF-induced responses in vivo. These results document that both p55 and p75 are constitutively shed in substantial amounts in vivo and suggest that the process of constitutive TNF receptor shedding plays an important role in regulating TNF activity under physiologic conditions.

Ligand-induced Formation of p55 and p75 Tumor Necrosis Factor Receptor Heterocomplexes on Intact Cells

The p55 and p75 tumor necrosis factor receptors are known to mediate their effects on cells through distinct signaling pathways. Under certain circumstances, the two classes of TNF receptors cooperate with each another to produce enhanced cellular responses. The only molecular mechanism proposed thus far to explain this effect is the process of "ligand passing," whereby TNF is concentrated at cell surfaces by binding to p75 and then following dissociation from this receptor class binds with high efficiency to p55. Using the in vivo model of TNF-induced TNF receptor shedding we have uncovered a novel ligand-dependent interaction of the two TNF receptors that occurs upon exposure of cells to TNF. Using TNF receptor-specific monoclonal antibodies that bind TNF receptors in the presence or absence of ligand, we report that TNF induces the formation of heterocomplexes consisting of both p55 and p75 TNF receptors. Whereas immunoprecipitates from untreated or human TNF-treated cells formed with either p55 or p75 TNF receptor-specific monoclonal antibodies contained only the relevant TNF receptor class, anti-p55 or anti-p75 precipitated both receptor types from murine TNF-treated cells. Ligand-induced complex formation was transient, occurred at physiologically relevant concentrations of TNF, and occurred with receptors lacking intracellular domains or that contained irrelevant transmembrane domains. Formation of TNF receptor heterocomplexes may therefore 1) define a novel molecular mechanism of ligand passing and/or 2) contribute to cooperative TNF receptor signaling via the juxtaposition of the intracellular domains of the two receptor classes and the signaling proteins that they recruit.