SMART MODE:

NORMAL GENOMIC

• Zhang J et al.
• Modulation of T-cell responses to alloantigens by TR6/DcR3.
• J Clin Invest. 2001; 107: 1459-68
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• TR6 (DcR3) is a new member of the TNF receptor (TNFR) family that lacks a transmembrane domain in its sequence, indicating that it is a secreted molecule. TR6 can bind to FasL and prevent FasL-induced apoptosis; it can also associate with LIGHT, another TNF family member. The role of TR6 in immune responses was investigated in this study. According to flow cytometry, recombinant human TR6-Fc binds to human LIGHT expressed on 293 cells or on activated human T cells and competes with the LIGHT receptor TR2 for the binding to LIGHT on these cells. Human TR6 could cross-react with mouse LIGHT in immunoprecipitation. TR6-Fc also downregulates cytotoxic T lymphocyte activity in vitro and graft-versus-host responses in mice. Moreover, TR6-Fc modulates lymphokine production by alloantigen-stimulated mouse T cells. TR6-Fc ameliorated rejection response to mouse heart allograft. These results indicate that TR6 can dampen T-cell responses to alloantigens. Such regulatory effects of TR6 probably occur via interference with interaction between pairs of related TNF and TNFR family members, LIGHT/TR2 being one of the possible candidate pairs.

• Baumgarten G, Knuefermann P, Nozaki N, Sivasubramanian N, Mann DL, Vallejo JG
• In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4.
• J Infect Dis. 2001; 183: 1617-24
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• Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.

• Bobe J, Goetz FW
• Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary.
• Comp Biochem Physiol B Biochem Mol Biol. 2001; 129: 475-81
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• Using degenerative primers, partial cDNAs of a TNF (tumor necrosis factor) receptor and two TNF ligands were obtained by PCR of zebrafish and trout cDNAs, or cDNA libraries. These fragments were then used to screen cDNA libraries of appropriate tissues to obtain clones containing full coding sequences. A zebrafish cDNA was obtained that presumably codes for a 438 amino acid ovarian TNF receptor (OTR) that was identified as a death-domain-containing member of the TNF receptor family. On Northern blots, the OTR cDNA hybridized with a 3.4-kb transcript that is abundant in the zebrafish ovary but lightly detected in all other tissues tested. A zebrafish cDNA presumably coding for a 214 amino acid protein with sequence similarity to mammalian TRAIL (TNF-related apoptosis inducing ligand), was also isolated. In addition, a fragment of the brook trout TRAIL homologue was obtained. Finally, a full-length brook trout cDNA, that presumably codes for a 255 amino acid protein with sequence similarity to mammalian TNF-alpha and lymphotoxin-alpha, was isolated. This study is the first report of a death-domain-containing TNF receptor and the first published report of a TNF ligand in fish.

• Bazzoni F, Regalia E
• Triggering of antitumor activity through melanoma-specific transduction of a constitutively active tumor necrosis factor (TNF) R1 chimeric receptor in the absence of TNF-alpha.
• Cancer Res. 2001; 61: 1050-7
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• Tumor necrosis factor-alpha (TNF-alpha) has been intensively studied because of the specific toxicity of this cytokine toward cells that undergo malignant transformation. However, its proinflammatory and immunoregulatory properties always represented a drawback to the TNF-alpha administration in cancer therapy. In this study, we describe an adenovirus-based strategy in which the tumoricidal activity of TNF-alpha can be selectively triggered to eradicate tumors without administering TNF-alpha. This strategy might allow us to prevent TNF-alpha effects on normal tissues and, therefore, to bypass its systemic toxic effects. We inserted the constitutively active version of the Mr 55,000 TNF receptor, which was shown previously (F. Bazzoni et al., Proc. Natl. Acad. Sci. USA, 92: 5376-5380, 1995) to be capable of killing cells upon expression in the absence of its ligand, into a replication-deficient adenovirus, and under the control of a melanoma-specific promoter/enhancer element. We show that, upon infection, the recombinant gene reaches high level of expression in melanoma cell lines and triggers apoptosis by activating the caspase cascade. Expression and function of this receptor is restricted to melanoma cell lines, because morphology, viability, and proliferation of other cell types exposed to the recombinant adenovirus infection are not affected. We show for the first time that a TNF-like apoptotic response can be triggered in the absence of TNF-alpha and can be selectively confined to specific cellular targets. Killing activity and tissue specificity of the recombinant TNF receptor adenovirus, together with the advantage of triggering a TNF-like antitumor activity in the absence of TNF-alpha itself, are ideal features for a vector that might be the choice for gene therapy aimed to eradicate malignant cells.

• Vabulas RM et al.
• Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells.
• J Biol Chem. 2001; 276: 31332-9
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• Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic "danger" signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-kappaB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands.

• Prange-Kiel J, Kreutzkamm C, Wehrenberg U, Rune GM
• Role of tumor necrosis factor in preovulatory follicles of swine.
• Biol Reprod. 2001; 65: 928-35
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• The effects of tumor necrosis factor (TNF) on cultured porcine granulosa cells that were obtained from preovulatory follicles were studied with regard to following parameters: 1) TNF receptor type I expression, 2) progesterone receptor and transforming growth factor beta receptor type II (TbetaR II) as markers of luteinization, 3) proliferation, and 4) apoptosis. For comparative purposes the effects of TNF were also studied on insulin/forskolin-treated cells, as this treatment is well established to induce luteinization. Cytochemical methods followed by semiquantitative analysis were used. Our data show that TNF treatment upregulates TNF receptor type I expression in granulosa cells. TNF downregulates the expression of TbetaR II of insulin/forskolin-stimulated and of unstimulated cells. The progesterone receptor is also downregulated by the cytokine after insulin/forskolin-induced luteinization. Supplementation of the medium with TNF leads to increased proliferation and at the same time it induces apoptosis. Our results indicate that TNF exerts an inhibitory influence on luteinization and that TNF influences the balance between follicular growth (proliferation) and atresia (apoptosis).

• Mountz JD
• Re: "collagen-induced arthritis in TNF receptor-1-deficient mice: TNF receptor-2 can modulate arthritis in the absence of TNF receptor 1".
• Clin Immunol. 2001; 99: 305-7

• Pridmore AC et al.
• A lipopolysaccharide-deficient mutant of Neisseria meningitidis elicits attenuated cytokine release by human macrophages and signals via toll-like receptor (TLR) 2 but not via TLR4/MD2.
• J Infect Dis. 2001; 183: 89-96
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• Meningococcal disease severity correlates with circulating concentrations of lipopolysaccharide (LPS) and proinflammatory cytokines. Disruption of the lpxA gene of Neisseria meningitidis generated a viable strain that was deficient of detectable LPS. The potency of wild-type N. meningitidis to elicit tumor necrosis factor (TNF)-alpha production by human monocyte-derived macrophages was approximately 10-fold greater than that of the lpxA mutant. Killed wild-type N. meningitidis and its soluble products induced interleukin (IL)-8 and TNF-alpha secretion by transfected HeLa cells expressing Toll-like receptor (TLR) 4/MD2, but the lpxA mutant was inactive via this pathway. In contrast, both strains induced IL-8 promoter activity in TLR2-transfected HeLa cells. These data provide evidence that N. meningitidis contains components other than LPS that can elicit biological responses via pathways that are independent of the TLR4/MD2 receptor system, and TLR2 is one of these alternate pathways. These findings have implications for future therapeutic strategies against meningococcal disease on the basis of the blockade of TLRs and the modulation of LPS activity.

• Thompson JS et al.
• Baff-r, a newly identified tnf receptor that specifically interacts with baff.
• Science. 2001; 293: 2108-11
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• B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

• Utsumi T et al.
• Transmembrane TNF (pro-TNF) is palmitoylated.
• FEBS Lett. 2001; 500: 1-6
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• Human transmembrane tumor necrosis factor (pro-TNF) was examined for protein acylation. The cDNA encoding pro-TNF was expressed in both COS-1 cells and Sf9 cells and metabolic labeling with [(3)H]myristic or [(3)H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [(3)H]palmitic acid. The [(3)H]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site-directed mutagenesis of the two cysteine residues residing in the leader sequence of pro-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains of pro-TNF. Thus, pro-TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.

• Jones EY
• The tumour necrosis factor receptor family: life or death choices.
• Curr Opin Struct Biol. 2000; 10: 644-8
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• The past twelve months have seen a renewal of interest in the therapeutic potential of members of the tumour necrosis factor receptor family and their cytokine ligands. This biomedical interest has spawned a number of structural studies, which have significantly deepened our understanding of the molecular basis for the function of these cell-surface signalling systems. The fresh data have revealed unexpected mechanisms conferring ligand-receptor specificity and have highlighted the structural requirements for the initiation of intracellular signalling.

• Sarrias MR et al.
• The three HveA receptor ligands, gD, LT-alpha and LIGHT bind to distinct sites on HveA.
• Mol Immunol. 2000; 37: 665-73
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• The herpes virus entry mediator A (HveA), a member of the tumor necrosis factor receptor (TNFR) superfamily, interacts with three different protein ligands; lymphotoxin-alpha (LT-alpha) and LIGHT (LIGHT stands for lymphotoxin homolog, which exhibits inducible expression and competes with HSV glycoprotein D for HveA and is expressed on T-lymphocytes) from the host and the herpes simplex virus (HSV) surface glycoprotein gD. It has been reported that the gD binding site on HveA is located within the receptor's two N-terminal CRP domains, and that gD and LIGHT compete for their binding to HveA. However, whether these ligands interact with the same or different sites on the receptor is unclear. We analyzed and compared the sites of interaction between HveA and its TNF ligands, by using two recombinant forms of the receptor, comprising the full-receptor ectodomain (HveA (200t)) and its two first CRP domains (HveA (120t)), as well as several monoclonal antibodies recognizing HveA. Two HveA peptide ligands (BP-1 and BP-2) that differentially inhibit binding of soluble gD and LT-alpha to the receptor were also used to demonstrate that gD, LIGHT and LT-alpha bind to distinct sites on the receptor. Our results suggest that binding of a ligand to HveA may alter the conformation of this receptor, thereby affecting its interaction with its other ligands.

• Navarre WW, Zychlinsky A
• Pathogen-induced apoptosis of macrophages: a common end for different pathogenic strategies.
• Cell Microbiol. 2000; 2: 265-73
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• Microbe-macrophage interactions play a central role in the pathogenesis of many infections. Several bacterial pathogens induce apoptosis specifically in macrophages, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Macrophage death caused by Shigella flexneri and Salmonella spp. has been shown to result in the release of pro-inflammatory cytokines. Conversely, Yersinia spp. induce apoptosis by suppressing the signalling pathways that lead to the production of tumour necrosis factor (TNF)-alpha, a cytokine essential for the control of this infection. It is likely that there are a variety of reasons why macrophages are particularly susceptible to pathogen-induced apoptosis. One reason may be the expression of surface receptors that recognize highly conserved bacterial components, such as lipopolysaccharide (LPS) and bacterial lipoproteins (BLPs). These receptors have recently been shown to activate pro-apoptotic signalling pathways. The roles of macrophage apoptosis in different disease processes are discussed.

• Long Q, Huang H, Shafizadeh E, Liu N, Lin S
• Stimulation of erythropoiesis by inhibiting a new hematopoietic death receptor in transgenic zebrafish.
• Nat Cell Biol. 2000; 2: 549-52

• Xu X, Nash P, McFadden G
• Myxoma virus expresses a TNF receptor homolog with two distinct functions.
• Virus Genes. 2000; 21: 97-109
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• Myxoma virus, a member of the poxvirus family of DNA viruses, encodes many virulence factors to combat and evade the host immune responses. Among the virus-encoded immuno-modulators is M-T2, a tumor necrosis factor receptor (TNF-R) homologue. M-T2 is secreted as monomeric and dimeric species that bind and inhibit rabbit TNF in a species-specific manner. Deletion analysis indicates that the anti-TNF function is mediated by the first three of four cysteine rich domains (CRDs) of M-T2. In addition, the intracellular form of M-T2 has the ability to block virus-induced apoptosis in lymphocytes, and the first two CRDs appear to be sufficient for this function. Although the mechanisms for the anti-TNF and anti-apoptotic functions of M-T2 are not yet fully defined, we postulate that these dual activities of M-T2 are mediated through different functional motifs and abrogate distinct cellular responses to virus infection.

• Sheikh MS, Fornace AJ Jr
• Death and decoy receptors and p53-mediated apoptosis.
• Leukemia. 2000; 14: 1509-13
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• Recently, several tumor necrosis factor receptor 1 (TNF-R1) and Fas-related death receptors have been discovered and include DR3, DR4, DR5 and DR6. These receptors contain an extracellular region containing varying numbers of cysteine-rich domains and an intracellular region that contains the death domain. The death receptors are activated in a ligand-dependent or independent manner and transduce apoptotic signals via their respective intracellular death domains. In addition to death receptors, several decoy molecules have also been identified and include DcR1/TRID, DcR2/TRUNDD, DcR3 and osteoprotegrin (OPG). The decoy molecules do not transduce apoptotic signals but rather compete with the death receptors for ligand binding and thereby inhibit ligand-induced apoptosis. Recent evidence suggests that p53 upregulates the expression of death receptors Fas and DR5, and thus, may mediate apoptosis in part via Fas and/or DR5. However, p53 also regulates the expression of TRAIL decoy receptors DcR1/TRID and DR2/TRUNDD. Although the significance of p53-dependent regulation of decoy receptors remains unclear, evidence suggests that DcR1/TRUNDD appears to inhibit 53-mediated apoptosis. It is, therefore, possible that p53 may blunt its DR5-dependent apoptotic effects by controlling the levels of decoy receptors.

• Tribouley C et al.
• Characterization of a new member of the TNF family expressed on antigen presenting cells.
• Biol Chem. 1999; 380: 1443-7
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• The TNF family is involved in the regulation of the immune system, and its members have been implicated in a variety of biological events such as apoptosis, cell proliferation, differentiation and survival. Here we present a new member of the TNF family, tumor necrosis factor superfamily member 20 (TNFSF20) that we have identified from the expressed sequence tag (EST) database and characterized. The human protein is a 285 amino acid long type II transmembrane protein and is 19% homologous to TNF in its extra-cellular domain. TNFSF20 is expressed at the surface of antigen presenting cells such as cells of the macrophagemonocyte lineage and dendritic cells. After treatment with bacterial lipopolysaccharide (LPS), TNFSF20 expression is downregulated at the surface of the expresssing cells, suggesting that the membrane-bound protein gets cleaved, and that a soluble factor is released in the extra-cellular compartment. The soluble form of the recombinant TNFSF20 induces proliferation of resting peripheral blood monocytes (PBMC) and cell death of activated lymphocytes. TNFSF20 might therefore play a critical role in the regulation of cell-mediated immune responses.

• Medzhitov R, Janeway CA Jr
• Innate immune induction of the adaptive immune response.
• Cold Spring Harb Symp Quant Biol. 1999; 64: 429-35

• Piguet PF, Vesin C, Donati Y, Tacchini-Cottier F, Belin D, Barazzone C
• Urokinase receptor (uPAR, CD87) is a platelet receptor important for kinetics and TNF-induced endothelial adhesion in mice.
• Circulation. 1999; 99: 3315-21
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• BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

• Bajorath J
• Specificity of the tumor necrosis factor receptor superfamily.
• J Mol Graph Model. 1999; 17: 220-2

• Underhill DM et al.
• The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens.
• Nature. 1999; 401: 811-5
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• Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.

• Wallach D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP
• Tumor necrosis factor receptor and Fas signaling mechanisms.
• Annu Rev Immunol. 1999; 17: 331-67
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• Four members of the tumor necrosis factor (TNF) ligand family, TNF-alpha, LT-alpha, LT-beta, and LIGHT, interact with four receptors of the TNF/nerve growth factor family, the p55 TNF receptor (CD120a), the p75 TNF receptor (CD120b), the lymphotoxin beta receptor (LT beta R), and herpes virus entry mediator (HVEM) to control a wide range of innate and adaptive immune response functions. Of these, the most thoroughly studied are cell death induction and regulation of the inflammatory process. Fas/Apo1 (CD95), a receptor of the TNF receptor family activated by a distinct ligand, induces death in cells through mechanisms shared with CD120a. The last four years have seen a proliferation in knowledge of the proteins participating in the signaling by the TNF system and CD95. The downstream signaling molecules identified so far--caspases, phospholipases, the three known mitogen activated protein (MAP) kinase pathways, and the NF-kappa B activation cascade--mediate the effects of other inducers as well. However, the molecules that initiate these signaling events, including the death domain- and TNF receptor associated factor (TRAF) domain-containing adapter proteins and the signaling enzymes associated with them, are largely unique to the TNF/nerve growth factor receptor family.

• Jones SJ et al.
• TNF recruits TRADD to the plasma membrane but not the trans-Golgi network, the principal subcellular location of TNF-R1.
• J Immunol. 1999; 162: 1042-8
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• The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid. The subcellular interactions of TNF-R1 and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the trans-Golgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation. Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fraction-containing plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi in response to exogenous TNF at any time examined. These results suggest that although TNF-R1 is predominantly a Golgi-associated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the plasma membrane.

• Schneider P et al.
• BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.
• J Exp Med. 1999; 189: 1747-56
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• Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

• Corcoran AE, Scallon BJ, Trinh H, Chernajovsky Y, Ghrayeb J, Feldmann M
• Minimal tumor necrosis factor receptor binding protein: optimum biological activity of a truncated p55 soluble tumor necrosis factor receptor-IgG fusion protein.
• Eur Cytokine Netw. 1998; 9: 255-62
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• We have previously demonstrated, using expressed deletion constructs, that the fourth membrane proximal cysteine-rich repeat of the p55 TNF receptor (TNF-R) is not required for binding of tumour necrosis factor-alpha (TNF) or lymphotoxin-alpha (LT; tumour necrosis factor-beta). We and others have also shown that the soluble p55 TNF-R, rendered dimeric by fusion to an IgG backbone is extremely effective at neutralizing the harmful effects of TNF overproduction, such as in toxic shock. Here we address the question of how the TNF binding properties of the truncated TNF-R comprising the three distal cysteine-rich repeats (delta4 TNF-R), when fused with an IgG backbone, compare with those of the full length soluble receptor. We constructed several versions of the soluble delta4 TNF-R, on a complete IgG heavy chain backbone and on an IgG lacking the CH1 (first constant region) domain. The constructs were expressed with an Ig or native TNF receptor leader sequence and altered or native N terminal sequence, to compare efficiency of expression. When compared with a full length, soluble receptor Ig fusion protein, the affinity of all for TNF was identical, as were their activities in in vitro binding and cytotoxicity assays. In vivo studies showed that the delta4 and wild type fusion proteins afforded equivalent protection against LPS-induced lethality. However, the delta4 proteins exhibited a significantly lower affinity for LT, and reduced activity in LT binding and cytotoxicity assays. We conclude that the truncated TNF receptor IgG fusion protein is as effective at neutralizing TNF activity as the full length soluble receptor fusion protein. Its lower affinity for LT may make it a more selective agent in blocking the action of TNF, while causing less interference with the action of LT. Also its smaller size may make it a more useful therapeutic agent as it may be less immunogenic than the full length receptor.

• Baker SJ, Reddy EP
• Modulation of life and death by the TNF receptor superfamily.
• Oncogene. 1998; 17: 3261-70
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• The tumor necrosis factor receptor (TNFR) superfamily represents a growing family, with over 20 members having been identified thus far in mammalian cells. These proteins share significant homologies in their extracellular ligand binding domains and intracellular effector (death) domains. These receptors appear to transmit their signals via protein-protein interactions, which convey either a death or survival signal. Isolation and characterization of death domain containing proteins (TRADD, FADD/MORT-1, RIP), TRAF domain containing proteins (TRAF1-6) as well as new members and adaptor proteins such as DAXX have provided new insights to our understanding of signaling mechanisms associated with this family of receptors. While the death signals seem to be associated with the activation of both the caspase and JUN kinase pathways, the survival signals are mediated via the activation of the NF-kappaB pathway.

• Solorzano CC et al.
• Pharmacokinetics, immunogenicity, and efficacy of dimeric TNFR binding proteins in healthy and bacteremic baboon.
• J Appl Physiol. 1998; 84: 1119-30
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• Immunogenicity, pharmacokinetics, and therapeutic efficacy of three novel dimeric soluble tumor necrosis factor (TNF)-receptor I constructs [TNF-binding protein (bp)] were evaluated in 28 baboons, 12 of which were healthy and 16 were challenged with a lethal Escherichia coli bacteremia. The three constructs differed only in the number of extracellular domains of the TNF receptor I and were dimerized with polyethylene glycol. Although all three constructs had generally similar pharmacokinetics when administered to a naive animal, they differed quantitatively in their immunogenicity. Antibodies were detected more frequently, and titers were significantly higher (P < 0.05) in both healthy and septic baboons that received the 4.0-domain TNF-bp construct, compared with animals receiving the 2.6-domain construct. When the TNF-bp constructs were administered a second time (21 days later), the half-lives of the three constructs were significantly shorter in animals that had an antibody response after the first injection. In contrast, all three TNF-bp constructs were equally effective at improving outcome, blocking a systemic TNF-alpha response, and attenuating the cytokine responses when administered at a dose of 1.0 mg/kg body wt 1 h before a lethal E. coli infusion. The findings suggest that immunogenicity of TNF-bp constructs can be altered by changing the number of functional domains, without affecting their capacity to neutralize TNF-alpha and to abrogate TNF-mediated pathology.

• Lee EK, Kehrli ME Jr, Taylor MJ
• Cloning and sequencing of cDNA encoding bovine tumor necrosis factor (TNF)-receptor I.
• Vet Immunol Immunopathol. 1998; 61: 379-85
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• Tumor necrosis factor (TNF) is a multipotent cytokine produced by activated macrophages and lymphocytes and its activity is mediated by specific cell surface receptors, TNF-RI and TNF-RII. We have isolated and analyzed a cDNA encoding bovine TNF-RI gene and compared it with known TNF-RI sequences from other species. The cDNA sequence for the coding region of bovine TNF-RI shows 80% homology with porcine TNF-RI and 77% with human TNF-RI. The cDNA sequence of bovine TNF-RI codes for 471 amino acids and shows 75% and 67% identity with the amino acid sequences of porcine TNF-RI and human TNF-RI, respectively. The predicted bovine TNF-RI amino acid sequence consists of a signal peptide, an extracellular domain, a transmembrane region and a cytoplasmic tail. The extracellular region contains four repeated cysteine rich domains, which are conserved in all species. Northern blot results show that bovine TNF-RI gene is expressed in neutrophils and mononuclear leukocytes.

• Grell M, Wajant H, Zimmermann G, Scheurich P
• The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor.
• Proc Natl Acad Sci U S A. 1998; 95: 570-5
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• Tumor necrosis factor (TNF) can induce a variety of cellular responses at low picomolar concentrations. This is in apparent conflict with the published dissociation constants for TNF binding to TNF receptors in the order of 100-500 pM. To elucidate the mechanisms underlying the outstanding cellular sensitivity to TNF, we determined the binding characteristics of TNF to both human TNF receptors at 37 degrees C. Calculation of the dissociation constant (Kd) from the association and dissociation rate constants determined at 37 degrees C revealed a remarkable high affinity for TNF binding to the 60-kDa TNF type 1 receptor (TNF-R1; Kd = 1.9 x 10(-11) M) and a significantly lower affinity for the 80-kDa TNF type 2 receptor (TNF-R2; Kd = 4.2 x 10(-10) M). The high affinity determined for TNF-R1 is mainly caused by the marked stability of ligand-receptor complexes in contrast to the transient interaction of soluble TNF with TNF-R2. These data can readily explain the predominant role of TNF-R1 in induction of cellular responses by soluble TNF and suggest the stability of the TNF-TNF receptor complexes as a rationale for their differential signaling capability. In accordance with this reasoning, the lower signaling capability of homotrimeric lymphotoxin, compared with TNF, correlates with a lower stability of the lymphotoxin-TNF-R1 complex at 37 degrees C.

• Arch RH, Gedrich RW, Thompson CB
• Tumor necrosis factor receptor-associated factors (TRAFs)--a family of adapter proteins that regulates life and death.
• Genes Dev. 1998; 12: 2821-30

• Benigni F, Sacco S, Aloe L, Ghezzi P
• Intracerebroventricular injection of anti-Fas activates the hypothalamus-pituitary-adrenal axis and induces peripheral interleukin-6 and serum amyloid A in mice: comparison with other ligands of the tumor necrosis factor/nerve growth factor receptor superfamily.
• Am J Pathol. 1998; 153: 1377-81
• Display abstract

• Fas is a receptor of the tumor necrosis factor (TNF)/ nerve growth factor (NGF) receptor superfamily that mediates apoptosis and some inflammatory changes. As the central administration of TNF is known to activate the hypothalamus-pituitary-adrenal axis (HPAA) and to induce peripheral responses including induction of serum interleukin (IL)-6 and serum amyloid A (SAA), we investigated the effects of intracerebroventricular (i.c.v.) administration of agonist anti-Fas monoclonal antibody Jo2. Centrally administered anti-Fas (1 microg/mouse, i.c.v.) induced elevated levels of corticosterone, IL-6, and SAA comparable to those observed after i.c.v. administration of recombinant murine TNF. On the other hand, administration of murine NGF did not elevate serum corticosterone or IL-6, but induced SAA. Thus, Fas can trigger a centrally mediated anti-inflammatory response (HPAA activation) and induce a peripheral acute-phase response comparable to that induced with TNF, whereas NGF induces only acute-phase proteins.

• Paulli M et al.
• Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily.
• Hum Pathol. 1998; 29: 1223-30
• Display abstract

• The spectrum of CD30+ cutaneous lymphoproliferative disorders is characterized by the histology of a high-grade lymphoma but frequent clinical regression of skin lesions in lymphomatoid papulosis (LyP) and occasional regression in CD30+ large cell lymphomas (LCLs). A recent study shows that apoptosis may be a significant mechanism of regression of LyP (Arch Dermatol 133:828-833, 1997). Therefore, we studied expression of proteins that induce apoptosis, including CD27, CD40, CD95, and nerve growth factor receptor (NGF-R), as well as anti-apoptotic protein bcl-2 in skin lesions from 25 patients within the spectrum of CD30+ cutaneous lymphoma. Our results show consistent expression of CD95 (APO-1/Fas), but rare or absent expression of CD27, CD40, and NGF-R on tumor cells from both regressing LyP lesions and nonregressing CD30+ lymphomas. Bcl-2 was expressed at low levels in LyP and at high levels in pleomorphic CD30+ lymphomas. These results indicate that, in addition to CD30, CD95 expression is preferentially expressed at high levels in all cutaneous CD30+ lymphomas and suggest that CD95 may play a role in the regression of CD30+ skin lesions. Expression of bcl-2 appears to protect tumor cells from apoptosis in CD30+ lymphoproliferative disorders.

• Vinay DS, Kwon BS
• Role of 4-1BB in immune responses.
• Semin Immunol. 1998; 10: 481-9
• Display abstract

• 4-1BB is an inducible T cell surface receptor which belongs to the tumor necrosis factor receptor superfamily, a group of cysteine-rich cell-surface molecules. Both human and mouse 4-1BB recently received HLDA nomenclature. Naive T cells lack 4-1BB, which is not only induced upon T cell activation, but also remains on activated T cells. The natural ligand for 4-1BB, 4-1BBL is also induced and is found on activated antigen-presenting cells. Cross-linking of the 4-1BB molecule by agonistic antibody transmits a distinct and potent co-stimulatory signal leading to the activation and differentiation of CD4(+) and CD8(+) cells. 4-1BB transmits signals through the TRAF2-NIK pathway and activates NF-kappaB. Signals relayed through 4-1BB inhibit activation-induced cell death and rescue the immune system during the post-CD28 phase. Antibodies to the 4-1BB molecule can increase GVHD, accelerate the rejection of cardiac allograft and skin transplants, and eradicate established tumors. Interference with the 4-1BB-4-1BBL pathway may be of therapeutic use in the treatment of HIV infection. 4-1BB-deficient mice show dysregulated immune responses and mount elevated Ig responses to T-dependent antigens.

• Yasuda H
• [Osteoclastogenesis inhibitory factor (OCIF)]
• Seikagaku. 1998; 70: 385-90

• Boussaud V, Soler P, Moreau J, Goodwin RG, Hance AJ
• Expression of three members of the TNF-R family of receptors (4-1BB, lymphotoxin-beta receptor, and Fas) in human lung.
• Eur Respir J. 1998; 12: 926-31
• Display abstract

• Receptors belonging to the tumour necrosis factor receptor (TNF-R) superfamily are implicated in a variety of important biological processes. Although messenger ribonucleic acid coding for many of these receptors has been detected in lung, little is known about their expression in this organ. In this study, immunohistochemical techniques were used to evaluate the expression of three receptors of this family (4-1BB, lymphotoxin-beta receptor (LTbeta-R), and Fas) in normal human lung, lung carcinomas and by tuberculous and sarcoid granulomas. The 4-1BB receptor was uniformly expressed by endothelial cells of small vessels and by basal epithelial cells within pseudostratified bronchial epithelium. LTbeta-R expression by parenchymal cells was limited to those of epithelial origin (bronchial and bronchiolar epithelium, hyperplastic alveolar epithelium and lung carcinomas). Fas was present on both fibroblasts and epithelial cells (bronchial and alveolar epithelium and most, but not all, carcinomas), but not on endothelial cells. A subpopulation of T-lymphocytes expressed these receptors, but only Fas was detected on normal alveolar macrophages and epithelioid cells. Thus, 4-1BB, LTbeta-R, and Fas have characteristic and only partially overlapping patterns of expression in the lung. The findings should facilitate further evaluation of their role in lung homeostasis and pathology.

• Loenen WA
• CD27 and (TNFR) relatives in the immune system: their role in health and disease.
• Semin Immunol. 1998; 10: 417-22
• Display abstract

• Relatives of the tumor necrosis factor receptor family are proving of crucial importance for an effective immune response as well as maintenance of homeostasis. The reviews in this issue summarise accumulating evidence for an emerging intricate interplay between these receptors themselves and with the CD28 pathway. It is hypothesised, that the lesser known CD27 receptor may overlap and synergise with CD40 and other relatives and may regulate the T-cell mediated activation cascade and control lymphocyte expansion by facilitating maturation and cell cycle progression. The diagnostic and prognostic value of the occurrence of these receptors and their soluble forms is becoming increasingly apparent, and recent data may herald new strategies for T cell manipulation via combinations of these receptors to ameliorate or prevent immune disease, coronary heart disease, transplant rejection, graft-versus-host disease, viral infections, and to promote tumor eradication.

• Chirinos-Rojas CL, Steward MW, Partidos CD
• A peptidomimetic antagonist of TNF-alpha-mediated cytotoxicity identified from a phage-displayed random peptide library.
• J Immunol. 1998; 161: 5621-6
• Display abstract

• Phage-displayed peptide libraries represent a vast collection of peptide sequences that can be used to identify novel therapeutic molecules. In this report, a 15-mer phage-displayed peptide library was used to identify potential TNF-alpha antagonists. After direct interaction of recombinant human TNF-alpha with the library, four randomly selected phage clones were shown to inhibit in a dose-dependent fashion both mouse and human TNF-alpha-induced cytotoxicity in vitro. DNA sequencing of the positive clones revealed a common amino acid sequence that does not bear any structural similarity to the known primary structures of the extracellular domains of either 55-kDa or 75-kDa TNF receptors. This sequence was synthesized, and the peptidomimotope was shown i) to bind to the recombinant human TNF-alpha using surface plasmon resonance (biosensor) technology and ii) to inhibit both recombinant mouse and human TNF-alpha-induced cytotoxicity in vitro in a dose-dependent fashion. These findings highlight the potential of phage-displayed random peptide libraries for the identification of novel low molecular antagonistic molecules that can block the biologic activities of TNF-alpha.

• Stroka DM et al.
• Expression of a negative dominant mutant of human p55 tumor necrosis factor-receptor inhibits TNF and monocyte-induced activation in porcine aortic endothelial cells.
• Transplant Proc. 1997; 29: 882-882

• Libby P, Mitchell RN
• Cytokines score a knockout. Harnessing gene targeting to gain insight into the pathogenesis of myocarditis.
• Circulation. 1997; 95: 551-2

• Wu GS et al.
• KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene.
• Nat Genet. 1997; 17: 141-3

• Gattorno M, Picco P, Buoncompagni A, Stalla F, Pistoia V
• Tumor necrosis factor receptor p75 correlates better than p55 with disease activity in juvenile rheumatoid arthritis: comment on the article by Mangge et al.
• Arthritis Rheum. 1996; 39: 883-4

• Gruss HJ
• Molecular, structural, and biological characteristics of the tumor necrosis factor ligand superfamily.
• Int J Clin Lab Res. 1996; 26: 143-59
• Display abstract

• The tumor necrosis factor receptor superfamily at present consists of ten different transmembrane (type I) glycoproteins with characteristic limited sequence homology for the cysteine-rich repeats in the extracellular domain. In parallel the tumor necrosis factor ligand super-family has been recognized by discovery of ligands for all members of the receptor superfamily. These molecules are also transmembrane (type II) glycoproteins, with the exception of lymphotoxin-alpha which is the only entirely secreted protein of the tumor necrosis factor-like proteins. Several members of the ligand superfamily, including tumor necrosis factor and CD95L also exist in a biologically active soluble form. The tumor necrosis factor ligand superfamily contains at present ten different proteins. In addition, NGFR p75 binds to a second family of proteins (neurotrophins). These nerve growth factor-like dimeric soluble molecules are basic neurotrophic factors and the five members (NGF, BDNF, NT-3, NT-4, NT-5) are not related to the tumor necrosis factor superfamily ligands. The members of the tumor necrosis factor ligand superfamily (TNF, LT-alpha, LT-beta, CD27L, CD30L, CD40L, CD95L, 4-IBB, OX40L, TRAIL) share common biological activities, but some properties are shared by only some ligands, while others are unique. The diverse biological activities triggered through tumor necrosis factor receptors have been linked to the regulation of cellular activation, including immune responses and inflammatory reactions, but also with the pathology of a series of human diseases.

• Rink L, Kirchner H
• Recent progress in the tumor necrosis factor-alpha field.
• Int Arch Allergy Immunol. 1996; 111: 199-209
• Display abstract

• Tumor necrosis factor (TNF is used synonymously with TNF-alpha) is a pleiotropic cytokine. The effects of TNF were first described in the last century, but the soluble factor was discovered in 1975. Now TNF is one of the most important cytokines investigated by scientists from different fields. This short review briefly summarizes the state of the art and adds the news of the last 2 years. The progress in the field of TNF is focussed on gene organization and transcription, gene polymorphism, biochemistry of TNF, TNF receptors and the biological role of TNF in autoimmune diseases, infectious diseases, transplantation, cancer and TNF functions on endothelial cells.

• Barbara JA, Van ostade X, Lopez A
• Tumour necrosis factor-alpha (TNF-alpha): the good, the bad and potentially very effective.
• Immunol Cell Biol. 1996; 74: 434-43
• Display abstract

• When the tumour necrosis factor-alpha (TNF-alpha) gene was cloned the protein became available for use in clinical trials as an antineoplastic agent. However, side effects have severely limited its application in cancer treatment. Studies on the species specificity of TNF have indicated that the p75 TNF receptor (TNFR75) may play an important role in the generation of these side effects in humans. Using human TNF mutants with selective receptor-binding properties it has been demonstrated in neutrophils and endothelium that TNFR75 is involved in the mediation of the proinflammatory activity of TNF by facilitating the p55 TNF receptor (TNFR55). However, only TNFR55 appears to be involved in mediating TNF cytotoxicity. Therefore the potential exists for the successful reintroduction of TNF-alpha, in the form of TNFR55-selective mutants, into the clinical arena with the promise of reduced side effects.

• Hintzen RQ, van Lier RA
• [Signals for cellular selection for life and death by a new family of cytokines]
• Ned Tijdschr Geneeskd. 1996; 140: 182-6

• Ferran C et al.
• Expression of a truncated form of the human p55 TNF-receptor in bovine aortic endothelial cells renders them resistant to human TNF.
• Transplant Proc. 1996; 28: 618-9

• Hattori A, Hayashi K, Kohno M
• Tumor necrosis factor (TNF) stimulates the production of nerve growth factor in fibroblasts via the 55-kDa type 1 TNF receptor.
• FEBS Lett. 1996; 379: 157-60
• Display abstract

• The role of the two types of tumor necrosis factor (TNF) receptors, TNF-R1 and TNF-R2, in mediating the capacity of TNF to stimulate nerve growth factor (NGF) production in fibroblasts has been investigated. Although Swiss 3T3 cells express both the TNF-R1 and TNF-R2, an agonistic anti-TNF R1 antibody, but not an agonistic anti-TNF-R2 antibody, increases the NGF mRNA level and stimulates the production of NGF protein in the cells. Treatment of the cells with a combination of anti-TNF-R2 antibody and interleukin-1 beta (IL-1 beta) does not increase the NGF production induced by IL-1 beta alone, although TNF's activity to stimulate NGF production is markedly enhanced by IL-1 beta. Furthermore, simultaneous addition of the both antibodies does not increase the NGF production above that observed with the anti-TNF-R1 antibody, indicating that TNF-R1 alone mediates the TNF's activity to stimulate NGF production in fibroblasts.

• Tewari M, Dixit VM
• Recent advances in tumor necrosis factor and CD40 signaling.
• Curr Opin Genet Dev. 1996; 6: 39-44
• Display abstract

• The past year has witnessed remarkable advances in the understanding of signaling by the two TNF (tumor necrosis factor) receptors, TNFR1 and TNFR2, and the related CD40 receptor. Adaptor molecules (termed TRAFs) have been identified that associate with TNFR2 and CD40 and function to modulate the signaling pathways. Significantly, TRAF2 mediates the activation of NF-kappa B by both receptors. Furthermore, a molecule called TRADD has been identified that associates with the cytoplasmic segment of TNFR1. TRADD, which contains a novel 'death domain' that binds to the corresponding death domain in the cytoplasmic segment of TNFR1, can mediate both activation of NF-kappa B and induction of apoptosis, the two major responses signaled by TNFR1.

• Neve R, Kissonerghis M, Clark J, Feldmann M, Chernajovsky Y
• Expression of an efficient small molecular weight tumour necrosis factor/lymphotoxin antagonist.
• Cytokine. 1996; 8: 365-70
• Display abstract

• TNF-alpha and lymphotoxin are proinflammatory cytokines that are non-homologous in sequence, have similar homotrimeric structure and they exert their biological activity by aggregating two types of shared cell surface receptors. The natural inhibitors of TNF and lymphotoxin are the shed extracellular domains of the p55 and p75 TNF receptors. However recombinant inhibitors composed of the extracellular domains of p75 or p55 receptors dimerized on IgG backbone have been shown to be much more effective. We have produced a dimeric form of the human p75 TNF receptor extracellular domain based on the structure of the native soluble shed receptor. The dimer was engineered by genetically linking the monomeric forms with a polyglycine-serine linker. Biochemical characterization showed that this dimeric TNF receptor elutes from a TNF affinity column at a lower pH than the monomeric form. Biological assay revealed this novel antagonist to be as efficient as a dimer based on an immunoglobulin backbone. However this new dimer is smaller, stable, and could have greater penetration into tissues.

• Adams LB, Mason CM, Kolls JK, Scollard D, Krahenbuhl JL, Nelson S
• Exacerbation of acute and chronic murine tuberculosis by administration of a tumor necrosis factor receptor-expressing adenovirus.
• J Infect Dis. 1995; 171: 400-5
• Display abstract

• Tumor necrosis factor (TNF) plays a pivotal role in inflammatory phenomena that culminate in either pathogenesis or resistance in mycobacterial disease. The regulatory role of TNF in murine tuberculosis was examined by administering a recombinant adenovirus encoding a fusion protein consisting of the human 55-kDa TNF receptor extracellular domain and the mouse IgG heavy chain domain (AdTNFR). During acute infections with Mycobacterium tuberculosis, AdTNFR pretreatment induced elevated mycobacterial burdens of 1 log10 in the tissues of H37Ra-infected mice and 2 log10 (spleen and liver) and 4 log10 (lungs) in H37Rv-infected mice. In mice infected chronically with H37Rv, AdTNFR treatment induced a 3-log10 increase of M. tuberculosis in the lungs, in which a tuberculous bronchopneumonia developed with numerous acid-fast bacilli visible in alveoli and bronchi. Administration of AdTNFR may serve as a useful model for studying the pathogenesis and chemotherapy of progressive primary tuberculosis.

• Warzocha K, Bienvenu J, Coiffier B, Salles G
• Mechanisms of action of the tumor necrosis factor and lymphotoxin ligand-receptor system.
• Eur Cytokine Netw. 1995; 6: 83-96
• Display abstract

• In the past few years, a number of experimental observations have provided more insight into the mechanisms of action of tumor necrosis factor (TNF)/lymphotoxin (LT) ligand-receptor system. This system consists of three ligands, TNF, LT alpha (LT alpha) and LT beta (LT beta), and three membrane-associated receptors, p55, p75 and LT beta-receptor (LT beta-R). Like TNF, LT alpha is a secreted protein which in solution forms a homotrimer molecule, with a conformation similar to that of TNF. LT beta is a transmembrane protein that provides the membrane anchor for the attachment to the cell surface of the heteromeric complex of LT alpha and LT beta. This complex retains a structure related to TNF and LT alpha homotrimers, with the homology regions interacting in a heterotypic fashion. The LT alpha 1:LT beta 2 heteromer has been found to be a predominant form of surface LT. The biological effects of TNF and LT alpha homotrimers are mediated by p55 and p75 receptors, while the heteromeric complex of LT alpha/LT beta transduces its cellular signal via LT beta-R. Membrane-associated receptor affinities as well as final biological effects of TNF/LT can be modulated by the influence of naturally occurring soluble receptors, derived from the cell surface by proteolytic cleavage. The multimerization of receptor cytoplasmic domains upon TNF/LT ligation is postulated to activate the intracellular signal-transduction pathways. One of them is the activation of phospholipase A2 (PL-A2) resulting in the production of arachidonic acid (AA) and other metabolites, including leukotriens, phosphatidycholine-specific phospholipase C (PC-PLC) with subsequent production of diacylglycerol (DAG) and activation of protein kinase C (PKC). As a third signaling pathway, TNF/LT employ the sphingomyelinase (SMase)-mediated hydrolysis of membrane sphingomyelin (SM) to ceramide. The final link in the TNF/LT signaling is activation of nuclear transcription factors, such as NF-kappa B, AP-1, interferon regulatory factors-1 and -2 (IRF-1, IRF-2), and NF-GMa. Since induction of AP-1, IRF-1 and IRF-2 as well as NF-GMa proceeds through translational event, the posttranslational TNF/LT-driven activation of NF-kappa B remains the only cellular event identified so far that serves as a direct target in their signaling cascade.

• Schuchmann M et al.
• Functional discrepancies between tumor necrosis factor and lymphotoxin alpha explained by trimer stability and distinct receptor interactions.
• Eur J Immunol. 1995; 25: 2183-9
• Display abstract

• Tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) are closely related cytokines which bind with nearly identical affinities to the same pair of cell surface receptors, p55 and p75TNFR. Therefore it is assumed that TNF and LT alpha are redundant cytokines. This study, however, demonstrates that TNF and LT alpha differ significantly with regard to their mitogenic and cytotoxic potentials. LT alpha's superior mitogenic effect could be explained by its formation of a more stable trimer. In contrast to the TNF trimer, which disintegrated under physiological conditions into biologically inactive monomers, the LT alpha trimer remained stable for several days. Accordingly, LT alpha more effectively induced fibroblast growth which demands long-term presence of the cytokine. TNF's superior cytotoxicity, which requires only short-term impact of the cytokine, could be attributed to a distinct interaction with the human p55TNFR. This was demonstrated in NIH 3T3 cells transfected with the human p55TNFR, where cytotoxicity is mediated exclusively by the transfected receptor. Although the p55ATNFR had virtually identical affinities for TNF and LT alpha, as defined by Scatchard analysis, it nevertheless discriminated between binding of each cytokine and showed a 200-fold enhanced cytotoxicity mediated by TNF.

• Song HY, Dunbar JD, Zhang YX, Guo D, Donner DB
• Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor.
• J Biol Chem. 1995; 270: 3574-81
• Display abstract

• The yeast-based two hybrid has been used to identify a novel protein that binds to the intracellular domain of the type 1 receptor for tumor necrosis factor (TNFR-1IC). The TNF receptor-associated protein, TRAP-1, shows strong homology to members of the 90-kDa family of heat shock proteins. After in vitro transcription/translation and 35S labeling, TRAP-1 was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, showing that the two proteins directly interact. The ability of deletion mutants of TNFR-1 to interact with TRAP-1 was tested using the two hybrid system. This showed that the amino acid sequences that mediate binding are diffusely distributed outside of the domain in the C terminus of TNFR-1IC that signals cytotoxicity. The 2.4-kilobase TRAP-1 mRNA was variably expressed in skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta. TRAP-1 mRNA was also detected in each of eight different transformed cell lines. Identification of TRAP-1 may be an important step toward defining how TNFR-1, which does not contain protein tyrosine kinase activity, transmits its message to signal transduction pathways.

• Suter B, Pauli U
• Cloning of the cDNA encoding the porcine p55 tumor necrosis factor receptor.
• Gene. 1995; 163: 263-6
• Display abstract

• We have utilized RT-PCR to clone the porcine p55TNFR cDNA, encoding the 55-kDa tumor necrosis factor receptor (TNFR), encompassing the entire coding region and most of the 3' untranslated region. PCR was performed using total cellular RNA of porcine kidney cell line 15 [PK(15)] and primers for the human p55TNFR. Since the length of the entire fragment was over 2000 bp, we fused two amplified subfragments with the help of a restriction endonuclease. The entire fragment was cloned and its amino acid (aa) sequence was compared to the human, rat and mouse p55TNFR. This comparison revealed identities of 79, 71 and 72%, respectively. The highest identities of 90, 80 and 85% were detected in the so called "death domain" for the human, rat and mouse sequences, respectively. This domain is crucial for the cytotoxic signal transduction of p55TNFR.

• Winston BW, Riches DW
• Activation of p42mapk/erk2 following engagement of tumor necrosis factor receptor CD120a (p55) in mouse macrophages.
• J Immunol. 1995; 155: 1525-33
• Display abstract

• Cross-linking of the dichotomous cell surface receptors for TNF-alpha (CD120a (p55) and CD120b (p75)) induces the activation of a variety of macrophage responses that mediate the role of this cell in inflammation and host defense. Although significant progress has been made in understanding the role that lipid second messengers play in mediating the action(s) of this multifunctional cytokine, less is known about the role of specific kinases in TNF-alpha-initiated signaling. We show that exposure of mouse macrophages to TNF-alpha stimulates a rapid and transient tyrosine phosphorylation and activation of p42mapk/erk2. By contrast, p44mapk/erk1 was found to be constitutively phosphorylated, with minimal further response to TNF-alpha. To investigate the use of CD120a (p55) and CD120b (p75) in the activation of p42mapk/erk2 in mouse macrophages, we determined the effects of blocking Ab on the activation of p42mapk/erk2 in response to TNF-alpha. In addition, we independently cross-linked each receptor with specific agonistic Ab, which have previously been shown to mimic the effects of TNF-alpha. Collectively, the results from these experiments indicate that cross-linking of CD120a (p55), but not that of CD120b (p75), was both necessary and sufficient for the activation of p42mapk/erk2 in mouse macrophages.

• Adam D, Kessler U, Kronke M
• Cross-linking of the p55 tumor necrosis factor receptor cytoplasmic domain by a dimeric ligand induces nuclear factor-kappa B and mediates cell death.
• J Biol Chem. 1995; 270: 17482-7
• Display abstract

• We have fused the cytoplasmic domain of the p55 tumor necrosis factor (TNF) receptor to the extracellular and transmembrane domain of the mouse platelet-derived growth factor (PDGF) receptor. Mouse mammary gland epithelial (NMuMG) cells were stably transfected with the PDGFR-TR55 chimeric receptor. These cells lack endogenous PDGF receptor expression and do not respond to PDGF. In the PDGFR-TR55 transfectants, PDGF elicited a cytotoxic response, which is indistinguishable from that induced by the wild type p55 TNF receptor. In addition, PDGF-induced activation of the PDGFR-TR55 chimeric receptor resulted in nuclear translocation of NF-kappa B. The data presented suggest that cross-linking of the p55 TNF receptor cytoplasmic domain by a dimeric ligand such as PDGF is sufficient to generate cellular responses that do not differ from those observed with the trimeric ligand TNF.

• Braxton CC et al.
• Parenteral nutrition alters monocyte TNF receptor activity.
• J Surg Res. 1995; 59: 23-8
• Display abstract

• The route of nutrient provision has been reported to influence some aspects of the host inflammatory response in both patient populations and normal subjects. The tumor necrosis factor receptor system is a complex regulatory mechanism that modulates the bioavailability of tumor necrosis factor (TNF). We sought to determine whether maintenance on total parenteral nutrition (TPN) can alter host response to endotoxin challenge, specifically as it relates to the TNF receptor system. Seventeen healthy men were randomized to receive either TPN (n = 8) or a defined formula enteral diet (ENT, n = 9) prior to intravenous infusion of endotoxin (Lot EC-5, 20 U/kg). The subjects that received 1 week of antecedent TPN exhibited an increased heart rate and temperature and decreased mean arterial pressure post-LPS compared to those subjects maintained on enteral nutritional support. The TPN subjects also exhibited comparatively higher TNF and interleukin-6 levels in response to endotoxin. Monocyte TNF receptor levels decreased in both groups post-LPS, but TPN subjects exhibited consistently greater expression of this functional membrane-associated TNF receptor. After LPS, soluble tumor necrosis factor receptor II (sTNFr II, p75) peaked three times higher in TPN subjects than in ENT subjects. Conversely, sTNFr I (p55) was higher in the enterally fed group. From these studies it appears that antecedent TPN not only influences clinical manifestations of endotoxin but also modulates the regulation of all associated TNF receptors and shedding of soluble receptors.

• Abe Y, Yamauchi K, Kimura S
• 75- but not 55-kDa tumor necrosis factor receptor is active in the homotypic aggregation and proliferation of human lymphokine-activated T killer (T-LAK) cells in vitro.
• J Leukoc Biol. 1995; 57: 462-8
• Display abstract

• Lymphotoxin (LT) and tumor necrosis factor (TNF) play important roles in the maturation and growth of human lymphokine-activated T killer (T-LAK) cells in vitro. The role of 55- and 75-kDa TNF receptor (TNF-R) in the aggregation and proliferation of T-LAK cells was investigated using agonistic anti-TNF-R rabbit polyclonal antibodies. Human peripheral blood T cells and T-LAK cells predominantly express 75-kDa TNF-R. The proliferation of T-LAK cells during the generation phase is supported by innate LT and TNF. In this phase, the proliferation was upregulated by anti-75- but not 55-kDa TNF-R antibody. Homotypic aggregation of T-LAK cells was induced by LT, TNF, and anti-75- but not by anti-55-kDa TNF-R antibody. The increase of homotypic aggregation was accompanied by up-regulation of intercellular adhesion molecule 1 but not lymphocyte function-associated antigen 1 expression on cells and by an elevation of membrane fluidity, both of which were up-regulated by anti-75- but not 55-kDa TNF-R antibody. Interestingly, LT and TNF suppressed the proliferation of mature T-LAK cells. Anti-75- but not 55-kDa TNF-R antibody suppressed the proliferation, mimicking LT and TNF. These findings indicated that 75- but not 55-kDa TNF-R is biologically active in the modulation of aggregation and proliferation of human T-LAK cells in vitro.

• Boldin MP et al.
• Self-association of the "death domains" of the p55 tumor necrosis factor (TNF) receptor and Fas/APO1 prompts signaling for TNF and Fas/APO1 effects.
• J Biol Chem. 1995; 270: 387-91
• Display abstract

• Signaling by the p55 tumor necrosis factor (TNF) receptor and by the structurally related receptor Fas/APO1 is initiated by receptor clustering. Data presented here and in other recent studies (Wallach, D., Boldin, M., Varfolomeev, E. E., Bigda, Y., Camonis, H.J. and Mett, I. (1994) Cytokine 6, 556; Song, H.Y., Dunbar, J.D., and Bonner, D.B. (1994) J. Biol. Chem. 269, 22492-22495) indicate that part of that region within the intracellular domains of the two receptors that is involved in signaling for cell death, as well as for some other effects (the "death domain", specifically self-associates. We demonstrate also the expected functional consequence of this association; a mere increase in p55 TNF receptor expression, or the expression just of its intracellular domain, is shown to trigger signaling for cytotoxicity as well as for interleukin 8 gene induction, while expression of the intracellular domain of Fas/APO1 potentiates the cytotoxicity of co-expressed p55 TNF receptor. These findings indicate that the p55 TNF and Fas/APO1 receptors play active roles in their own clustering and suggest the existence of cellular mechanisms that restrict the self-association of these receptors, thus preventing constitutive signaling.

• Bazzoni F, Alejos E, Beutler B
• Chimeric tumor necrosis factor receptors with constitutive signaling activity.
• Proc Natl Acad Sci U S A. 1995; 92: 5376-80
• Display abstract

• Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

• Hess S, Kurrle R, Lauffer L, Riethmuller G, Engelmann H
• A cytotoxic CD40/p55 tumor necrosis factor receptor hybrid detects CD40 ligand on herpesvirus saimiri-transformed T cells.
• Eur J Immunol. 1995; 25: 80-6
• Display abstract

• The B cell activation molecule CD40 and the p55 tumor necrosis factor receptor (p55TNFR) belong to the same family of structurally conserved proteins. We constructed a chimeric receptor consisting of the CD40 extracellular and transmembrane domains and the p55TNFR intracellular domain. This receptor hybrid retained the biological activity and the ligand specificity of the respective wild-type receptor domains. Thus it exerted a marked cytotoxic effect in three different transfected cell lines after activation not only with anti-CD40 antibody but also with CD40 ligand (CD40L) in soluble and membrane-bound forms. Using hybrid-transfected baby hamster kidney cells we demonstrated that herpesvirus saimiri-transformed human CD4+ T lymphocytes constitutively express bioactive CD40 ligand on their surface. The hybrid receptor-based assay was highly specific for CD40 activating reagents and more sensitive than an assay measuring CD40-mediated B cell rescue from apoptosis. Hence CD40/p55TNFR transfectants may be useful for dissecting CD40L-mediated events in T-B cell interactions, and also to detect a defective CD40L molecule in putative hyper-IgM syndrome patients.

• Heldin CH
• Dimerization of cell surface receptors in signal transduction.
• Cell. 1995; 80: 213-23

• Ward CW, Hoyne PA, Flegg RH
• Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor.
• Proteins. 1995; 22: 141-53
• Display abstract

• The insulin receptor (INSR) and epidermal growth factor receptor (EGFR) are representatives of two structurally related subfamilies of tyrosine kinase receptors. Using the Wisconsin GCG sequence analysis programs, we have demonstrated that the cysteine-rich regions of INSR and EGFR conform to the structural motif found in the tumor necrosis factor receptor (TNFR) family. The study also revealed that these regions were not composed of simple repeats of eight cysteine residues as previously proposed and that the second Cys-rich region of EGFR contained one fewer TNFR repeat than the first. The sequence alignments identified two cysteine residues in INSR that could be responsible for the additional disulfide bonds known to be involved in dimer formation. The published data on the alignments for the fibronectin type III repeat region of the INSR together with previous cysteine mutagenesis studies indicated that there were two disulfide bonds linking the alpha and beta chains of the INSR, but only one alpha-beta linkage in the insulin-like growth factor 1 receptor (IG1R). Database searches and sequence alignments showed that the TNFR motif is also found in the cysteine-rich repeats of laminins and the noncatalytic domains of furin-like proteases. If the starting position of the repeat is altered the characteristic laminin repeat of eight cysteine residues can be shown to consist of a TNFR-like motif fused to the last half of an EGF-like repeat. The overlapping regions of these two motifs are known to have identical disulfide bonding patterns and similar protein folds.

• Grell M et al.
• The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor.
• Cell. 1995; 83: 793-802
• Display abstract

• The 60 kDa tumor necrosis factor receptor (TNFR60) is regarded as the major signal transducer of TNF-induced cellular responses, whereas the signal capacity and role of the 80 kDa TNFR (TNFR80) remain largely undefined. We show here that the transmembrane form of TNF is superior to soluble TNF in activating TNFR80 in various systems such as T cell activation, thymocyte proliferation, and granulocyte/macrophage colony-stimulating factor production. Intriguingly, activation of TNFR80 by membrane TNF can lead to qualitatively different TNF responses such as rendering resistant tumor cells sensitive to TNF-mediated cytotoxicity. This study demonstrates that the diversity of TNF effects can be controlled through the differential sensitivity of TNFR80 for the two forms of TNF and suggests an important physiological role for TNFR80 in local inflammatory responses.

• Rydberg J, Miorner H, Chandramuki A, Lantz M
• Assessment of a possible imbalance between tumor necrosis factor (TNF) and soluble TNF receptor forms in tuberculous infection of the central nervous system.
• J Infect Dis. 1995; 172: 301-4
• Display abstract

• Distributions of tumor necrosis factor (TNF) and its soluble receptor forms, R55-BP and R75-BP, were analyzed in the cerebrospinal fluid of patients with severe acute or chronic central nervous system infections. Tuberculous infections were associated with high ratios of R55-BP and R75-BP to TNF, 27.2 and 28.0, respectively, suggesting a small biologically active fraction of TNF. The opposite was found in subjects with acute bacterial meningitis. They had large fractions of biologically active TNF and thus low ratios of R55-BP and R75-BP to TNF, 3.7 and 4.0, respectively. It is hypothesized that chronic infectious diseases, such as tuberculous infections, may be associated with inadequate production of TNF and a concomitant relative increase of soluble TNF receptors, which may prolong the disease.

• Schumacher M et al.
• Dependence of soluble tumor necrosis factor receptor 1 and 2 values on the severity and duration of ischemic heart disease.
• Circulation. 1995; 92: 3362-3

• Beutler B, van Huffel C
• Unraveling function in the TNF ligand and receptor families.
• Science. 1994; 264: 667-8

• Baum PR et al.
• Identification of OX40 ligand and preliminary characterization of its activities on OX40 receptor.
• Circ Shock. 1994; 44: 30-4

• Cosman D
• A family of ligands for the TNF receptor superfamily.
• Stem Cells. 1994; 12: 440-55
• Display abstract

• Recent progress in the definition of molecules involved in immune regulation has led to the discovery of a number of type I membrane glycoproteins with a distinctive, cysteine-rich, repetitive domain structure within their extracellular regions. Because the prototype members of this family are receptors for cytokines (tumor necrosis factor [TNF] and nerve growth factor [NGF]), it was expected that the ligands for the other receptors would possess cytokine-like activities. This prediction has been fulfilled by the cloning of cDNA encoding a series of type II membrane glycoproteins, with homology to TNF, that bind to, and signal through, their cognate receptors. While the biological role of some of these ligand-receptor pairs remains obscure, at least two members of the family, CD40 and Fas, have proven their importance. The human X-linked immunodeficiency, hyper IgM syndrome, is the result of mutations in the CD40 ligand gene, and the Fas and Fas ligand genes are mutated in two mouse strains, lpr and gld, that develop autoimmune disease. These findings, together with other evidence, point to key roles of CD40/CD40 ligand interactions in immune activation, particularly in T-dependent B cell responses, and of Fas/Fas ligand in apoptosis and peripheral tolerance. These molecules, as well as the other ligands of the family, share the property of costimulation of T cell proliferation and are all expressed by activated T cells. More detailed analysis of the expression patterns of ligands and receptors on lymphocyte subpopulations will be necessary to define their different roles in immune activation and suppression.

• Baum PR et al.
• Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34.
• EMBO J. 1994; 13: 3992-4001
• Display abstract

• A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.

• Theze J
• Cytokine receptors: a combinative family of molecules.
• Eur Cytokine Netw. 1994; 5: 353-68

• Powell EE, Wicker LS, Peterson LB, Todd JA
• Allelic variation of the type 2 tumor necrosis factor receptor gene.
• Mamm Genome. 1994; 5: 726-7

• Rodseth LE et al.
• Two crystal forms of the extracellular domain of type I tumor necrosis factor receptor.
• J Mol Biol. 1994; 239: 332-5
• Display abstract

• The soluble extracellular domain of human type I tumor necrosis factor receptor (sTNFrI) is a 161 residue polypeptide found in serum and urine. This domain tightly binds tumor necrosis factors (TNF) alpha and beta and, as part of the whole receptor, initiates the powerful biological effects of TNF. The extracellular domain, typical of other TNF receptor superfamily members, comprises four cysteine-rich motifs. We have obtained two crystal forms of the sTNFrI. One crystal form is grown at pH 3.7 with MgSO4 as the precipitant. These crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 78.5 A, b = 85.5 A and c = 67.5 A. A data set to 2.0 resolution has been collected for these crystals. Tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions a = 69.0 A and c = 185.5 A are obtained using methylpentanediol as precipitant at pH 8.5. Data to 2.8 A have been measured from these crystals. It appears that both unit cells may contain two molecules in the asymmetric unit. These crystal structures of sTNFrI may reveal possible conformational differences between receptor localized on the cell surface (high pH), the receptor in the endosomal compartments (low pH) and the receptor in a complex with tumor necrosis factor beta. An accurate structure of the receptor and an understanding of its mechanism will provide a basis for rational drug design.

• Rabizadeh S, Bredesen DE
• Is p75NGFR involved in developmental neural cell death?
• Dev Neurosci. 1994; 16: 207-11
• Display abstract

• The tumor necrosis factor receptor superfamily includes twelve members, at least two of which--tumor necrosis factor receptor I and FAS/Apo-1--induce cell death following ligand binding. This review summarizes data suggesting that two other members of the family--p75NGFR and CD40--achieve a similar effect in the inverse fashion; they induce apoptosis constitutively when unbound by their respective ligands, with the induction of apoptosis being inhibited by the binding of their respective ligands. The potential roles that such receptors may play in development and pathological processes are discussed.

• Smith CA, Farrah T, Goodwin RG
• The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death.
• Cell. 1994; 76: 959-62

• Kuhnert P, Kemper O, Wallach D
• Cloning, sequencing and partial functional characterization of the 5' region of the human p75 tumor necrosis factor receptor-encoding gene (TNF-R).
• Gene. 1994; 150: 381-6
• Display abstract

• A 1887-bp region at the 5' flank of the human p75 tumor necrosis factor receptor (p75 TNF-R)-encoding gene was found to be active in driving expression of the luc (luciferase-encoding) reporter gene, suggesting that it contains the promoter for the receptor. Rather unexpectedly, a 1827-bp region at the 3' end of the first intron of the p75 TNF-R gene also displayed promoter activity. This activity may be artefactual, reflecting only the presence of an enhancer in this region; yet it also raises the possibility that p75 TNF-R is controlled by more than one promoter and that it encodes various forms of the receptor, or even other proteins. We present here the nucleotide sequences of the 5' flanking and intron regions. Possible implications for the transcriptional regulation of the p75 TNF-R gene are discussed.

• Mancini I, Bosseloir A, Hooghe-Peters E, Heinen E, Simar L
• Follicular dendritic cells do not produce TNF-alpha nor its receptor.
• Adv Exp Med Biol. 1993; 329: 431-6

• Slowik MR, De Luca LG, Fiers W, Pober JS
• Tumor necrosis factor activates human endothelial cells through the p55 tumor necrosis factor receptor but the p75 receptor contributes to activation at low tumor necrosis factor concentration.
• Am J Pathol. 1993; 143: 1724-30
• Display abstract

• Tumor necrosis factor-alpha (TNF-alpha) interacts with two distinct membrane receptor proteins, p55 and p75, which are variably expressed on different cell types. We have examined the function of p55 and p75 on human endothelial cells (EC). Both receptor types are detected on cultured EC by FACS analysis. A mutagenized recombinant human TNF (R32W-TNF), which binds selectively to p55, is equipotent with human recombinant wild-type TNF (wt-TNF) in upregulating several different leukocyte adhesion molecules as well as class I major histocompatibility complex molecules. R32W-TNF also fully desensitizes EC to wt-TNF, as assessed by inhibition of re-induction of endothelial leukocyte adhesion molecule-1 (ELAM-1). At low wt-TNF concentrations, induction of ELAM-1 is partly inhibited by blocking monoclonal antibodies to either p55 or p75 and to a greater extent by a combination of both monoclonal antibodies. In contrast, ELAM-1 induction by R32W-TNF is only inhibited by anti-p55. We conclude that both TNF receptors (p55 and p75) can contribute to TNF-induced activation of EC, but that signaling through p55 is sufficient.

• Kalthoff H, Roeder C, Brockhaus M, Thiele HG, Schmiegel W
• Tumor necrosis factor (TNF) up-regulates the expression of p75 but not p55 TNF receptors, and both receptors mediate, independently of each other, up-regulation of transforming growth factor alpha and epidermal growth factor receptor mRNA.
• J Biol Chem. 1993; 268: 2762-6
• Display abstract

• The expression and cytokine-mediated regulation of the two different receptors for tumor necrosis factor, TNF-R-75 and TNF-R-55, was investigated in human malignant epithelial cell lines. Here we show that cells treated with TNF-alpha up-regulate the TNF-R-75 mRNA and protein levels. No changes were seen regarding the level of TNF-R-55 transcripts. Phospholipase and protein kinase C inhibitors abrogated the signal transduction pathway of TNF-mediated TNF-R-75 mRNA up-regulation which proceeded in the absence of transcriptional activation. This process was also elicited by an agonistic antibody binding specifically to TNF-R-55. Ligand binding assays using specific inhibitory antibodies showed a marked shift in active binding sites from p55 to p75 without significant changes in the total binding for TNF-alpha after up-regulation of p75 TNF-R. This ligand-induced regulation of one of the corresponding receptors has so far only been detected in malignant epithelial cells and not in hematopoietic cell lines. In our search for a specific function we were able to show that p75 is the specific receptor for TNF-mediated up-regulation of transforming growth factor alpha mRNA, whereas p55 is the signal transducer for TNF-induced up-regulation of epidermal growth factor receptor mRNA. This is the first demonstration of an exclusive function of TNF-R-75 in cells of epithelial origin.

• Egido J et al.
• Role of tumor necrosis factor-alpha in the pathogenesis of glomerular diseases.
• Kidney Int Suppl. 1993; 39: 5964-5964

• Suda T, Takahashi T, Golstein P, Nagata S
• Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family.
• Cell. 1993; 75: 1169-78
• Display abstract

• The Fas antigen (Fas) belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family, and it mediates apoptosis. Using a soluble form of mouse Fas, prepared by fusion with human immunoglobulin Fc, Fas ligand was detected on the cell surface of a cytotoxic T cell hybridoma, PC60-d10S. A cell population that highly expresses Fas ligand was sorted using a fluorescence-activated cell sorter, and its cDNA was isolated from the sorted cells by expression cloning. The amino acid sequence indicated that Fas ligand is a type II transmembrane protein that belongs to the TNF family. The recombinant Fas ligand expressed in COS cells induced apoptosis in Fas-expressing target cells. Northern hybridization revealed that Fas ligand is expressed in activated splenocytes and thymocytes, consistent with its involvement in T cell-mediated cytotoxicity and in several nonlymphoid tissues, such as testis.

• Naylor MS, Stamp GW, Foulkes WD, Eccles D, Balkwill FR
• Tumor necrosis factor and its receptors in human ovarian cancer. Potential role in disease progression.
• J Clin Invest. 1993; 91: 2194-206
• Display abstract

• The gene for tumor necrosis factor, TNF, was expressed in 45 out of 63 biopsies of human epithelial ovarian cancer. In serous tumors, there was a positive correlation between level of TNF expression and tumor grade. TNF mRNA was found in epithelial tumor cells and infiltrating macrophages, whereas TNF protein localized primarily to a subpopulation of macrophages within and in close proximity to tumor areas. mRNA and protein for the p55 TNF receptor gene localized to the tumor epithelium and tumor, but not to stromal macrophages. The p75 TNF receptor was confined to infiltrating cells. Cells expressing TNF mRNA were also found in ovarian cancer ascites and TNF protein was detected in some ascitic fluids. In 2 out of 12 biopsies of normal ovary, TNF mRNA was detected in a minority of cells in the thecal layer of the corpus luteum. Serum levels of TNF and its soluble receptor did not correlate with extent of TNF expression in matched biopsies. Northern and Southern analysis revealed no gross abnormality of the TNF gene. The coexpression of TNF and its receptor in ovarian cancer biopsies suggests the capacity for autocrine/paracrine action. TNF antagonists may have therapeutic potential in this malignancy.

• Morimoto H, Bonavida B
• [TNF and TNF receptors: structure, mechanism of action, role in disease and therapy]
• Nippon Rinsho. 1993; 51: 1885-90
• Display abstract

• In this article, the topics on TNF and TNF receptors presented in the 4th International Conference on Tumor Necrosis Factor and Related Cytokines (Netherlands, May, 1992) are briefly summarized in Japanese, based on Dr. Benjamin Bonavida's report (Cancer Update, supplement 8, 1992) with permission. Following categorized themes were discussed in the meeting: 1) regulation of TNF synthesis, especially at the transcriptional level, 2) TNF receptors, including type I (55 kd), type II (75 kd) and soluble forms, 3) TNF cytotoxicity and immunomodulation with considerations for mechanisms, 4) TNF and infectious diseases such as Plasmodium falciparum infection, 5) TNF and inflammatory diseases such as rheumatoid arthritis and 6) TNF and cancer, in attempts to overcome drug resistance and TNF toxicity.

• Takeda K, Iwamoto S, Takeda M
• Roles of two tumor necrosis factor receptors in induction of differentiation of ML-1 cells.
• Anticancer Res. 1993; 13: 883-6
• Display abstract

• The individual roles of two types of TNF receptors (55 kDa and 75 kDa) in induction of differentiation of human myeloblastic leukemia ML-1 cells were investigated using three monoclonal antibodies. The antibody htr-9, which recognizes the 55 kDa receptor, induced differentiation of ML-1 cells. Utr-1, which recognizes the 75 kDa receptor, blocked 125I-TNF binding by about 80% and inhibited by about 80% the TNF-induced NBT reducing activity. Htr-5 recognizes the 55 kDa receptor, blocked 125I-TNF binding by about 20% and inhibited by about 60% the TNF-induced NBT reducing activity. The data suggest that either of the two TNF receptors alone can mediate signals for the differentiation of ML-1 cells, and that simultaneous stimulation of both receptors will induce differentiation more effectively.

• Kemper O, Wallach D
• Cloning and partial characterization of the promoter for the human p55 tumor necrosis factor (TNF) receptor.
• Gene. 1993; 134: 209-16
• Display abstract

• An 809-bp region at the 5' flank of the human p55 tumor necrosis factor receptor (TNF-R)-encoding gene was found to be active in driving expression of the cat reporter gene, indicating that it contains a functional promoter. Deletion analysis showed that the promoter activity is present in the region between nucleotides (nt) -385 and -207; the sequence upstream from this region (nt -809 to -385) has an inhibitory effect. The promoter for the p55 TNF-R resembles housekeeping-type promoters in that it drives transcription from multiple start points (tsp) and lacks canonical TATA and CAAT box motifs. The cluster of tsp lies in a region which is particularly C+T rich. In this region, upstream from and near to the furthest upstream tsp, two closely located TCC repeat motifs were identified. These motifs also appear twice in the promoter for the epidermal growth factor receptor, where they were shown to be essential for promoter activity. The human p55 TNF-R promoter shows an overall resemblance, yet also some marked dissimilarities, to the recently described promoter for the mouse p55 TNF-R.

• Tartaglia LA, Pennica D, Goeddel DV
• Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor.
• J Biol Chem. 1993; 268: 18542-8
• Display abstract

• To understand the role of the 75-kDa tumor necrosis factor (TNF) receptor in non-lymphoid cells, the cytotoxic signaling and ligand binding activities of the 55-kDa (TNF-R1) and 75-kDa (TNF-R2) TNF receptors were investigated using agonist and antagonist antibodies specific for the two receptor types. This study indicates that although TNF-R2 can significantly reduce the TNF concentration required for cell killing, the mechanism by which this is accomplished is not through the generation of an intracellular signal by TNF-R2. Instead, TNF-R2 regulates the rate of TNF association with TNF-R1, possibly by increasing the local concentration of TNF at the cell surface through rapid ligand association and dissociation. We propose that other cell-surface receptors, such as the low affinity p75 nerve growth factor receptor, may utilize an analogous "ligand passing" mechanism.

• Grell M, Scheurich P, Meager A, Pfizenmaier K
• TR60 and TR80 tumor necrosis factor (TNF)-receptors can independently mediate cytolysis.
• Lymphokine Cytokine Res. 1993; 12: 143-8
• Display abstract

• The human rhabdomyosarcoma cell line KYM-1 coexpresses high numbers of both tumor necrosis factor-alpha (TNF) receptors, TR60 and TR80, and is highly sensitive to TNF-induced cytotoxicity. We show here that each receptor type can mediate cytotoxicity on its own on selective stimulation. This was achieved using receptor specific antibodies, able to compete with ligand binding to the respective receptor molecules. Thus, the TR60-specific monoclonal antibody H398 was strongly agonistic, i.e., cytotoxic, in the presence of a secondary cross-linking immunoglobulin. Selective stimulation of TR80 by antibody-mediated receptor crosslinking also induced strong cytotoxicity in KYM-1 cells. Interestingly, when both receptor subsets were stimulated in parallel by limited receptor cross-linking, additive cytolytic effects were observed. In each case the respective Fab fragments showed no agonistic activity. When the natural ligand TNF was used to induce cytolysis, blocking studies with receptor specific Fab fragments indicated that the main cytotoxic effect of TNF is mediated via TR60, although these receptors represent only about 8% of the total TNF receptor number.

• Trefzer U et al.
• The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-alpha and by ultraviolet B radiation.
• J Clin Invest. 1993; 92: 462-70
• Display abstract

• In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha.

• Tchorzewski H et al.
• The effects of tumor necrosis factor (TNF) derivatives on TNF receptors.
• Cytokine. 1993; 5: 125-32
• Display abstract

• The pleiotropic cytokine TNF has been implicated in the regulation of many immune and inflammatory responses in vivo, and in addition exerts a wide range of effects on target cells in vitro. However, although two cell surface receptors for TNF have been identified, and their cDNAs cloned, the amino acid residues necessary for the biological activity of TNF have not been characterized. We have therefore constructed derivatives of TNF termed 'muteins', in which the first 3 to 7 amino acids of native TNF-alpha have been replaced, using synthetic cDNA expressed in E. coli. In the present study we compare the effects of native TNF-alpha and muteins III, IV, V and VI in several different in vitro systems and in one in vivo model. We observed binding to the p75 TNF receptor on Jijoye Burkitt lymphoma cells with native TNF-alpha and mutein III alone, whereas the p55 TNF receptor on the human epithelioid carcinoma cell line HeLa bound TNF-alpha, mutein III and mutein V. Muteins IV and VI failed to recognize either TNF receptor. WEHI 164 fibrosarcoma cells were killed by muteins III, V and VI. Human umbilical vein endothelial cells responded to native TNF-alpha and to muteins III, IV and V, but not to mutein VI, by increasing the surface expression of ICAM-1 antigen and secretion of the cytokines GM-CSF and IL-6. All four compounds were pro-inflammatory in a mouse in vivo model. The results presented in this report confirm that N-terminal amino acids are critical for both receptor binding and biological activity of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

• Pfeffer K et al.
• Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.
• Cell. 1993; 73: 457-67
• Display abstract

• The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules. Thymocyte development and lymphocyte populations are unaltered, and clonal deletion of potentially self-reactive T cells is not impaired. However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the host's defense against microorganisms and their pathogenic factors.

• Winzen R, Wallach D, Kemper O, Resch K, Holtmann H
• Selective up-regulation of the 75-kDa tumor necrosis factor (TNF) receptor and its mRNA by TNF and IL-1.
• J Immunol. 1993; 150: 4346-53
• Display abstract

• In cells of the fibroblastoid line SV-80, rapid down-modulation of TNF binding in response to TNF itself, or to IL-1, was followed by a gradual recovery of binding, which occurred even in the continuous presence of the cytokines. Untreated cells carried mainly the 55-kDa receptor species. In cells treated with TNF or IL-1, the 55-kDa TNF-R, although increasing after initial down-modulation, remained lower than before treatment. Expression of the 75-kDa TNF-R species upon treatment with either cytokine was markedly increased. Both TNF and IL-1 also induced a strong increase of the mRNA for the 75-kDa receptors, whereas the amount of mRNA for the 55-kDa receptors decreased. The effects of TNF on cell surface expression of the TNF-R could not be blocked with antibodies to IL-1, nor could the effects of IL-1 be blocked with antibodies to TNF, indicating that each cytokine affects the cell surface expression of the receptors independently of the other. Applying both cytokines together resulted in much stronger increase in expression of the 75-kDa TNF-R than applying each alone. Similar changes in cell surface expression and mRNA levels of the two TNF-R as observed in SV-80 cells were also found in TNF and IL-1-treated human foreskin fibroblasts. It is suggested that these sustained changes in the pattern of receptor expression contribute to the adjustment of the cellular response to TNF when formation of TNF and IL-1 takes place over a prolonged period.

• Leithauser F et al.
• Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells.
• Lab Invest. 1993; 69: 415-29
• Display abstract

• BACKGROUND: APO-1 is a 48 kilodalton transmembrane, cysteine-rich glycoprotein identical with the Fas antigen which belongs to the nerve growth factor/tumor necrosis factor receptor superfamily. Cross-linking of APO-1 induces apoptotic cell death in sensitive cells. EXPERIMENTAL DESIGN: As suggested by our preliminary results, APO-1 expression is not restricted to cells of the hematopoietic lineage. We therefore investigated APO-1 expression in normal human tissues and in various epithelial and nonepithelial tumors. RESULTS: We show by immunohistochemistry that APO-1 is a non-lineage antigen constitutively expressed in a variety of epithelial cells. This includes the basal layers of various squamous epithelia, transitional epithelium and columnar epithelium of the biliary tract and intestine. Among the epithelial cell types of the reproductive system of both genders, APO-1 expression is complex. Except the satellite cells of autonomic ganglia, all cells of the nervous tissue are APO-1-negative. Among mesenchymal cells, constitutive APO-1 expression is rare but detectable in various kinds of activated cells, e.g. fibroblasts, osteoblasts, and subpopulations of endothelial cells. Within the immune system, APO-1 is broadly distributed among histiocytic cells but restricted to minor subpopulations of peripheral T and B cells. Immature T cells, i.e., thymocytes, do not express detectable APO-1-antigen. Expression of APO-1 was induced in phytohemagglutinin activated T cells and in a mammary carcinoma cell line by interferon-gamma alone and in combination with tumor necrosis factor alpha. Consistently, there was an in situ induction of APO-1 in several types of glandular epithelium in microtopographic association with lymphohistiocytic infiltrates. This inflammation-associated APO-1 induction went along with increased expression of this molecule within the lymphocytic compartment of the lesion. In tumors. APO-1 expression was heterogeneous. In comparison to their normal counterparts, some tumors showed abnormal hypo-expression or loss of APO-1. However, abnormal neo-expression was also found. CONCLUSIONS: Tissue distribution, in vitro expression, and reaction upon cytokine-induced activation suggest that APO-1 might not only transmit apoptotic signals but might play a more general role in growth control.

• Higuchi M, Aggarwal BB
• P80 form of the human tumor necrosis factor receptor is involved in DNA fragmentation.
• FEBS Lett. 1993; 331: 252-5
• Display abstract

• Two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. TNF is known to cause DNA fragmentation in certain tumor cell lines but the role of p60 and p80 in this action is not understood. In the present study, we examined the role of these receptors in TNF-induced DNA fragmentation. Treatment of U-937 cells with phorbol ester caused downregulation of both types of TNF receptors and this was accompanied by disappearance of the TNF-induced DNA fragmentation. The removal of phorbol ester led to two time-dependent events: (1) the rapid regeneration of the p80 form but not the p60 form of the TNF receptor; and (2) the reappearance of TNF-induced DNA fragmentation. These results suggest that the p80 receptor could mediate the TNF-induced DNA fragmentation.

• Bloom JW, Bettencourt JD, Mitra G
• Epitope mapping and functional analysis of three murine IgG1 monoclonal antibodies to human tumor necrosis factor-alpha.
• J Immunol. 1993; 151: 2707-16
• Display abstract

• Immunologic and enzyme digest epitope mapping techniques were used to compare and contrast the interactions of three mouse mAb, designated A10G10, A6, and B6, with human TNF-alpha. These antibodies have previously been shown to protect mouse and human cells from TNF toxicity. ELISA showed that the antibodies recognize predominantly conformational epitopes, and that native TNF binds at least two molecules of the same mAb. Enzyme digestion of native TNF bound to each of the mAb in turn showed that each of the antibodies binds to or sterically masks a large fraction of the exposed surface of TNF. These epitope mapping data were compared with four putative TNF cell receptor binding sites presented in the literature. The results are consistent with the hypothesis that these antibodies prevent TNF-induced cell death by binding to or sterically masking the TNF receptor binding site.

• Hsu KC, Chao MV
• Differential expression and ligand binding properties of tumor necrosis factor receptor chimeric mutants.
• J Biol Chem. 1993; 268: 16430-6
• Display abstract

• The receptors for tumor necrosis factor (TNF) are represented by two transmembrane proteins, p55TNFR and p75TNFR, which are members of a family of cell surface molecules, including the Fas antigen, CD30, CD40, OX40, a Shope fibroma virus protein, and the low affinity p75 nerve growth factor receptor. A common structural feature is a sequence of 40 amino acids that is found in adjacent repeated domains, with 6 cysteine residues in a conserved register. To assess the functional significance of this cysteine-rich domain (CRD), we have constructed chimeric receptors between each TNF receptor and the low affinity nerve growth factor receptor. The chimeric receptor cDNAs were expressed efficiently in COS-1 and 3T3 fibroblasts, as assessed by affinity cross-linking, cell surface biotinylation and immunoprecipitation, and equilibrium binding. Receptors with two CRD of either TNF receptor were incapable of binding TNF, whereas receptors with all four CRD retained the ability to bind TNF with wild type affinity. These results, in conjunction with previous deletion mutation studies, suggest that TNF binding to each receptor requires all four cysteine-rich repeats. Furthermore, analysis of chimeric receptors containing domains of p55TNFR suggests that cytoplasmic sequences directly influence the levels of receptor expression.

• Kalinina AR, Sadovnikov VB
• [Exogenous iron as a factor determining the effect of tumor necrosis factor (TNF) on the Raji cell line]
• Dokl Akad Nauk. 1993; 329: 515-8

• Yang Y, Yelavarthi KK, Chen HL, Pace JL, Terranova PF, Hunt JS
• Molecular, biochemical, and functional characteristics of tumor necrosis factor-alpha produced by human placental cytotrophoblastic cells.
• J Immunol. 1993; 150: 5614-24
• Display abstract

• Previous studies have shown that the TNF-alpha gene is transcribed and translated in fully differentiated human placental syncytiotrophoblast. In this study, TNF transcripts were identified by in situ hybridization in cytotrophoblastic cells, a progenitor subpopulation that proliferates rapidly in early gestation tissues. To establish molecular and biochemical characteristics of cytotrophoblastic TNF and to evaluate potential utilization, experiments were conducted on two cytotrophoblastic cell lines, Jar and JEG-3. Northern blot hybridization and immunocytochemical tests showed that Jar and JEG-3 cells contained TNF mRNA and specific protein. Enzyme immunoassays demonstrated production of TNF, and immunoprecipitation experiments showed that Jar cell TNF protein was the same molecular mass as macrophage TNF. DNA synthesis in both lines was promoted by rTNF, and experiments employing 17-mer TNF antisense and sense oligonucleotides showed specific inhibition of DNA synthesis by antisense sequences. Both p60 and p80 TNF-R mRNA were present in the choriocarcinoma cell lines, and DNA synthesis was inhibited by antibody to the p60 TNF-R. Although the two lines were similar in many respects, Jar cells produced more TNF and demonstrated a greater reliance on TNF for their growth. Collectively, the results indicate that: 1) the TNF gene is expressed in both normal and malignant cytotrophoblast; 2) certain molecular, immunologic, and biochemical characteristics of trophoblast-derived TNF are similar to macrophage TNF; and 3) the p60 TNF-R facilitates utilization of TNF as an autocrine growth factor by choriocarcinoma cells. Although TNF apparently serves important functions in cytotrophoblast during the course of placental development that might include promotion of proliferation and invasion, constitutive expression of this gene in neoplastic cells could account in part for the remarkable ability of trophoblastic tumors to overcome host defenses.

• Schwalb DM et al.
• Identification of a new receptor subtype for tumor necrosis factor-alpha.
• J Biol Chem. 1993; 268: 9949-52
• Display abstract

• Two distinct receptors, which bind both tumor necrosis factor-alpha and tumor necrosis factor-beta (TNF-alpha and TNF-beta), have been previously identified and cloned from transformed cells. The present study identifies a novel receptor subtype in normal human liver which binds TNF-alpha but not TNF-beta. TNF-alpha but not TNF-beta competes for 125I-TNF-alpha binding and incorporation into affinity-labeled complexes in human liver plasma membranes (HLPM). Antisera to the cloned receptors competed for 125I-TNF-alpha binding to plasma membranes isolated from various transformed cell lines but not to HLPM. However, mRNAs corresponding in size to both known TNF receptors were detected in liver RNA, making it likely that post-transcriptional modifications account for the TNF-alpha specificity of HLPM. These observations suggest that the effects of TNF-alpha and TNF-beta on some normal tissues may be more distinct than previously realized.

• Pennica D et al.
• Biochemical characterization of the extracellular domain of the 75-kilodalton tumor necrosis factor receptor.
• Biochemistry. 1993; 32: 3131-8
• Display abstract

• An expression plasmid encoding the extracellular domain of the 75-kDa human tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable cell line secreting soluble TNF-R2 (sTNF-R2). Purified sTNF-R2 was resolved by SDS-PAGE into one band of approximate M(r) 43,000, consistent with a molecular weight of 36,000 +/- 4800 obtained by sedimentation equilibrium analysis. The apparent molecular weight observed by gel filtration chromatography was approximately 136,000. Glycosylation analysis revealed that Asn-149 is fully glycosylated, while Asn-171 is incompletely glycosylated (approximately 50%), and that a proline-, serine-, and threonine-rich region (residues 175-234) contains O-linked carbohydrate structures. Scatchard analysis of [125I]TNF-alpha and [125I]TNF-beta binding to sTNF-R2 gave dissociation constants (Kd) of 0.3 and 0.75 nM, respectively, comparable to those observed for intact cell-surface TNF-R2. The sTNF-R2 was found to block the cytotoxicity of both TNF-alpha and TNF-beta in a murine L-M cell assay. The sizes of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes determined by gel filtration chromatography were approximately 322 and 204 kDa, respectively. The stoichiometry of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes were examined by size-exclusion chromatography, sedimentation equilibrium, and cross-linking. The data from these studies suggest that at least two molecules of sTNF-R2 can bind to a single TNF-alpha or TNF-beta trimer.

• Brakebusch C, Nophar Y, Kemper O, Engelmann H, Wallach D
• Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor.
• EMBO J. 1992; 11: 943-50
• Display abstract

• The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.

• Austgulen R, Liabakk NB, Espevik T
• [Secretion of soluble receptors for tumor necrosis factor. An immunologic buffer mechanism during normal pregnancy?]
• Tidsskr Nor Laegeforen. 1992; 112: 3545-7
• Display abstract

• High concentrations of tumour necrosis factor may threaten the well-being of the foetus. Secretion of soluble cytokine receptors has been suggested as a mechanism for regulating cytokine activity in vivo. In this study pregnancy-related materials were examined for the presence of soluble receptors (p55 and p75) for tumour necrosis factor. In contrast to control materials, serum and urine samples from pregnant women and newborns contained high concentrations of p55, and p55 was highly expressed by villous syncytiotrophoblasts. Thus, the present data suggest that shedding of specific receptors may contribute to the regulation of tumour necrosis factor activity throughout a normal pregnancy.

• Gonen B, Kahana O, Witz IP
• In vivo tumorigenicity and in vitro sensitivity to tumor-necrosis-factor alpha mediated killing of c-Ha-ras-transformed cells.
• Cancer Immunol Immunother. 1992; 35: 388-94
• Display abstract

• Cellular subclones of high and low tumorigenicity obtained from a mouse c-Ha-ras-transformed clone, were examined for their sensitivity to tumor-necrosis-factor (TNF)-mediated cytotoxicity. Cells of the highly tumorigenic subclones showed a significantly enhanced resistance to the cytotoxic effect of TNF plus cyclohexamide (CHI) as compared to cells of the low-tumorigenic subclones. The enhanced resistance to TNF+CHI was not due to a lower expression of TNF receptors on the cells. The c-Ha-ras-transfected cells were transformed and maintained in culture only (C cells). In vivo passage of cells of the initially low-tumorigenic c-Ha-ras subclones through the mouse significantly enhanced the tumorigenic potential of these CTC cells (culture/tumor/culture). In correlation with their enhanced tumorigenicity, the CTC cells were highly resistant to TNF-mediated cytotoxicity as compared to C cells of the same subclone. Furthermore, the involvement of TNF in determining the tumorigenic phenotype of the c-Ha-ras-transformed cells was demonstrated in a more direct manner. Cells of a c-Ha-ras-transformed low-tumorigenic, highly TNF-sensitive subclone were selected by repeated cycles of in vitro exposure to TNF alpha. As a result, a stable, highly TNF-resistant population of cells emerged. These TNF-resistant cells caused more tumors in mice as compared to their original TNF-sensitive cells. These results show that the resistance to the cytotoxic effect of TNF plus cyclohexamide may be involved, at least partially, in the tumorigenic potential of c-Ha-ras-transformed cells and suggest a possible role for TNF in the enhancement of the tumorigenic potential of these cells in mice.

• Austgulen R, Espevik T, Mecsei R, Scott H
• Expression of receptors for tumor necrosis factor in human placenta at term.
• Acta Obstet Gynecol Scand. 1992; 71: 417-24
• Display abstract

• The biological effects of tumor necrosis factor (TNF) are mediated through its interaction with high affinity receptors on target cells. Secretion of soluble cytokine receptors has been suggested as a mechanism of regulating cytokine activity in vivo. In a previous study we detected soluble TNF receptors (TNFRs) in amniotic fluid and urine samples from pregnant women, suggesting that secretion of soluble TNFRs may provide a mechanism for protection of the fetus against TNF action during pregnancy. In the present study, TNFR containing cells in cryostat sections from normal placentas at term were evaluated by monoclonal antibodies against the 55 kD--and the 75 kD TNFR in an indirect immunofluorescence technique. The 55 kD TNFR was expressed by the villous syncytiotrophoblasts, by vascular endothelial cells, by some decidual cells and by occasional cells in the placental stroma. Staining for the 75 kD TNFR was confined to the vascular endothelial cells, a relatively small number of stromal cells and decidual cells, whereas the villous syncytiotrophoblasts were negative. The abundant expression of TNFRs in placental tissue suggests: 1. That a considerable number of the placental cells are receptive to the regulatory activities of TNF; 2. That placental cells may be the cellular origin of soluble TNFRs secreted during pregnancy.

• Adolf GR, Fruhbeis B
• Monoclonal antibodies to soluble human TNF receptor (TNF binding protein) enhance its ability to block TNF toxicity.
• Cytokine. 1992; 4: 180-4
• Display abstract

• A soluble extracellular fragment of the human 55-60 kDa tumor necrosis factor receptor (sTNF-R I), originally isolated from urine, binds both TNF-alpha and TNF-beta and blocks the activity of these cytokines in biological assays. Three monoclonal antibodies (mAbs) raised against sTNF-R I (TBP-1, -2 and -6) as well as a mAb developed by immunization with the intact receptor (H398) were analysed for their epitope specificities in ELISAs and for biological activity in cytotoxicity assays on murine L-M cells. TBP-2 and H398 bind to related epitopes on sTNF-R I; they compete with TNF-alpha for binding and block the protective effect of sTNF-R I in the bioassay. MAbs TBP-1 and TBP-6 recognize two further, independent epitopes; both bind sTNF-R I in the presence of an excess of TNF-alpha. Both TBP-1 and TBP-6 markedly enhance the ability of sTNF-R I to protect cells against the cytotoxic activities of TNF-alpha and TNF-beta, but have no activity in the absence of sTNF-R I. Fab fragments show much lower activity. We propose that the ability of certain mAbs to enhance the protective activity of sTNF-R is due to a steric hindrance phenomenon.

• Ininns EK et al.
• Growth of the endometrial adenocarcinoma cell line AN3 CA is modulated by tumor necrosis factor and its receptor is up-regulated by estrogen in vitro.
• Endocrinology. 1992; 130: 1852-6
• Display abstract

• We demonstrate that tumor necrosis factor (TNF) has a biphasic effect on the growth of the human endometrial adenocarcinoma cell line AN3 CA in vitro. Low levels (0.2-5 pg/ml) of TNF were moderately growth stimulatory (up to 20% enhancement), while levels over 100 pg were growth inhibitory (up to 45% inhibition). Northern blot analysis showed expression of the 75-kilodalton (kDa) TNF receptor mRNA, but no expression of the 55-kDa TNF receptor mRNA or TNF mRNA. The growth of these cells was not directly affected by physiological concentrations (10(-7)-10(-9) M) of 17 beta-estradiol (E2). However, [125I]TNF binding studies and Scatchard analysis showed that 18-h coculture with 10(-8) M E2 increased the number of TNF receptors expressed on these cells 3-fold. Quantitative mRNA analysis confirmed that 75-kDa TNF receptor mRNA expression increased within 4 h of incubation with E2. These observations suggest an interaction between the endocrine and the immune systems, with an important implication for the homeostasis of endometrial tissues.

• Wong GH, Tartaglia LA, Lee MS, Goeddel DV
• Antiviral activity of tumor necrosis factor is signaled through the 55-kDa type I TNF receptor [corrected]
• J Immunol. 1992; 149: 3350-3
• Display abstract

• Agonist antibodies (Ab) to the two TNF receptors, TNF-R1 (55 kDa) and TNF-R2 (75 kDa), have been shown to signal many of the distinct functions induced by TNF-alpha. We have found that anti-TNF-R1, but not anti-TNF-R2, Ab trigger antiviral activity in human hepatoma Hep-G2 cells and enhance the antiviral activity of IFN-gamma in human lung fibroblast A549 cells. Likewise, anti-human-TNF-R1 Ab had antiviral enhancing activity on murine L929 cells engineered to express human TNF-R1. However, L929 cells that express human TNF-R1 lacking most of the intracellular domain fail to respond to anti-human-TNF-R1 Ab. This demonstrates that the intracellular domain of TNF-R1 is necessary to generate antiviral activity. TNF-R1 but not TNF-R2 also signals killing of virus-infected cells by TNF-alpha. Thus, all the known antiviral activities of TNF-alpha are mediated through TNF-R1.

• Aggarwal BB
• Comparative analysis of the structure and function of TNF-alpha and TNF-beta.
• Immunol Ser. 1992; 56: 61-78

• Tsujimoto M, Oku N
• Regulation of TNF receptors.
• Immunol Ser. 1992; 56: 149-60

• Garret M, McHendry-Rinde B, Spickofsky N, Margolskee RF
• Isolation of a clone which induces expression of the gene encoding the human tumor necrosis factor receptor.
• Gene. 1992; 111: 215-22
• Display abstract

• Tumor necrosis factor (TNF) is a cytokine with pleiotropic effects upon cell growth, inflammation and immunologic responsiveness. High-affinity TNF receptors (TNFRs) of 55 and 75 kDa are found in many cell types. Using an Epstein-Barr virus (EBV)-based mammalian expression library, we have isolated a clone from human lymphoblastoid transfectants that induces overexpression of the TNFR-encoding gene (TNFR). Transfectants overproducing the TNFR were isolated by multiple rounds of sorting on a fluorescence-activated cell sorter using fluorescent TNF ligand binding as the selection procedure. Among the sorted transfectants were cells producing approx. 150,000 receptors per cell (Kd of approx. 1 nM). These cells have multiple copies of the TNFR gene present as extrachromosomal plasmids. These cells also overproduced the mRNA for TNFR. Low-Mr EBV episomes were isolated from these overproducing cells and used to transform Escherichia coli. One of the colonies isolated contained a plasmid encoding a portion of the noncoding region of the TNFR gene. Transfection of human lymphoblastoid cells with this DNA gave rise to high-level production of TNFR. Fluorescent TNF bound to these transfectants is fully and specifically displaced by an excess of TNF. The rescued clone contains approx. 10 kb of human genomic DNA including the 3'-untranslated region of TNFR and several Alu sequences; apparently during the selection procedure in human cells, recombination occurred to rescue a portion of the TNFR gene. Transient transfection was used to narrow down the region responsible for TNFR induction to 5.2 kb. The mechanism by which this clone induces TNFR expression has not been determined.

• Barinaga M
• Viruses launch their own 'star wars'.
• Science. 1992; 258: 1730-1

• Tartaglia LA, Goeddel DV
• Tumor necrosis factor receptor signaling. A dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor.
• J Biol Chem. 1992; 267: 4304-7
• Display abstract

• To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.

• Kraus T et al.
• Evaluation of soluble tumor necrosis factor receptors after orthotopic liver transplantation.
• Transplant Proc. 1992; 24: 2539-41

• Xenos ES et al.
• Effect of tumor necrosis factor alpha and of the soluble tumor necrosis factor receptor on insulin secretion of isolated islets of Langerhans.
• Transplant Proc. 1992; 24: 2863-4

• He CJ, Kanfer A
• Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli.
• Kidney Int. 1992; 41: 1170-4
• Display abstract

• Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.

• Heilig B, Mapara M, Bargou R, Fiehn C, Dorken B
• TNF alpha therapy activates human B-lymphoma cells in vivo and may protect myelopoiesis.
• Leuk Res. 1992; 16: 769-73
• Display abstract

• In vitro TNF alpha induces proliferation and expression of predominantly type A TNF-receptors on B-lymphoma cells. In a pilot study we treated 2 patients with refractory B lymphomas with two courses of TNF alpha and consecutive aggressive chemotherapy (high dose Ara-C and mitoxantrone). TNF alpha was applied on days 1-4, chemotherapy on days 2-4 (TNF-AraM). The TNF-AraM therapy was repeated on day 43. Both patients responded to therapy. TNF alpha therapy induced expression of the 75 kD TNF receptor and the interleukin 2 receptor (CD25) and weakly the 55 kD TNF receptor on leukemic B lymphocytes. Interleukin 3, interleukin 6 and GM-CSF were induced and measured in the serum of patients. The mean time of severe granulocytopenia (less than 0.5/nl) was 9 days (range 8-10 days), the mean time of thrombocytopenia (less than 20/nl) was 5 days (range 2-6 days). In 7 patients, who were treated with high dose Ara-C and mitoxantrone (AraM) mean time of granulocytopenia (less than 0.5/nl) was 23 days (range 18-34 days), and of thrombocytopenia (less than 20/nl) was 13.3 (range 3-27 days). We conclude that TNF alpha can activate tumor B cells in vivo and may exhibit also myeloprotective effects when applied before aggressive chemotherapy.

• Dayer JM
• Immunomodulating functions of tumor necrosis factor and interleukin 1 inhibitors.
• J Rheumatol Suppl. 1992; 32: 59-60
• Display abstract

• Cytokines play a crucial role in the inflammatory and immune responses. The activity of cytokines is counterbalanced by specific inhibitors with some functioning as receptor antagonists. Inhibitors to interleukin 1 and tumor necrosis factor may have therapeutic potential in conditions such as inflammatory arthritis, diabetes mellitus, disseminated intravascular coagulopathy and septic shock. The ability to modulate host defenses with cytokines and cytokine antagonists may also have applications in the fields of transplantation, oncohematology and immunodeficiency.

• Rothe J, Gehr G, Loetscher H, Lesslauer W
• Tumor necrosis factor receptors--structure and function.
• Immunol Res. 1992; 11: 81-90
• Display abstract

• Tumor necrosis factors (TNFs) have been a focus of research for well over a decade now. The identification and recent molecular cloning of two different types of cell-surface TNF receptors will shed further light on the mode of action of these pleiotropic cytokines. In the present article, we summarize the data on the biochemistry and structure of the receptors and focus on the molecular cloning of the respective cDNAs. The nucleotide sequences of the receptor genes revealed that both TNF receptors belong to the still growing nerve growth factor receptor gene family. The function and origin of TNF inhibitory proteins as well as receptor-mediated signal transduction are discussed.

• Durkop H, Latza U, Hummel M, Eitelbach F, Seed B, Stein H
• Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease.
• Cell. 1992; 68: 421-7
• Display abstract

• In man, Hodgkin's disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.

• Miles DW, Aderka D, Engelmann H, Wallach D, Balkwill FR
• Induction of soluble tumour necrosis factor receptors during treatment with interleukin-2.
• Br J Cancer. 1992; 66: 1195-9
• Display abstract

• Interleukin-2 (IL-2) treatment induces other cytokines such as tumour necrosis factor (TNF) TNF may mediate some of the anti-tumour activity of IL-2, but conversely, may contribute to its dose limiting toxicities. Cleaved extracellular domains of the p55 and the p75 TNF receptors (sTNF-R1 and R2) bind to and inhibit the biological activity of TNF in vitro, but may also act as carrier molecules. We have assayed TNF and sTNFR-1 and 2 in the plasma of advanced cancer patients, before and during treatment with IL-2. Plasma levels of TNF in 22 patients were not significantly different from 25 normal controls, but levels of sTNFR-1 and sTNFR-2 were higher (P < 0.001). Levels of TNF and both its soluble receptors were significantly increased in 13 patients receiving IL-2 therapy. Maximum induced levels of sTNFR-1 and sTNFR-2 correlated closely with maximum induced levels of TNF (P < 0.001), but peak levels of sTNFR-1 and two were achieved 24-48 h after peak TNF. Levels of TNF and sTNF-Rs did not correlate with toxicity. Treatment with IL-2 leads not only to induction of TNF but also soluble binding proteins at levels which may modulate its biological activity.

• Kern P et al.
• Soluble tumor necrosis factor receptors correlate with parasitemia and disease severity in human malaria.
• J Infect Dis. 1992; 166: 930-4
• Display abstract

• Elevated serum or plasma concentration of immunoreactive tumor necrosis factor (TNF) is consistently detected in patients with malaria. TNF levels correlate with high parasitemia and clinical severity but not always with outcome. Since the effects of TNF may be neutralized by soluble TNF receptors, sera of 30 nonimmune patients with malaria were analyzed before and during antimalarial therapy. High concentrations of receptors R1 (55 kDa) and R2 (75 kDa) were detected immunologically in all sera of untreated patients. Levels of immunoreactive TNF correlated closely with levels of soluble TNF R1 and R2 (r = .75 and .59, respectively). In contrast, sera lacked cytotoxic activity against target cells in the TNF bioassays. Soluble TNF receptor levels remained elevated for days after treatment. These results suggest that excessive release of TNF induced by the asexual stage of malaria parasites is controlled by a subsequent shedding of soluble TNF receptors that may bind and deactivate biologically functional TNF.

• Grazioli L et al.
• Inhibitory effect of suramin on receptor binding and cytotoxic activity of tumor necrosis factor alpha.
• Int J Immunopharmacol. 1992; 14: 637-42
• Display abstract

• The effect of suramin on the binding of human Tumor Necrosis Factor alpha (huTNF alpha) to specific cell-surface receptors as well as on its cytotoxic activity in vitro was investigated. Suramin inhibited both activities in a dose-dependent manner. Experiments designed to discriminate if suramin exerted its inhibitory activity on the ligand or on the receptor showed that the ligand (huTNF alpha) was the most likely target for suramin in this system. These results may explain, in part, the immunosuppressive activities of suramin that have been observed in vivo and suggest that suramin could be useful in those disease states in which hyperproduction of huTNF alpha has been shown to play a pathogenic role.

• Aderka D et al.
• Variation in serum levels of the soluble TNF receptors among healthy individuals.
• Lymphokine Cytokine Res. 1992; 11: 157-9
• Display abstract

• Soluble forms of the two receptors for tumor necrosis factor (TNF) are present in human sera at concentrations that increase greatly in various disease states as well as varying among healthy individuals. Measurements of the soluble TNF receptor (sTNF-R) concentrations in healthy individuals at time lapses of 3 months (17 individuals) or 1 year (51 individuals) showed a significant correlation between the first and the second measurements from each individual, implying that individual differences are stable. Since the sTNF-Rs are believed to function as physiological attenuators of TNF activity, these steady individual differences may contribute to differences in the severity of the harmful effects of TNF in disease states.

• Pandita R, Pocsik E, Aggarwal BB
• Interferon-gamma induces cell surface expression for both types of tumor necrosis factor receptors.
• FEBS Lett. 1992; 312: 87-90
• Display abstract

• 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.

• Beutler B
• Application of transcriptional and posttranscriptional reporter constructs to the analysis of tumor necrosis factor gene regulation.
• Am J Med Sci. 1992; 303: 129-33

• Galloway CJ et al.
• Anti-tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti-idiotypic antibody.
• Eur J Immunol. 1992; 22: 3045-8
• Display abstract

• Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. A10G10 is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen-binding site of A10G10 was raised and affinity purified over an A10G10 column. The resultant anti-idiotypic antibody recognized not only A10G10 but also both TNF receptors. It showed TNF agonist activity in two different TNF bioassays, and competed with several anti-TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand-specific receptor in the absence of the receptor itself.

• Zhang XM, Weber I, Chen MJ
• Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship.
• J Biol Chem. 1992; 267: 24069-75
• Display abstract

• In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.

• Greenblatt MS, Elias L
• The type B receptor for tumor necrosis factor-alpha mediates DNA fragmentation in HL-60 and U937 cells and differentiation in HL-60 cells.
• Blood. 1992; 80: 1339-46
• Display abstract

• Tumor necrosis factor-alpha (TNF) binds to two specific cell surface receptors, types A and B, which are both present on HL-60 and U937 cells, and induces monocytoid differentiation in HL-60 cells and early DNA fragmentation in HL-60 and U937 cells. To further define the receptors' roles, we studied how monoclonal antibodies (MoAbs) against each receptor affected TNF-induced cellular responses. HTR-9, an MoAb against the type B (low affinity, 55 Kd) receptor, reproduced all of these effects in a dose-dependent manner. UTR-1, an MoAb against the type A (high affinity, 75 Kd) receptor, had no effect in saturating doses, but supersaturating doses enhanced DNA fragmentation threefold. TNF and interferon gamma (IFN-gamma) synergistically induced morphologic differentiation and monocytic antigen expression, while the antitype B receptor MoAb was synergistic for morphologic response, but not antigen expression. Our results indicate that (1) the type B receptor mediates some responses to TNF in HL-60 and U937 cells, (2) the type A receptor does not stimulate these responses, (3) the TNF molecule is not necessary for some of these actions, and (4) TNF-induced morphologic changes and surface antigen expression in HL-60 cells may be regulated by separate postreceptor pathways.

• Gullberg U, Lantz M, Lindvall L, Olsson I, Himmler A
• Involvement of an Asn/Val cleavage site in the production of a soluble form of a human tumor necrosis factor (TNF) receptor. Site-directed mutagenesis of a putative cleavage site in the p55 TNF receptor chain.
• Eur J Cell Biol. 1992; 58: 307-12
• Display abstract

• Soluble forms of receptors for tumor necrosis factor (TNF) might be important for regulating the actions of TNF. Site-directed in vitro mutagenesis was employed to examine the processing of the p55 tumor necrosis factor receptor chain (TNF-R55) into soluble TNF-binding protein (TNF-R55-BP). An Asn/Val sequence close to the transmembrane region in TNF-R55 was indicated as a putative cleavage site for proteolytic processing. By mutagenesis, Asn and Val were replaced with Gly and Ala, respectively. Expression in Chinese hamster ovary (CHO) cells resulted in identical binding of ligand to mutated receptors as compared to receptors not subjected to mutagenesis. Turnover rates of receptors as judged by disappearance of TNF binding capacity after inhibition of de novo protein synthesis and downregulation in response to incubation with phorbol esters were also identical between wild-type and mutated receptors. However, mutations of the cleavage site resulted in a decreased spontaneous and phorbol ester-induced release of soluble receptor (TNF-R55-BP) which was almost abolished when both Asn and Val were mutated. Our results clearly demonstrate the importance of an Asn/Val sequence for proteolytic processing of the TNF-R55 into soluble TNF-R55-BP and indicate that phorbol ester-induced downregulation of the TNF-R55 may be dissociated from proteolytic cleavage of the receptor.

• Aderka D, Engelmann H, Maor Y, Brakebusch C, Wallach D
• Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors.
• J Exp Med. 1992; 175: 323-9
• Display abstract

• The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs follows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.

• Nicod LP
• [Therapeutic perspectives of cytokines in respiratory allergy conditions]
• Allerg Immunol (Paris). 1992; 24: 197-8
• Display abstract

• Among the cytokines playing a role in asthma, IL-1 and TNF alpha or beta are important agents to be considered. IL-1 TNF alpha are produced by antigen-presenting cells. TNF alpha and beta are released by activated lymphocytes. Recently a natural inhibitor of IL-1 produced by monocytes or macrophages in the form of a 22-25 kD protein, which binds competitively to IL-1 receptors has been cloned and sequenced. This inhibitor is called the IL-1 receptors antagonist. Two types of TNF soluble receptors (55 and 75 kD) can be liberated from the surface of macrophages and neutrophils. These soluble receptors block both TNF alpha and beta activity. The respective roles of these cytokine inhibitors in the physiopathology of asthma need to be further defined, but new therapeutic perspectives are opening.

• Tartaglia LA, Goeddel DV
• Two TNF receptors.
• Immunol Today. 1992; 13: 151-3
• Display abstract

• In the past few years, considerable progress has been made on the identification and characterization of tumor necrosis factor (TNF) receptors. The relative roles played by the two receptor types in signaling the diverse functions of TNF are less clear. Here, Louis Tartaglia and David Goeddel summarize progress to date and propose a model of TNF receptor triggering that reconciles the seemingly conflicting data.

• Ruddle NH
• Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).
• Curr Opin Immunol. 1992; 4: 327-32
• Display abstract

• Distinctions between tumor necrosis factor, TNF-alpha, and lymphotoxin, TNF-beta, have previously been based on the differences between their protein sequences, biological activity, and molecular regulation. In the past year, elucidation of the molecular nature of the two molecules and the interactions with common receptors has emphasized their similarities, although profound differences continue to emerge with regard to their mode of production, transcription rates, mRNA half-lives, and the importance of various DNA regulatory sequences. A role for both TNF-alpha and TNF-beta has recently been suggested with regard to disease, particularly multiple sclerosis. The past year has also seen the description of an extensive microsatellite polymorphic system which should provide a more definitive understanding of the association of the TNF locus with disease.

• Kruppa G, Thoma B, Machleidt T, Wiegmann K, Kronke M
• Inhibition of tumor necrosis factor (TNF)-mediated NF-kappa B activation by selective blockade of the human 55-kDa TNF receptor.
• J Immunol. 1992; 148: 3152-7
• Display abstract

• The numerous biologic activities of TNF appear mediated by two types of specific cell surface receptors of 55 to 60 kDa (TR55) and 75 to 80 kDa (TR75) molecular mass, respectively. The role of TR55 in the activation of the nuclear transcription factor kappa B (NF-kappa B) was investigated using an antagonistic, mAb, H398, specific for the human TR55. The human leukemic T cell line, Jurkat, which expresses both types of receptors at comparable levels, was used to test for NF-kappa B activation by electrophoretic mobility shift assays using as a probe an oligonucleotide encompassing the two tandemly arranged kappa B sites of the HIV-1 LTR enhancer. mAb H398 is shown to efficiently block not only TNF- but also lymphotoxin-mediated activation of NF-kappa B. Furthermore mAb H398 also impeded TNF- or lymphotoxin-mediated activation of chloramphenicol acetyl transferase gene expression from the HIV-1-LTR as determined by transient transfection assays. These findings indicate that both, induction of NF-kappa B binding to DNA, and transcriptional activity can be efficiently inhibited by selective blockade of TR55. Finally it is shown, that human TR55 confers NF-kappa B inducibility when expressed in the mouse pre-B cell line 70Z/3, which does not respond to TNF in its parental state. Together, the results of this study indicate that TR55 is both necessary and sufficient for mediating TNF activation of NF-kappa B.

• Santis AG, Campanero MR, Alonso JL, Sanchez-Madrid F
• Regulation of tumor necrosis factor (TNF)-alpha synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway.
• Eur J Immunol. 1992; 22: 3155-60
• Display abstract

• The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-alpha/TNF receptor system in human T lymphocytes has been investigated. It has been found that both TNF-alpha synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover, TNF-alpha secretion was also observed by activation of T lymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2-activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75-kDa receptor form, whereas they lacked detectable levels of the 55-kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75-kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55-kDa TNF receptor. In addition, we have found that TNF-alpha enhanced the proliferative response triggered by the mixture of anti-CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF-alpha/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.

• Gehr G, Braun T, Lesslauer W
• Cytokines, receptors, and inhibitors.
• Clin Investig. 1992; 70: 64-9
• Display abstract

• Cytokines are endogenous mediators in inflammatory and immunologic host defense reactions. In various diseases cytokines produced in excess cause systemic or local toxic effects. Cytokines therefore are tightly controlled by regulation of their biosynthesis and release and by counteracting mechanisms which limit their activities. Two new cytokine inhibitory mechanisms have recently been discovered. First, the generation of soluble receptors which compete with cellular receptors for cytokine binding has been recognized as a general phenomenon. Second, a receptor antagonist polypeptide binding to the receptor but not eliciting biological activity has been discovered in the IL-1 system. These polypeptides, when expressed in various recombinant forms, are not only research tools but may find also direct clinical use.

• Goillot E et al.
• Tumor necrosis factor as an autocrine growth factor for neuroblastoma.
• Cancer Res. 1992; 52: 3194-200
• Display abstract

• Recombinant tumor necrosis factor (TNF) stimulates the proliferation of two neuroblastoma cell lines, SKNFI and SKNBE, in both serum-free medium and fetal calf serum-supplemented medium but has no effect in medium without insulin. This effect is very similar with TNF doses ranging from 5 to 500 ng/ml but depends on the duration of treatment; when cells are treated for 168 h with TNF, the maximal index of proliferation is observed between 120 and 144 h of treatment. The two neuroblastoma cell lines express type A and type B TNF receptors and contain TNF protein; however, TNF is undetectable in culture supernatants. Treatment of the two neuroblastoma cell lines with a rabbit polyclonal antibody to TNF for 96 h fully inhibits [3H]thymidine incorporation; less than 5% viable cells are left in the samples after treatment. A combination of two monoclonal antibodies against type A and type B TNF receptors also inhibits over 85% of the [3H]thymidine incorporation by the two cell lines after 96 h of treatment; the use of a single antibody has a partial effect, suggesting that both receptors are functional on the neuroblastoma cell lines. Taken together, these results show that TNF is an autocrine growth factor for the two neuroblastoma cell lines SKNFI and SKNBE. The results described above have been confirmed on two other neuroblastoma cell lines, IRM32 and CLB-PE.

• Higuchi M, Aggarwal BB
• Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of recombinant P60 and P80 receptors.
• Biochem Biophys Res Commun. 1992; 182: 638-43
• Display abstract

• 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.

• Porteu F, Nathan CF
• Mobilizable intracellular pool of p55 (type I) tumor necrosis factor receptors in human neutrophils.
• J Leukoc Biol. 1992; 52: 122-4
• Display abstract

• The distribution of type I (p55) and type II (p75) tumor necrosis factor receptors (TNF-Rs) in human polymorphonuclear neutrophils (PMNs) was analyzed by Western blotting of subcellular fractions obtained by centrifugation of PMN cavitates on Percoll density gradients. In resting PMNs, the p55 receptor was associated with both gamma and beta fractions, enriched in plasma membranes and specific granules, respectively, whereas the p75 TNF-R was located only in the gamma fraction. Intracellular p55 TNF-R bound 125I-labeled TNF in a ligand blot assay and migrated as a diffuse band of 46-60 kd, similar to the plasma membrane receptor. Activation of PMNs with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), in conditions that induced the selective release of the tertiary granule marker gelatinase, caused the shedding of most of membrane p55 TNF-R as a 28-kd soluble form but had little effect on the distribution of the receptors in beta fractions. By contrast, when specific granule secretion was induced in the presence of cytochalasin B, a marked decrease in reactivity of beta fractions with anti-p55 TNF-R antibody was observed. At the same time, the amount of soluble 28-kd fragment found in the supernatant was increased. Thus, the intracellular pool of p55 TNF-R is associated with functional secretory granules and is redistributed in response to PMN activation.

• Brownell E et al.
• Chromosome assignments of the human TNF p55 and p75 receptor genes.
• J Exp Zool. 1992; 262: 340-2
• Display abstract

• At least two different receptor molecules have been described that are capable of binding tumor necrosis factor alpha, a cytokine that plays an important role in inflammation and antitumor activity. Comparative analyses at the nucleotide sequence level suggest that these receptors are members of a newly defined protein family that also includes human and rat nerve growth factor receptors. In this study, we determine the chromosome assignments of the human TNF alpha receptor genes, one of which may have evolved as part of a conserved Hox locus-containing chromosome segment.

• Gehr G, Gentz R, Brockhaus M, Loetscher H, Lesslauer W
• Both tumor necrosis factor receptor types mediate proliferative signals in human mononuclear cell activation.
• J Immunol. 1992; 149: 911-7
• Display abstract

• TNF is a highly pleiotropic cytokine. The recent identification of two distinct cellular receptors for TNF may provide explanations for the many different TNF activities. We have investigated the expression of the two receptor types, TNFR alpha (75 kDa) and TNFR beta (55 kDa), in human PBMC. Both receptors were found simultaneously expressed by cytofluorimetric, radioligand binding and Northern analysis of naive as well as PHA-activated PBMC. The expression levels in the CD14+ and CD14- subsets were different. Both receptors were strongly expressed in the CD14+ subset. The expression of the receptors in the CD14-, CD3+, CD4+, and CD8+ subsets was lower and similar among these subsets, but TNFR alpha was expressed at higher level than TNFR beta. To dissect the functional roles of the two receptors, we studied the growth factor activity of TNF in the late proliferative responses of PBMC to PHA. In the first approach, the activity of either receptor was blocked by neutralizing, receptor type specific antibodies. In a second approach, the ligand, TNF, was inhibited by a neutralizing antiserum, and the cells were restimulated using type-specific anti-TNFR antibodies with agonistic activity. It was found that both receptor types mediated signals required for proliferative responses of PBMC to PHA from day 4 to day 8 in culture. The cell responses to the activation of either receptor type appeared to be independent, because one receptor could not compensate for the reduction in cell activation caused by blocking the other receptor type.

• Van Zee KJ, Kohno T, Fischer E, Rock CS, Moldawer LL, Lowry SF
• Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo.
• Proc Natl Acad Sci U S A. 1992; 89: 4845-9
• Display abstract

• Tumor necrosis factor alpha (TNF alpha), a primary mediator of systemic responses to sepsis and infection, can be injurious to the organism when present in excessive quantities. Here we report that two types of naturally occurring soluble TNF receptors (sTNFR-I and sTNFR-II) circulate in human experimental endotoxemia and in critically ill patients and demonstrate that they neutralize TNF alpha-induced cytotoxicity and immunoreactivity in vitro. Utilizing immunoassays that discriminate between total sTNFR-I and sTNFR-I not bound to TNF alpha, we show that sTNFR-I-TNF alpha complexes may circulate even in the absence of detectable free TNF alpha. To investigate the therapeutic possibilities of sTNFR-I, recombinant protein was administered to nonhuman primates with lethal bacteremia and found to attenuate hemodynamic collapse and cytokine induction. We conclude that soluble receptors for TNF alpha are inducible in inflammation and circulate at levels sufficient to block the in vitro cytotoxicity associated with TNF alpha levels observed in nonlethal infection. Administration of sTNFR-I can prevent the adverse pathologic sequelae caused by the exaggerated TNF alpha production observed in lethal sepsis.

• Higuchi M, Aggarwal BB
• Microtiter plate radioreceptor assay for tumor necrosis factor and its receptors in large numbers of samples.
• Anal Biochem. 1992; 204: 53-8
• Display abstract

• We developed a rapid radioreceptor assay for tumor necrosis factor (TNF) and its receptors by utilizing serum-coated 96-well plates. This assay has an advantage over the traditional tube assay in speed, number of simultaneous samples, and amount of material needed for the assay. As many as 200 samples were assayed in less than 3 h. The usefulness of this assay to determine TNF and its cell surface receptors and to screen for TNF inhibitors in large numbers of biological fluids from patients is demonstrated. This assay may also prove useful for determining the activity of competitive antagonists of TNF.

• Higashi N
• [Function, molecular structure and gene expression regulation of tumor necrosis factor and lymphotoxin]
• Nippon Rinsho. 1992; 50: 1939-44
• Display abstract

• Tumor necrosis factor (TNF, also called TNF-alpha) and lymphotoxin (LT, TNF-beta) have 32% homology in their primary structures, and it has recently been clarified that they also have closely related tertially structures. These two related cytokines share various biological functions, such as cytotoxic effect, immunomodulating effect, etc. although they produce different effects on several lymphoid, endothelial, and other cellular targets. In this paper, the structures of the two cytokines and their receptor binding sites are described. Furthermore, the structures of the two types of TNF receptors, as well as their role in TNF mediated signal transduction, and possible second messengers responsible for TNF action are also discussed.

• Winzen R et al.
• Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells.
• J Immunol. 1992; 148: 3454-60
• Display abstract

• Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.

• Chang MJ, Pourbohloul SC, Yu WD, Furmanski P, Johnson CS
• Differential effect in vitro of tumor necrosis factor-alpha (TNF) on normal and virus-infected erythroid progenitors from Friend virus (FVA)-infected mice.
• Exp Hematol. 1992; 20: 1271-7
• Display abstract

• In vivo administration of tumor necrosis factor-alpha (TNF) suppresses both normal and Friend virus (FVA)-infected erythroid progenitor cells (CFU-E). To examine the mechanism of erythroid suppression by TNF, we examined TNF's direct effect on normal and virus-infected cells in vitro. Productively infected fibroblast cell lines, fresh acute virus-infected spleen cells, and virus-infected CFU-E were sensitive, whereas uninfected CFU-E were resistant to TNF cytotoxicity in vitro. When FVA-infected erythroblasts were depleted from the spleen cell population in vitro with antivirus antibodies, TNF suppression of the remaining (uninfected) cells was abrogated. In contrast, both normal and virus-infected macrophage progenitor cells and immature erythroid progenitor cells were equally sensitive to TNF cytotoxicity in vitro. Normal erythroblasts had significantly fewer TNF receptors than FVA-infected erythroblasts, which also were morphologically less mature. These results suggest that TNF can differentially suppress late-stage virus-infected erythroid progenitors in vitro.

• Piguet PF, Grau GE, Vesin C, Loetscher H, Gentz R, Lesslauer W
• Evolution of collagen arthritis in mice is arrested by treatment with anti-tumour necrosis factor (TNF) antibody or a recombinant soluble TNF receptor.
• Immunology. 1992; 77: 510-4
• Display abstract

• Immunization of DBA/1 mice with type II collagen within complete Freund's adjuvant leads to arthritis, lasting more than 3 months. Injection of anti-tumour necrosis factor (TNF) IgG, 2 and 3 weeks after immunization prevented the development of arthritis in the following months. This treatment had no effect when started 2 months after induction of the disease. A soluble form of the human recombinant TNF receptor type-beta (rsTNFR-beta), continuously infused at a rate of 20 micrograms/day during the second and third week after immunization, also had a long-term protective effect. Anti-TNF antibody had no effect upon the production of anti-type II collagen antibodies. These results indicate that TNF is critically involved in an early phase of this arthritis.

• Ding AH, Porteu F
• Regulation of tumor necrosis factor receptors on phagocytes.
• Proc Soc Exp Biol Med. 1992; 200: 458-65

• Wiegmann K, Schutze S, Kampen E, Himmler A, Machleidt T, Kronke M
• Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades.
• J Biol Chem. 1992; 267: 17997-8001
• Display abstract

• The numerous biological activities of tumor necrosis factor (TNF) appear mediated by two types of receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular mass. To test TR55 for its individual role in signaling across the membrane, a cDNA coding for the human TR55 was stably expressed in murine 70Z/3 pre-B cells, which lack binding sites for, and proved nonresponsive to human TNF. The transfected TR55 showed high affinity ligand binding and active internalization. It is demonstrated that the TNF signaling cascade, i.e. stimulation of protein kinase C, sphingomyelinase, and phospholipase A2, production of the second messengers diacylglycerol and ceramide, can occur completely through exclusive binding of TNF to TR55. The p55 TNF-binding site functions as an autonomous TNF receptor that mediates key signal transduction pathways, which may control the majority of TNF actions.

• Aderka D, Engelmann H, Wysenbeek AJ, Levo Y
• The possible role of tumor necrosis factor (TNF) and its natural inhibitors, the soluble-TNF receptors, in autoimmune diseases.
• Isr J Med Sci. 1992; 28: 126-30

• Abdolrasulnia R, Shepherd VL
• Purification of type I and type II tumor necrosis factor receptors from human lung tissue.
• Am J Respir Cell Mol Biol. 1992; 7: 42-8
• Display abstract

• Two receptors for tumor necrosis factor-alpha (TNF) were purified from detergent-solubilized human lung tissues by adsorption to TNF-Sepharose, followed by elution with low pH. By SDS-PAGE analysis, the two proteins had molecular weights of 75 and 55 kD. Using a soluble receptor assay, a binding affinity of approximately 1.2 nM was calculated for the isolated lung receptors. Each protein, isolated by electroelution from polyacrylamide gels, specifically bound TNF. Antibodies raised against the mixture of type I and II receptors bound specifically to both purified receptors by immunoblot analysis. Both the 75- and 55-kD receptors could be precipitated from 125I-surface-labeled or 35S-methionine-labeled U937 cells using TNF-Sepharose or anti-receptor antibodies. In addition, the anti-TNF receptor antibodies partially blocked binding of TNF to U937 cells and specifically immunoprecipitated 125I-TNF cross-linked to its receptors on U937 cells. These results demonstrate that both type I and II TNF receptors can be isolated from human lung tissue by ligand affinity chromatography, and that U937 cells express both TNF receptor types.

• Meager A
• Fourth International Congress on Tumour Necrosis Factor and Related Cytokines. Meeting at Koningshof, Veldhoven, The Netherlands 2-6 May 1992.
• Cytokine. 1992; 4: 413-7

• Andus T et al.
• High concentrations of soluble tumor necrosis factor receptors in ascites.
• Hepatology. 1992; 16: 749-55
• Display abstract

• Ascites and plasma concentrations of soluble tumor necrosis factor receptors p55 and p75 were measured in a prospective study in 34 patients (35 occasions of ascites) with hepatic (5 infected and 21 uninfected) and malignancy-related (9) ascites. All patients had high concentrations of both soluble tumor necrosis factor receptors in ascites and plasma; these were about 500 times higher than the corresponding tumor necrosis factor-alpha concentrations. Ascites levels of soluble tumor necrosis factor receptors p55 and soluble tumor necrosis factor receptors p75 were significantly elevated in patients with malignancy-related (p55: 26.0 +/- 8.6 ng/ml; p75: 20.5 +/- 17.4 ng/ml; mean +/- S.D.) and infected ascites (p55: 25.1 +/- 10.9 ng/ml, p75: 22.6 +/- 11.0 ng/ml) compared with patients with uncomplicated hepatic ascites (p55: 10.1 +/- 4.4 ng/ml; p75: 6.0 +/- 2.6 ng/ml). Patients with infected or malignancy-related ascites also showed higher soluble tumor necrosis factor receptor concentrations in plasma than did patients with plain hepatic ascites. Successful antibiotic treatment of peritonitis reduced soluble tumor necrosis factor receptor p55 and p75 ascites levels in three patients from 24.2 +/- 15.2 ng/ml to 10.7 +/- 1.9 ng/ml and from 20.2 +/- 14.4 ng/ml to 7.5 +/- 1.8 ng/ml, respectively. Soluble tumor necrosis factor receptors p55 and p75 at cutoff levels of 16.5 ng/ml and 9.5 ng/ml, respectively, differentiated between infected or malignant and plain hepatic ascites with diagnostic accuracies of 94% and 89%, respectively. They did not differentiate between infected and malignant ascites. The concentrations of soluble tumor necrosis factor receptor p55 were usually higher in ascites than in plasma in all subgroups of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

• Cope AP, Brennan FM
• Cytokine measurements in biological fluids.
• Br J Rheumatol. 1992; 31: 721-2

• Volpes R, van den Oord JJ, De Vos R, Desmet VJ
• Hepatic expression of type A and type B receptors for tumor necrosis factor.
• J Hepatol. 1992; 14: 361-9
• Display abstract

• We have examined the in-situ distribution of type A and type B receptors for tumor necrosis factor (TNF) in normal and diseased human liver biopsy specimens. In normal liver tissue, no or very small amounts of TNF receptors were found. In acute and chronic inflammatory liver diseases, a strong up-regulation of the expression of both TNF receptors was found on hepatocytes, bile duct epithelium, sinusoidal lining cells and mononuclear inflammatory cells. With immunoelectronmicroscopy, all these cells showed cytoplasmic, in addition to membranous staining, suggesting active synthesis of the receptor or, alternatively, internalization of the receptor and its ligand. This up-regulated expression of both type A and type B receptors for TNF was similar in acute and chronic active hepatitis, and was not related to the etiology of the liver disease, nor restricted to areas of liver inflammation. Our results indicate that hepatocytes, sinusoidal endothelial cells, bile duct epithelial cells and mononuclear inflammatory cells, by displaying receptors for TNFs, represent target cells for both these cytokines. Up-regulated expression of type A and type B receptors for TNFs endows these cells with augmented responsiveness for the pleiomorphic biological activities of these cytokines during liver injury.

• Farrah T, Smith CA
• Emerging cytokine family.
• Nature. 1992; 358: 26-26

• Fuchs P, Strehl S, Dworzak M, Himmler A, Ambros PF
• Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13 [corrected]
• Genomics. 1992; 13: 219-24
• Display abstract

• Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.

• Pennica D et al.
• Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.
• Biochemistry. 1992; 31: 1134-41
• Display abstract

• An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

• Owen-Schaub LB, Yonehara S, Crump WL 3rd, Grimm EA
• DNA fragmentation and cell death is selectively triggered in activated human lymphocytes by Fas antigen engagement.
• Cell Immunol. 1992; 140: 197-205
• Display abstract

• Fas is a mouse monoclonal antibody-defined cell surface antigen of an unknown physiologic function. Previous studies demonstrated that the anti-Fas antibody mediated apoptosis in those cells sensitive to tumor necrosis factor (TNF) and, further, triggered the co-downregulation of tumor necrosis factor receptors (TNF-Rs). These findings led to speculation that Fas may be associated with TNF-Rs. The present studies were undertaken as an extension of our previous work on the obligate requirement for TNF in development and maintenance of cytotoxic lymphocytes and were designed to analyze the expression and consequences of Fas engagement in these cells. Herein, we demonstrate that, in contrast to TNF-R expression, both resting and IL-2-activated lymphocytes express Fas. In accordance with previous studies using tumor cell lines, lymphocytes rapidly downregulate TNF-Rs after treatment with anti-Fas. The ability of anti-Fas to mediate apoptotic cell death in lymphocytes, however, was dependent upon the status of cellular activation. For example, lymphocytes activated in IL-2 for longer than 4 days underwent rapid DNA fragmentation and cell death after anti-Fas treatment. Despite their expression of Fas, nonactivated lymphocytes and those activated for periods less than 4 days were refractory to antibody-mediated cell killing. Because anti-Fas-mediated lethality is selective for chronically activated lymphocytes, Fas may prove to be an appropriate target for immunosuppressive intervention.

• Heller RA, Song K, Freire-Moar J
• Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.
• Biotechniques. 1992; 12: 3032345-3032345
• Display abstract

• The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.

• Spinas GA, Keller U, Brockhaus M
• Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxinemia.
• J Clin Invest. 1992; 90: 533-6
• Display abstract

• Serial plasma samples from human volunteers obtained after intravenous administration of Escherichia coli endotoxin were analyzed for the presence of circulating soluble tumor necrosis factor receptors (sTNFR). A four- to fivefold increase of type A (p75) and type B (p55) sTNFR was observed 3 h after endotoxin challenge. Pretreatment of the volunteers with ibuprofen before the injection of endotoxin resulted in a slight increase (3.87 +/- 0.2 vs. 3.27 +/- 0.3 ng/ml) and temporal shift of sTNFR-A release concurrent to a marked augmentation of TNF levels (603 +/- 118 vs. 338 +/- 56 pg/ml) as compared to the group without ibuprofen pretreatment. There was a significant correlation between peak sTNFR-A levels and peak TNF levels in the individual probands (r = 0.52, P = 0.04). On the contrary, release kinetics and plasma concentrations of sTNFR-B were identical in both groups (7.38 +/- 0.69 vs. 7.44 +/- 0.33 ng/ml) and no correlation with individual TNF levels was observed. The amount of sTNFR liberated upon endotoxin challenge was not sufficient to block TNF-mediated cytotoxic effects. Our data indicate that the release in vivo of type A and type B sTNFR upon a short exposure to endotoxin is regulated differently.

• Branellec D, De Cremoux P, Barreau P, Calvo F, Chouaib S
• Tumor necrosis factor-mediated cell lysis in vitro: relationship to cAMP accumulation and guanine nucleotide-binding proteins.
• Eur J Immunol. 1992; 22: 963-7
• Display abstract

• We have investigated the modulation of tumor necrosis factor (TNF)-mediated tumor cell lysis by cAMP. Among a panel of human breast tumor cell lines, MCF7 and MDA MB 231 were shown to be, respectively, sensitive and resistant to TNF-mediated cell lysis in vitro. 125I-labeled TNF-binding experiments demonstrated that both cell lines bind TNF, indicating that the differential sensitivity to TNF was not related to TNF receptor expression. To study the relationship between TNF-mediated cell lysis and cAMP accumulation, cAMP measurement was performed following TNF treatment. Our data show that TNF alone did not induce an enhancement of intracellular cAMP accumulation either in the TNF-sensitive or in the TNF-resistant cell line. Experiments in which cells were exposed to forskolin revealed that this cAMP elevating drug was efficient in enhancing the sensitivity to TNF of MCF7 cell line. This potentiating effect of forskolin was maximal for suboptimal concentrations of TNF (10 ng/ml), reaching up to 100% when forskolin was added at 100 microM. However, co-stimulating with forskolin of either MDA MB 231 or a TNF-resistant MCF7 clone (MCF7-R-A1) did not induce any reversal of resistance to TNF. We further assessed the interaction of TNF with transmembrane signalling and the possible involvement of guanine nucleotide-binding proteins (G-proteins). Bacterial toxin-catalyzed ADP ribosylation of MCF7 and MDA MB 231 membranes was, therefore, performed. Using cholera toxin, we demonstrate that TNF treatment did not quantitatively alter the activity of stimulatory G-proteins either in MCF7 or MDA MB 231 cell line. In contrast, pertussis toxin-catalyzed ADP ribosylation experiments suggest a functional coupling of TNF receptors to a 40-kDa pertussis toxin-sensitive G-protein in the TNF-sensitive MCF78 cell line but not in the TNF-resistant MDA MB 231 cell line. Taken together, these data indicate that cAMP might play a role in TNF-mediated cell lysis and are in support of the involvement of a pertussis toxin-sensitive G-protein in TNF-mediated MCF7 cells lysis.

• Decker K
• [Tumor necrosis factor alpha]
• Schweiz Rundsch Med Prax. 1992; 81: 1044-7
• Display abstract

• TNF-alpha (Cachectin) is a cytokine (tissue hormone) synthesized and secreted as a trimeric polypeptide; major producers are the cells of the RES after stimulation, e.g. by endotoxin. Its synthesis is strictly regulated. TNF elicits a great number of cell-specific responses. IL-1 and probably additional mediators cooperate in these effects. TNF is instrumental not only in the pathogenesis of inflammation, infections and some cell injuries, but also in the unspecific immune response, tumor toxicity and tissue homeostasis. TNF binding to specific receptors on cell surfaces initiates of secondary intracellular signals that, in turn, mediate the metabolic responses of the target cells. Pharmacological intervention may be envisaged at the level of synthesis (glucocorticoids, anti-oxidants) or interaction of the cytokine with its receptors.

• Smith RA, Baglioni C
• Characterization of TNF receptors.
• Immunol Ser. 1992; 56: 131-47

• Loetscher H
• Purification and structural properties of two distinct TNF receptors on human cells.
• Immunol Ser. 1992; 56: 161-87

• Kronke M, Schutze S, Scheurich P, Pfizenmaier K
• TNF signal transduction and TNF-responsive genes.
• Immunol Ser. 1992; 56: 189-216

• Pocsik E, Higuchi M, Aggarwal BB
• Down-modulation of cell surface expression of p80 form of the tumor necrosis factor receptor by human immunodeficiency virus-1 tat gene.
• Lymphokine Cytokine Res. 1992; 11: 317-25
• Display abstract

• Tumor necrosis factor-alpha (TNF-alpha) induces the expression of human immunodeficiency virus type-1 (HIV-1) in vitro in chronically infected cells of T and monocytic origin. The tat protein from the HIV-1 virus has been shown to be essential for HIV replication and in the immunosuppression associated with the virus infection. Previous studies in our laboratory have shown that HIV-1 tat gene induces TNF-beta (lymphotoxin) in human B-lymphoblastoid cells (Sastry et al., 1990, J. Biol. Chem. 265, 20091-20093). In an attempt to characterize further the relationship between the host and HIV-1, we investigated the effect of the functional HIV-1 tat gene on the expression of TNF receptors in a human B lymphoblastoid cell line (Raji). We report here that Raji cells transfected with HIV-1 tat gene express fewer cell surface TNF receptors than control cells. At least a 5-fold decrease in the receptor number without any significant change in receptor affinity was observed. The decrease in TNF receptors in tat-transfected Raji cells (Raji-tat cells) was found not to be due to receptor occupancy by the autocrine production of TNF-beta. The decrease in the cell surface expression of TNF receptors in Raji-tat cells was also found to be not due to a decrease in the gene expression of the receptor. The kinetics, amount of TNF binding and its internalization were temperature dependent, and it was different in Raji-tat cells than in the control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

• Grosen EA et al.
• Blocking factors (soluble membrane receptors) for tumor necrosis factor and lymphotoxin detected in ascites and released in short-term cultures obtained from ascites and solid tumors in women with gynecologic malignancy.
• Lymphokine Cytokine Res. 1992; 11: 347-53
• Display abstract

• Studies were conducted to identify and establish the cell and tissue source of blocking factors (BF), materials that inhibit the bioactivity of human TNF and LT in vitro. Ascites and samples of solid tumors were collected from women with various gynecologic malignancies. Supernatants were collected from cultures of tumor and ascites cells after 24, 48, and 72 h. Cell-free ascites (CFA) and culture supernatants were tested for their ability to block human recombinant TNF and LT-induced lysis of L929 cells in vitro. Levels of soluble forms of the 55- and 75-kDa TNF/LT receptors were measured by ELISA assay in the same samples. CFA and culture supernatants contained TNF/LT blocking factors and high levels of one or both soluble 55- and 75-kDa TNF/LT membrane receptors. Levels of BF bioactivity and receptors appeared rapidly, peaked at 24 h, and declined thereafter. Soluble TNF/LT receptors may be the active BF in these samples, and tumor tissues and ascitic cells may be a source of these receptors in the ascites fluid of these patients.

• Lie BL et al.
• Identification of the binding site of 55kDa tumor necrosis factor receptor by synthetic peptides.
• Biochem Biophys Res Commun. 1992; 188: 503-9
• Display abstract

• We have synthesized a series of peptides, which cover almost the whole range of the N-terminal extracellular domain of human 55kDa TNF receptor (55kDa TNF-R). The peptides were examined for the binding activity to TNF by solid phase binding assay and for the inhibition of TNF cytotoxicity to mouse L-M cells. The peptide 159-178 exhibited remarkably higher binding activity to TNF than other peptides did. The specificity of the TNF binding to the peptides was confirmed by their inability to bind other cytokines. The peptide 159-178 also inhibited TNF cytotoxicity. These results indicate that the specific binding site of 55kDa TNF-R to TNF might reside within the peptide segment of amino acid numbers 159 to 178 in the N-terminal extracellular domain.

• Marsters SA, Frutkin AD, Simpson NJ, Fendly BM, Ashkenazi A
• Identification of cysteine-rich domains of the type 1 tumor necrosis factor receptor involved in ligand binding.
• J Biol Chem. 1992; 267: 5747-50
• Display abstract

• The extracellular portion of the type 1 (p55) and type 2 (p75) tumor necrosis factor (TNF) receptors contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs). This pattern is found also in several other cell surface proteins, including the p75 nerve growth factor receptor and the CD40, 4-1BB, OX40, Fas, and CD27 antigens. To investigate whether CRDs play a role in TNF binding, we have constructed soluble variants of the extracellular portion of human type 1 TNF receptor (sTNFR1), in which CRD1 (N-terminal) or CRD4 (C-terminal) was deleted by mutagenesis. These variants or a wild type sTNFR1 were linked in their C terminus to the hinge and Fc portion of IgG1 heavy chain to create sTNFR1-IgG chimeras (immunoadhesins). Deletion of either CRD1 or -4 did not cause any major perturbations in the structure of the sTNFR1 variants, as evidenced by their efficient expression and secretion from transfected cells, and by their binding to conformation-dependent monoclonal antibodies that recognize diverse epitopes on sTNFR1. The wild type sTNFR1 immunoadhesin exhibited high affinity binding to TNF alpha (Kd = 65 pM) and TNF beta (Kd = 640 pM). Deletion of CRD4 resulted in about a 10-fold reduction in affinity for TNF alpha (Kd = 660 pM) and for TNF beta (Kd = 5.7 nM). In contrast, deletion of CRD1 resulted in a complete loss of binding to TNF alpha and to TNF beta. These results indicate that CRD4 is important but not necessary for TNF binding, while CRD1 is required. In addition, the results suggest some overlap between the TNFR1 binding sites for TNF alpha and TNF beta, despite low amino acid sequence homology between these cytokines.

• Vandenabeele P et al.
• Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma.
• J Exp Med. 1992; 176: 1015-24
• Display abstract

• We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines. Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF. After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF. The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1. In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells. This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies. These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

• Waage A, Liabakk N, Lien E, Lamvik J, Espevik T
• p55 and p75 tumor necrosis factor receptors in patients with chronic lymphocytic leukemia.
• Blood. 1992; 80: 2577-83
• Display abstract

• We studied the expression of the two tumor necrosis factor (TNF) receptors, p55 and p75, on B cells from patients with chronic lymphocytic leukemia (CLL), and the presence of soluble TNF receptors in serum. Expression of membrane-associated receptors was quantified by double labeling of peripheral blood mononuclear cells (PBMC) with monoclonal antibodies against CD19 and p55/p75 TNF receptors and flow cytometry. A high fraction of the CD19+ cells expressed the p55 receptor (44% +/- 34% [SD]) and p75 receptor (61% +/- 31%). In healthy controls, 0% to 1% of the CD19+ cells expressed the p55 receptor and 0% to 10% expressed the p75 receptor. Incubation of CD19+ cells with 10 ng/mL of TNF increased the incorporation of thymidine in 11 patients tested, and this was decreased to 65% (P < .05) by antibodies to the p55 receptor or the p75 receptor, and to 35% +/- 7% (P < .001) when both antibodies were combined. With an enzyme-linked immunoassay, we measured soluble TNF receptors in serum from CLL patients. The mean level of p55 receptors was increased to 12.9 +/- 8.9 ng/mL (P < .000001 v normal). The mean level of p75 receptors was increased to 13 +/- 24 ng/mL (P = .01 v normal). The membrane expression of the two receptors was positively correlated (r +/- 97, P < .01); however, there was no correlation between membrane expression and serum concentration of either receptor. Autologous serum containing high levels of soluble TNF receptors inhibited TNF-induced proliferation of CD19+ cells. In conclusion, we have demonstrated that neoplastic cells from patients with CLL have increased expression of p55 and p75 TNF receptors, and that both receptors mediate signal to proliferation. Furthermore, serum from CLL patients has elevated levels of soluble TNF receptors, which may counteract the proliferative effect of TNF.

• Cairns JA, Guy GR, Tan YH
• Interleukin-6 regulates the cytotoxic effect of tumour necrosis factor on U937 cells.
• Immunology. 1992; 75: 669-73
• Display abstract

• In this study, we demonstrate that low but not high concentrations of interleukin-6 (IL-6) potentiate the cytotoxic effect of tumour necrosis factor-alpha (TNF-alpha) on U937 cells, in a dose-dependent manner. Killing of U937 cells by 100 U/ml of TNF-alpha, was maximally potentiated by 50 U/ml of IL-6. No potentiation of cell killing was observed when the concentration of IL-6 was increased to 4000 U/ml. At a concentration of 50 U/ml, IL-6 up-regulated TNF receptor expression but no change in TNF receptor number was observed when the concentration of IL-6 was increased to 4000 U/ml. Low concentrations of IL-6 can also induce sub-cytotoxic doses of TNF-alpha (0.1 and 0.33 U/ml) to kill U937 cells. Up-regulation of TNF receptors by IL-6 is dependent on de novo protein synthesis since receptor induction is abolished in the presence of cycloheximide. Taken together the data suggest that the potentiation of cell killing observed by a combination of these lymphokines is mediated in part by IL-6-induced changes in TNF receptor expression.

• Balkwill FR
• Tumour necrosis factor and cancer.
• Prog Growth Factor Res. 1992; 4: 121-37
• Display abstract

• TNF is a cytokine whose diverse actions are dependent on the local microenvironment. As a member of the cytokine network, TNF plays an important role in infection and inflammation, but excessive and deregulated production can contribute to disease processes. Likewise in malignant disease, TNF may have a role in cancer therapy and contribute to host response against tumours, but it may also be involved in the progression and spread of the cancer. In experimental models, recombinant TNF can induce significant haemorrhagic necrosis, localised to the tumour vasculature and specific tumour immunity. Although the historical background and preclinical data are promising, systemic therapy with TNF in human cancer has proved highly toxic and is inactive against all tumour types so far tested. Local therapy, particularly isolated limb perfusion, has resulted in complete and long lasting tumour regressions with necrotic activity confined solely to the tumour vascular bed. However, in several animal models, TNF contributes to malignant progression and there is evidence that TNF may have autocrine or paracrine actions in human ovarian cancer.

• Hwu P, Schwarz S, Custer M, Smith CA, Mule JJ, Rosenberg SA
• Use of soluble recombinant TNF receptor to improve detection of TNF secretion in cultures of tumor infiltrating lymphocytes.
• J Immunol Methods. 1992; 151: 139-47
• Display abstract

• The accurate quantitation of picogram amounts of TNF is possible by ELISA and is useful in many areas of biomedical research, including studies of TNF release in vitro by stimulated lymphocytes and macrophages, and of serum levels in patients with cancer and sepsis. However, we show in this report that the detection of recombinant TNF standards by ELISA falls over time with incubation at 37 degrees C, and is further decreased when incubated with tumor infiltrating lymphocytes (TIL), making accurate quantitation difficult. We demonstrate that the soluble dimeric form of the TNF receptor can prevent this decrease, both in the presence and absence of TIL. In contrast, the soluble monomeric TNF receptor was much less effective in preventing this decrease. In addition, the dimeric but not the monomeric TNF receptor was found to inhibit bioactivity of TNF as measured by L929 cytotoxicity. The dimeric TNF receptor does not interfere with the detection of recombinant TNF standards by ELISA, and entirely stabilizes TNF levels incubated over 48 h at 37 degrees C in the presence and absence of TIL. This protection is specific, and the TNF receptor does not stabilize interferon-gamma. The dimeric form of the soluble TNF receptor has proven useful in detecting TNF released by TIL transduced with the TNF cDNA that are currently being used in studies of the gene therapy of cancer with TIL. The dimeric TNF receptor may also prove useful in the accurate quantitation of TNF released by stimulated lymphocytes and macrophages in vitro, and in the quantitation of serum TNF levels in patients.

• Corti A, Fassina G, Marcucci F, Cassani G
• Antigenic regions of tumor necrosis factor alpha and their topographic relationships with structural/functional domains.
• Mol Immunol. 1992; 29: 471-9
• Display abstract

• The immunogenic regions of human Tumor Necrosis Factor alpha (huTNF) have been mapped by studying the interaction between various mouse anti-huTNF sera and synthetic huTNF fragments, spanning the entire sequence of huTNF. Three main immunogenic regions were identified within residues 1-23, 95-116 and 137-157 of huTNF and two other less immunogenic regions within residues 117-136 and 37-55. The same huTNF regions were found to contain antigenic sites by binding studies with cognate anti-peptide sera. Competitive binding experiments with shorter synthetic subfragments provided evidence for the location of strong antigenic sites within residues 1-10, 17-23, 104-112 and 137-143. In particular the immunodominant site was found to be located within residues 104-112. huTNF regions corresponding to residues 24-36, 56-75, 76-94, and 147-157 resulted to be not or poorly antigenic. However, treatment of huTNF with Triton X-100 under conditions that partially dissociate the oligomeric quaternary structure resulted in the exposition of sites recognized by sera against peptides huTNF [56-75] and huTNF [76-94], suggesting that antigenic sites not accessible in the oligomeric huTNF are exposed in the dissociated form. The principal antigenic sites in the oligomeric molecule are localized in the flexible N-terminal part and in hydrophilic segments located in the "middle/top" region of the molecule, opposite to the C-terminus. Protein segments of the "bottom" region, close to the C-terminus, were poorly immunoreactive. Neutralization assays of TNF cytolytic activity on L-M cells showed that binding of antibodies to epitopes located in the "middle/top" regions of huTNF does not affect its cytolytic activity, supporting the hypothesis of a receptor binding site location at the "bottom" of TNF trimer.

• Deloukas P, Aigner L, Kania M, van Loon AP
• Construction of yeast artificial chromosome (YAC) clone banks covering three genome equivalents and isolation of YACs containing the human gene encoding tumor necrosis factor receptor beta.
• Gene. 1992; 110: 189-95
• Display abstract

• Yeast artificial chromosome (YAC) banks covering in total about three haploid genome equivalents were constructed using a human Epstein-Barr-virus-transformed B lymphocytic cell line. Two clone banks were made: 20,000 clones with average inserts of 350 kb in the pYAC4 vector and 9850 clones with average inserts of 180 kb using vectors pJS89 and pJS91. Direct comparison of pYAC4 with pJS89 and pJS91 showed pYAC4 to be the most suitable cloning vector. Two partial banks with average insert sizes of 220 kb for human endothelial cell DNA and epithelial HEp2 cell DNA were also constructed, each covering 10% of the haploid genome. A rapid, three-step PCR screening procedure for isolation of individual YAC clones was developed and used to identify two clones encoding TNF-R beta. These clones cover about 200 kb and have 170 kb in common. TNF-R beta is 9.3 kb long and contains two introns within the protein-coding sequence.

• Heller RA, Song K, Fan N, Chang DJ
• The p70 tumor necrosis factor receptor mediates cytotoxicity.
• Cell. 1992; 70: 47-56
• Display abstract

• Tumor necrosis factor alpha (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface receptors (TNFRs), proteins of 55 kd (p55) and 70-80 kd (p70), mediate TNF action. Mouse TA1 cells are not killed by human (h) TNF, but are killed by mouse (m) TNF alone. Since the mouse p70 TNFR is recognized only by mTNF, these results implicate p70 receptor action in TA1 cell killing. Human HeLa cells have mainly the p55 receptor and are not killed by hTNF alone. When transfected with the human p70 TNFR, HeLa p70 die within 24 hr. HeLa p70 cells also show reduced c-fos and manganous superoxide dismutase induction by TNF. NIH 3T3 mouse fibroblasts are sensitive to only mTNF, but overexpression of the human p70 receptor causes cell death by hTNF and increased sensitivity to mTNF. These results provide a direct function for the p70 TNFR in TNF-induced cytotoxicity.

• Vanhaesebroeck B et al.
• Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.
• J Immunol. 1992; 148: 2785-94
• Display abstract

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

• Schleiffenbaum B, Olgiati L, Fehr J
• TNF-specific deactivation of granulocytes in vivo: a possible mechanism of self-protection.
• Eur J Haematol. 1992; 49: 239-45
• Display abstract

• Granulocytes (PMN) have recently been shown to be specifically deactivated towards tumor necrosis factor (TNF) by preexposure to TNF itself and to other stimuli such as endotoxin and complement C5a in vitro. When TNF (200 micrograms/m2) was infused to a male volunteer, we found ex vivo an almost complete TNF-specific deactivation of PMN adhesion (57 +/- 3* vs 6 +/- 2%**), of exocytosis of secondary granules (37 +/- 1* vs 7 +/- 1%**) and of the respiratory burst as measured by hexose monophosphate shunt activation (74 +/- 1* vs 12 +/- 3** nM/10(7) PMN/45 min). Furthermore, up- and down-regulation, respectively, of the PMN surface adhesion molecules Mac-1 and LAM-1 became TNF-resistant. Similar to the results obtained in vitro, deactivation correlated with a decrease in the number of cellular TNF receptors. In PMN of 4 patients exposed to activated complement during hemodialysis, a state of TNF-specific cross-deactivation could also be demonstrated.

• Higuchi M, Aggarwal BB
• Modulation of two forms of tumor necrosis factor receptors and their cellular response by soluble receptors and their monoclonal antibodies.
• J Biol Chem. 1992; 267: 20892-9
• Display abstract

• Recently, two different receptors for human tumor necrosis factor (TNF) with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. In this report, we investigated the effect of the soluble forms of these receptors and monoclonal antibodies against them on ligand interaction, receptor down-regulation, and mediation of cellular response in U-937 cells. Our results indicate that p60 and p80 constitute 20-30 and 60-80% of the total TNF-binding sites on U-937 cells, respectively. However, by cross-linking, only the p80 form of the receptor could be detected. In contrast to unlabeled TNF, the anti-p60 and anti-p80 antibodies together only partially inhibited ligand binding, and this inhibition was not additive. Lack of additive inhibition of binding was found to be not due to stereo-chemical hindrance. TNF binding to cells can be completely displaced by soluble forms of either the p60 or p80 receptor. However, 100-fold more of the p80 than the p60 form of the soluble receptor is needed for equivalent displacement. Under optimum conditions, TNF and the anti-p80 and anti-p60 antibodies down-regulated 30, 80, and 20% of the TNF receptors, respectively. The anti-p60 and anti-p80 antibodies down-regulated not only their own receptors, but also reciprocal receptors, suggesting a cross-communication between the p60 and p80 forms of the TNF receptor. In spite of inhibiting as much as 80% of TNF binding, none of the receptor antibodies significantly inhibited the cytotoxic response to TNF in U-937 cells. Soluble forms of both receptors, however, completely abrogated the cellular response to TNF. Thus, overall, our results indicate that the antibodies against both receptors together inhibit the majority of the receptor-ligand interaction without any significant effect on the biological response to TNF.

• Pennica D et al.
• Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.
• J Biol Chem. 1992; 267: 21172-8
• Display abstract

• An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.

• Austgulen R, Liabakk NB, Brockhaus M, Espevik T
• Soluble TNF receptors in amniotic fluid and in urine from pregnant women.
• J Reprod Immunol. 1992; 22: 105-16
• Display abstract

• Secretion of soluble cytokine receptors has been suggested as a mechanism for regulation of cytokine activity in vivo. The present investigation was performed to study whether secretion of soluble TNF (tumor necrosis factor) receptors (TNFRs) might be associated with pregnancy. There are two known molecular species of the TNFR, the 55-kDa TNFR and the 75-kDa TNFR. The 75-kDa, as well as the 55-kDa TNFR, was detected in urine from pregnant women, whereas only the 75-kDa TNFR was detected in urine from the non-pregnant group. The concentration of TNFRs in urine increased towards term and was reduced in association with spontaneous delivery. The soluble forms of both TNFRs were also detected in amniotic fluid. Collectively, the data suggest that secretion of soluble TNFRs during pregnancy might be a defence mechanism for the protection of the fetus against TNF action.

• Winstanley FP
• Detection of TNF alpha receptors on ovine leucocytes by flow cytofluorimetry.
• Res Vet Sci. 1992; 52: 129-31
• Display abstract

• Tumour necrosis factor receptor activity was detected on sheep leucocytes using flow cytofluorimetry and commercially available fluorescent human tumour necrosis factor. The density of receptors per cell and the proportion of cells expressing TNF receptor activity was found to be in the following rank order: monocytes greater than granulocytes greater than lymphocytes.

• Schutze S, Machleidt T, Kronke M
• Mechanisms of tumor necrosis factor action.
• Semin Oncol. 1992; 19: 16-24
• Display abstract

• Tumor necrosis factor (TNF) is able to induce a great diversity of cellular responses via modulating the expression of a number of different genes. The multitude of TNF activities may be explained by both structural and functional heterogeneity in TNF receptors as well as by a diversification of postreceptor signal transduction pathways. Purification of TNF receptors has revealed two major, distinct binding proteins (TR60 and TR80). TR60 seems to be an essential component for TNF signaling; the functional role of TR80 remains to be elucidated. The pathway of postreceptor signal transduction involves phospholipase A2, a phosphatidylcholine-specific phospholipase C, protein kinase C, and other serine/threonine and tyrosine-specific protein kinases with as yet unknown function. At the receiving end of TNF signaling, induction of gene expression is mediated through activation of nuclear transcription factors, such as NFkB, AP-1, IRF-1, and NF-GMa.

• Okamoto T et al.
• Endogenous tumor necrosis factor exerts its protective function intracellularly against the cytotoxicity of exogenous tumor necrosis factor.
• Cancer Res. 1992; 52: 5278-81
• Display abstract

• Based on the findings that expression of endogenous tumor necrosis factor (enTNF), which is not present in TNF-susceptible cells, was generally observed in TNF-resistant cells and that TNF gene transfection gives rise to TNF resistance, the assumption was made that enTNF may be a protective protein against the cytotoxicity of exogenous TNF. However, it remains unknown whether the protection by enTNF is exerted in an intracellular or extracellular (autocrine) manner. We therefore transfected a nonsecretory human TNF gene (pTNF delta pro) into highly TNF-sensitive mouse tumorigenic fibroblasts (L-M cells) and investigated their TNF susceptibility. The transfectants expressed enTNF which was not secreted into the medium and acquired an appreciable degree of resistance to exogenous TNF. A significant increase in the manganous superoxide dismutase level was also noted in the transfectants. These findings suggest that enTNF exerts its protective function intracellularly by inducing manganous superoxide dismutase production.

• Hayakawa M, Hori T, Shibamoto S, Tsujimoto M, Oku N, Ito F
• Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.
• Arch Biochem Biophys. 1991; 286: 323-9
• Display abstract

• Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.

• Heilig B, Mapara M, Brockhaus M, Krauth K, Dorken B
• Two types of TNF receptors are expressed on human normal and malignant B lymphocytes.
• Clin Immunol Immunopathol. 1991; 61: 260-7
• Display abstract

• We analyzed the expression of the 75- and 55-kDa tumor necrosis factor (TNF) receptors on the surface of human normal and malignant B cells. Freshly isolated tonsillar B cells do not express detectable amounts of receptors. After 24 hr of stimulation with anti-IgM and low-molecular-weight (l.m.w.) BCGF, B cells express the 75-kDa receptor but not the 55-kDa receptor, but after 48 hr of stimulation both types of receptors are expressed. Three of 10 patients with B-chronic lymphatic leukemia or hairy cell leukemia (HCL) had a small population of TNF receptor positive tumor cells; after in vitro stimulation of tumor cells in 10/10 patients various patterns of 75- and 55-kDa TNF receptor expression were found. MAb UTR-1, which binds the 75-kDa receptor, inhibits the l.m.w. BCGF-induced [3H]thymidine incorporation of cultured tonsillar B cells and HCL cells. This may provide evidence that in B cells the 75 kDa TNF receptor molecule is functionally active.

• Baker E, Chen LZ, Smith CA, Callen DF, Goodwin R, Sutherland GR
• Chromosomal location of the human tumor necrosis factor receptor genes.
• Cytogenet Cell Genet. 1991; 57: 117-8
• Display abstract

• TNFR1 and TNFR2, the genes encoding the two forms of the human tumor necrosis factor receptor, were localized to normal human chromosomes by in situ hybridization and Southern blot analysis of a series of human x mouse hybrid cell lines. TNFR1 maps to 12p13 and TNFR2 maps to 1p36.

• Milatovich A, Song K, Heller RA, Francke U
• Tumor necrosis factor receptor genes, TNFR1 and TNFR2, on human chromosomes 12 and 1.
• Somat Cell Mol Genet. 1991; 17: 519-23
• Display abstract

• Tumor necrosis factor, TNF, is a 17-kDa protein secreted by macrophages and classified as a cytokine. TNF binds to high-affinity receptors on the cell surface and is involved in a wide variety of biological responses. There are at least two types of receptors, tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2). The genes for TNFR1 a 55-kDa protein, and TNFR2, a 70-kDa protein, have been mapped to human chromosomes 1 12 (12pter-cen) and (1pter-p32), respectively, by Southern blot analysis of human x Chinese hamster somatic cell hybrid panels. Recently, the corresponding genes in the mouse have been mapped to chromosomes 4 and 6 in regions that are conserved on human chromosomes 1 and 12.

• Ryffel B, Brockhaus M, Greiner B, Mihatsch MJ, Gudat F
• Tumour necrosis factor receptor distribution in human lymphoid tissue.
• Immunology. 1991; 74: 446-52
• Display abstract

• The nature and location of cells responding to tumour necrosis factor-alpha (TNF-alpha) were investigated in situ by immunohistochemistry using monoclonal antibodies (mAb) directed against the p75 and p55 proteins of the TNF receptor. Receptor expression was found in the thymus and secondary lymphoid tissues. In the thymus the p75 receptor was confined to medullary lymphoblasts and dendritic cells, which co-stain with the Tac protein of the interleukin-2 (IL-2) receptor. In lymph nodes and other secondary lymphoid tissues, the p75 receptor was expressed on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal centre dendritic reticulum cells (DRC), which is the main site of TNF-alpha production. TNF receptor (TNFR) proteins were up-regulated in reactive hyperplasia together with increased TNF-alpha expression. Surprisingly, no TNFR was detectable on non-lymphoid tissues. The species specificity of these TNFR antibodies was high: whereas the antibodies cross-reacted with epitopes in non-human primates, no immunoreactivity was detected in lower animal species, e.g. dog, rabbit and rodents. The data presented suggest that TNF-alpha, which is produced by germinal centre DRC, might regulate an in vivo immune response through autocrine and paracrine pathways, e.g. through the p55 and p75 receptor proteins, which are expressed at different sites of the lymphoid tissue.

• Aderka D et al.
• Increased serum levels of soluble receptors for tumor necrosis factor in cancer patients.
• Cancer Res. 1991; 51: 5602-7
• Display abstract

• Soluble forms of the two molecular species of the cell surface receptors for tumor necrosis factor (TNF) have been detected in normal urine. Using enzyme-linked immunosorbent assays for these soluble receptors, we determined their levels in the sera of 40 healthy subjects and 59 patients with solid tumors. The mean +/- SD concentrations of both the soluble type I (p55) and type II (p75) receptors were significantly higher in the cancer patients than in the healthy controls: 1.96 +/- 1.19 versus 0.79 +/- 0.19 ng/ml (P less than 0.001) and 6.43 +/- 4.8 versus 3.2 +/- 0.6 ng/ml (P less than 0.001), respectively. The incidence and the extent of the increase correlated with the staging of disease. Sera of the cancer patients had a marked inhibitory effect on the in vitro cytocidal activity of TNF. This inhibition was proportional to the content of soluble TNF receptors and could be fully abolished by the addition to the sera of specific antibodies against the receptors. Among the cancer patients, the incidence of increase in the concentrations of soluble TNF receptors (about 70%) greatly exceeded that of the serum carcinoembryonic antigen (about 26%), a commonly used cancer marker. The origin of the serum soluble TNF receptors in cancer patients and the physiological implications of their effect on TNF function remain to be elucidated.

• Gatanaga T, Hwang CD, Gatanaga M, Cappuccini F, Yamamoto RS, Granger GA
• The regulation of TNF receptor mRNA synthesis, membrane expression, and release by PMA- and LPS-stimulated human monocytic THP-1 cells in vitro.
• Cell Immunol. 1991; 138: 1-10
• Display abstract

• The regulation of the 55-kDa TNF receptor (TNF-R) mRNA synthesis, membrane expression, and TNF binding factor (BF) release was examined in resting and activated human monocytic THP-1 and human promyelocytic leukemia HL-60 cells in vitro. Cells were activated with phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS). TNF alpha cytolytic activity in the supernatant of THP-1 cells stimulated by PMA began to appear at 4 hr, reached a peak at 8 hr, and declined by 12 hr. For THP-1 cells stimulated with LPS, the peak of TNF alpha activity appeared at 4 hr and then declined. TNF alpha-binding sites on the cell membrane were down-regulated within 1 hr after PMA and LPS treatment and then reappeared 12 hr later. Fifty-five-kilodalton TNF-R mRNA expression during this time period did not correlate with the level of membrane TNF-binding site expression. Additional studies indicated the presence of a 30-kDa TNF-BF in the supernatants which appeared after 24 hr. These data suggest that activated THP-1 and HL-60 cells are capable of releasing TNF-BF into the supernatant and this material may be involved in the control of secreted TNF alpha activities.

• Rock CS, Lowry SF
• Tumor necrosis factor-alpha.
• J Surg Res. 1991; 51: 434-45

• Elsasser TH, Caperna TJ, Fayer R
• Tumor necrosis factor-alpha affects growth hormone secretion by a direct pituitary interaction.
• Proc Soc Exp Biol Med. 1991; 198: 547-54
• Display abstract

• Administration of 50, 250, and 1,250 ng/kg iv of recombinant bovine tumor necrosis factor-alpha (RBTNF) did not affect basal plasma concentrations of growth hormone (GH) or thyroid-stimulating hormone in male calves. However, when administered 30 min before challenge with 1 microgram/kg iv of thyrotropin-releasing hormone (TRH), 250 ng/kg of RBTNF increased the subsequent incremental GH response. At 1,250 ng/kg of RBTNF, GH response to TRH was significantly blunted. For each dose of RBTNF administered, the incremental change in plasma thyroid-stimulating hormone following TRH was not significantly different from control. To examine direct effects of RBTNF on pituitary function, fresh bovine pituitaries were sliced into 1-mm cubes and incubated with 0 or 10(-8), 10(-9), or 10(-10) M RBTNF. Additional cultures were treated with 10(-8) or 10(-9) M GH-releasing factor or 10(-8) M TRH and 0 or 10(-8) M RBTNF. Media GH increased in cultures with 10(-10) M RBTNF and declined linearly as RBTNF concentration increased. RBTNF blocked GH release from GH-releasing factor- and TRH-challenged pituitary slices. Membranes prepared from homogenized bovine pituitaries had specific saturable binding characteristics for monomeric 125I-RBTNF. Membranes treated with 4 M MgCl2 for 10 min and washed free of Mg2+ produced Scatchard plots fit to a two-site model (high affinity site Kd = 6.6 nM), while Scatchards of non-Mg(2+)-treated membranes fit a single site (Kd = 8.9 nM). Polyacrylamide gel electrophoresis separation of 125I-RBTNF cross-linked pituitary membranes showed specific binding of monomeric 125I-RBTNF to protein components ranging in molecular weight from 19,000 to 77,000. The data suggest that RBTNF has modulatory effects on the regulation of GH secretion acting directly at the pituitary through specific receptors.

• Nakamura S et al.
• Interactions between novel tumor necrosis factor-alpha mutants and receptors on tumor and normal cells.
• Agric Biol Chem. 1991; 55: 53-7
• Display abstract

• We prepared several TNF mutants by protein engineering techniques and compared their biological properties with those of the wild-type TNF. The mutant that lacked 7 N-terminal amino acids had higher cytotoxicity and higher binding activity to receptors on tumor cells. In contrast, the mutagenesis of Arg32 or Ala84, in combination with the deletion of 7 N-terminal amino acids, eliminated the cytotoxicity and the receptor binding. These mutants also lacked acute lethal toxicity in normal mice. Therefore, we concluded that the N-terminus, Arg32 and Ala84 of TNF might be concerned with binding to the TNF receptor. It was also suggested that the receptor molecules on tumor cells bound to the same or neighboring sites on TNF molecules as normal cell receptors.

• Soliven B, Szuchet S, Nelson DJ
• Tumor necrosis factor inhibits K+ current expression in cultured oligodendrocytes.
• J Membr Biol. 1991; 124: 127-37
• Display abstract

• The effects of tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated macrophages, on the electrical membrane properties of cultured adult ovine oligodendrocytes (OLGs) were investigated using the whole-cell voltage-clamp technique. Treatment with recombinant human TNF-alpha (rhTNF) for 24 to 72 hr produces (i) process retraction in some but not all OLGs, (ii) a reduction in the resting membrane potential with no significant change in membrane capacitance or input resistance over control cells and (iii) a decrease in the expression of both the inwardly rectifying and outward K+ current. The magnitude of the membrane potential change as well as K+ current inhibition was larger in cells with retracted processes. The electrophysiological effects of rhTNF were attenuated when rhTNF was neutralized with a polyclonal anti-rhTNF antibody. The binding of rhTNF to its receptor has been reported to increase GTP binding, to increase GTPase activity of a pertussis-sensitive G protein, and to produce an elevation in intracellular cAMP in other cell types. However, pretreatment of OLGs with activated pertussis toxin failed to attenuate or mimic the effects of rhTNF. Chronic exposure of OLGs to the membrane permeant analogue of cAMP, 8-bromo-cAMP, resulted primarily in an inhibition of the inwardly rectifying K+ current, an effect which was less than that produced by rhTNF alone and without any of the associated rhTNF-induced morphological changes. This indicates that the effects of rhTNF cannot be entirely accounted for by an elevation in intracellular cAMP. Cycloheximide (CHX), an inhibitor of protein synthesis, mimicked the effects of rhTNF; however, the effects of rhTNF and CHX were not additive. The finding that both ionic current expression and membrane potential were reduced in cells treated with rhTNF that appeared morphologically normal suggests that abnormal ion channel expression in OLGs precedes and may contribute to eventual myelin swelling and damage.

• Loetscher H et al.
• Two distinct tumour necrosis factor receptors--members of a new cytokine receptor gene family.
• Oxf Surv Eukaryot Genes. 1991; 7: 119-42

• Aggarwal BB
• Structure of tumor necrosis factor and its receptor.
• Biotherapy. 1991; 3: 113-20

• Van Bladel S, Libert C, Fiers W
• Interleukin-6 enhances the expression of tumor necrosis factor receptors on hepatoma cells and hepatocytes.
• Cytokine. 1991; 3: 149-54
• Display abstract

• We have studied the effect of interleukin-6 (IL-6) on the binding of tumor necrosis factor (TNF) to various cell lines. A significant increase (up to 250%) in binding was observed on rat hepatocytes and on the human hepatoma cell line HepG2, while no changes in the number of cells or cell morphology could be observed. Scatchard plot analysis showed that IL-6 enhanced the number of TNF receptors without affecting the receptor affinity. The effect reached plateau levels after approximately 6 h and at IL-6 concentrations of 10 ng/ml. It could be completely eliminated by cotreatment of cells with anti-IL-6 antibodies, but not by treatment with anti-interferon-gamma (IFN-gamma), suggesting that IFN-gamma, which can enhance TNF receptor expression on a variety of cells, was not a mediator in this IL-6 effect. Treatment with inhibitors of protein or RNA synthesis completely abolished the IL-6-induced increase, suggesting that IL-6 caused an enhanced transcription of TNF receptor mRNA. IL-1 had no effect on TNF binding to HepG2. However, when cells were cotreated with IL-1 and IL-6, IL-1 could completely abrogate the IL-6 effect.

• Jaattela M
• Biologic activities and mechanisms of action of tumor necrosis factor-alpha/cachectin.
• Lab Invest. 1991; 64: 724-42

• Mallett S, Barclay AN
• A new superfamily of cell surface proteins related to the nerve growth factor receptor.
• Immunol Today. 1991; 12: 220-3
• Display abstract

• In this article Susan Mallett and Neil Barclay discuss the molecular and functional features of a new superfamily of membrane proteins defined by the presence of cysteine-rich motifs originally identified in the low-affinity nerve growth factor receptor. This superfamily includes two lymphocyte proteins of unknown function and two receptors for tumor necrosis factor.

• Ware CF et al.
• Tumor necrosis factor (TNF) receptor expression in T lymphocytes. Differential regulation of the type I TNF receptor during activation of resting and effector T cells.
• J Immunol. 1991; 147: 4229-38
• Display abstract

• The expression of TNF-alpha receptors (TNFR) was examined on a CD4+ T cell hybridoma, transformed T cell lines, CTL clones, and activated T cells from peripheral blood to determine the basis of the immunomodulatory activity of TNF on T cell function. Analyses by ligand cross-linking and competitive binding assays with mAb to the 80-kDa receptor (TNFR-I), demonstrated that the TNFR-I was the predominant receptor expressed on activated CD4+ and CD8+ T cell subsets. However, on T cell leukemic lines, a second, non-TNFR-I binding site was identified, most likely the 55-kDa form (TNFR-II). Additional subsets of T cells were readily distinguished by their expression of TNFR-I and related members of the TNFR gene family (CD40 and CD27). Expression of the TNFR-I was dependent upon the state of T cell activation. Signaling through the TCR for Ag or IL-2R was sufficient to induce TNFR mRNA and protein expression in resting T cells. Multiple sizes of TNFR-I transcripts were detected during T cell activation; however, biosynthetic studies showed these multiple species encode a single protein of 80 kDa. These results, combined with the known ability of TNF to induce IL-2R expression, indicate that TNF and IL-2 form a reciprocating receptor amplification circuit. In contrast, differentiated effector T cells triggered through the TCR or protein kinase C initiated a rapid down-regulation (transmodulation) of the TNFR-I that preceded TNF or lymphotoxin secretion. The mechanism of transmodulation involved proteolytic processing of the mature 80-kDa receptor releasing a soluble 40-kDa fragment. This indicates that a TNF autocrine loop is not likely to form during the response of an effector T cell. Collectively, these results suggest that transcriptional and post-translational modification of the TNFR-I are important control points regulating the expression of this receptor during T cell activation.

• Matsuda M et al.
• Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.
• J Immunol. 1991; 147: 3837-41
• Display abstract

• Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.

• Hwang CD, Gatanaga M, Innins EK, Yamamoto RS, Granger GA, Gatanaga T
• A 20 amino acid synthetic peptide of a region from the 55 kDa human TNF receptor inhibits cytolytic and binding activities of recombinant human tumour necrosis factor in vitro.
• Proc R Soc Lond B Biol Sci. 1991; 245: 115-9
• Display abstract

• Tumour Necrosis Factor (TNF) and Lymphotoxin (LT) can exert a wide range of effects on cells and tissues and they are important effector molecules in cell mediated immunity. All these effects are induced subsequent to the binding of these cytokines to specific membrane receptors. Recently, two of these membrane receptors of 55 and 75 kDa, have been identified which share some amino acid (AA) homology in their N-terminal extracellular domains but differ in their intracellular domains. We synthesized two synthetic 20 AA peptides from hydrophilic regions of the N-terminal extracellular domains of the 55 kDa receptor; peptide A shares homology with both 55 and 75 kDa receptors, peptide B is unique. We found peptide B inhibits both the binding and cytolytic activity of recombinant human TNF when tested on murine L929 cells in vitro. Polyclonal antiserum generated against peptide B will block binding of 125I-labelled TNF to these cells in vitro. However, peptide A and antiserum prepared against peptide A are without effect in these same assay systems. These data suggest that the 20 AA sequences from AA 175 to 194 in the N-terminal extracellular domain of the 55 kDa TNF receptor are expressed on the cell surface and are involved in the binding of TNF.

• Peppel K, Crawford D, Beutler B
• A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.
• J Exp Med. 1991; 174: 1483-9
• Display abstract

• Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.

• Kiwit JC, Schmitz KH, Daum L, Reifenberger G, Roosen N
• Effects of recombinant human tumor necrosis factor on rodent gliomas and normal brain.
• J Neurosurg. 1991; 75: 597-603
• Display abstract

• In a study examining the possible therapeutic effects of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) on malignant gliomas without expression of tumor necrosis factor (TNF)-receptors, RG-2 glioma cells were tested in vitro as well as in a rat experimental glioma model. A growth inhibition assay revealed no inhibiting effect in vitro up to a concentration of 20 micrograms/ml rHuTNF-alpha. Receptor-binding studies showed that RG-2 cells did not present specific receptors for rHuTNF-alpha. The pharmacokinetics of rHuTNF-alpha after intravenous injection were studied with respect to serum, tissue, and brain tumor concentrations and showed increased glioma concentrations of (mean +/- standard error of the mean) 0.47 +/- 0.18 ng TNF/mg brain compared to 0.15 +/- 0.05 ng TNF/mg brain in the normal contralateral hemisphere. No therapeutic effect on solid RG-2 gliomas could be observed after stereotactic injection of 7.3 micrograms rHuTNF/10 microliter buffer solution into the tumor in 10 animals. Immunohistochemical studies after stereotactic injection of rHuTNF-alpha showed total disappearance of the substance after 24 hours without internalization into tumor cells. Stereotactic injection of 7.3 micrograms rHuTNF 10 microliters into normal brain resulted in marked inflammatory response around the injection track, including microvascular thrombosis. These results demonstrate that rHuTNF has neither direct nor indirect cytotoxic activity on RG-2 glioma cells. Furthermore, before clinical use of rHuTNF-alpha in malignant gliomas, the authors suggest that receptor studies be done in each patient. In receptor-positive patients undergoing treatment with rHuTNF-alpha, precautions should be taken to prevent local encephalitic reactions.

• Liabakk NB et al.
• Development of immunoassays for the detection of soluble tumour necrosis factor receptors.
• J Immunol Methods. 1991; 141: 237-43
• Display abstract

• Immunoassays were established for the detection of the 55 kDa and 75 kDa tumour necrosis factor receptor (TNFR) fragments present in urine. The immunoassays were based on pairs of monoclonal TNFR antibodies directed against different epitopes of the 55 kDa and 75 kDa TNFRs. The immunoassays were judged to be specific for unoccupied TNFR since the signals were inhibited by adding recombinant human or murine TNF-alpha, and to a lesser extent by rTNF-beta (LT). Other cytokines such as IL-1 beta, IL-2 or rIFN-gamma did not affect the signal. In a preliminary screening it was found that urines from febrile patients contained higher amounts of 55 kDa and 75 kDa TNFR fragments than did urine from non-febrile individuals. The immunoassays could be used to monitor the purification of the two types of TNFR from the same febrile urine. Furthermore, the sensitivity and the speed of the assay could be increased by the use of magnetic beads as a solid support in the assay.

• Goodwin RG et al.
• Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor.
• Mol Cell Biol. 1991; 11: 3020-6
• Display abstract

• Clones encoding the type 1 (p80) and type 2 (p60) forms of the murine receptors for tumor necrosis factor (TNF) were isolated by cross-hybridization using probes derived from the cloned human TNF receptors. Each of the murine receptors shows strong sequence homology to the corresponding human receptor (approximately 65% amino acid identity) throughout the molecule but only modest homology, limited to ligand-binding domains, between themselves. The ligand-binding characteristics of the recombinant murine receptors mirror those of the human homologs: both receptor types bind TNF-alpha and -beta with multiple affinity classes, and the ligands cross-compete. Analysis of the murine transcripts encoding these receptors revealed the presence of RNAs for one or both forms of the receptors in all cells examined. It was also demonstrated that for both types of human TNF receptor, variably sized transcripts are observed in different cells. The murine cDNAs were further used to determine the chromosomal locations of the TNF receptor genes. They are not linked, in contrast to the ligands, and map to chromosomes 4 (type 1) and 6 (type 2).

• Libert C, Van Bladel S, Brouckaert P, Shaw A, Fiers W
• Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor.
• J Immunol. 1991; 146: 2625-32
• Display abstract

• C57BL/cnb mice were found to be protected against a lethal combination of recombinant murine (m) TNF and GalN by pretreatment with several cytokines. At certain doses, rmTNF and human (h) TNF protected completely. The clearest protection was induced by rIL-1: all four rIL-1 species (both m and h, as well as alpha and beta) protected when given 12 h before the challenge. LPS and rmIFN-gamma protected weakly, whereas rmIL-6 and rhIL-6 did not protect at all. Also adrenocorticotropic hormone, dexamethasone, or dexamethasone in combination with rhIL-6 could not protect. A single IL-1 injection also completely protected mice against a lethal dose of mTNF in the absence of GalN sensitization. The desensitization by IL-1 cannot be explained by a faster clearance of the challenge TNF. In addition, we demonstrate that the IL-1-induced desensitization was only observed when a functioning liver was present, that IL-1-pretreated animals did not show decreased numbers of hepatocyte TNF receptors, and that the amount of TNF-induced IL-6 was not reduced.

• Vilcek J, Lee TH
• Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions.
• J Biol Chem. 1991; 266: 7313-6

• Turetskaia RL, Udalova IA, Kuprash DV, Terskikh AV, Nedospasov SA
• [Cloning and characteristics of the human genome locus containing genes of a second type of receptor (75 kDa) for tumor necrosis factor and lymphotoxin]
• Dokl Akad Nauk SSSR. 1991; 320: 1258-61

• Chamba A, Stockley RA, Burnett D
• Effects of neutrophil adherence on the characteristics of receptors for tumor necrosis factor-alpha.
• FEBS Lett. 1991; 282: 373-6
• Display abstract

• Human recombinant [125I]TNF-alpha was incubated with non-adherent human neutrophils, cells adherent to fibronectin-coated plastic, or adherent cells scraped into suspension (post-adherent). Binding of TNF to all cells increased with doses of added TNF but adherent cells bound little TNF. Binding of TNF by post-adherent cells was greater than when adherent, but still significantly less than that of non-adhered neutrophils, suggesting that TNF receptors were relocated on the adherent surface of neutrophils. Scatchard analysis showed that adherent cells expressed significantly fewer TNF receptors, but of higher affinity, than non-adherent cells. The results suggest that altered expression of TNF receptors might contribute to the differential effects of TNF on adherent and non-adherent neutrophils.

• Vanhaesebroeck B et al.
• Two discrete types of tumor necrosis factor-resistant cells derived from the same cell line.
• Cancer Res. 1991; 51: 2469-77
• Display abstract

• From the murine fibrosarcoma cell line L929s, which is sensitive to tumor necrosis factor (TNF)-mediated cell lysis, two discrete types of TNF-resistant variants were derived by TNF selection. Cells of the first type (named L929r1) were not sensitized to TNF cytotoxicity by cotreatment with either inhibitors of protein or RNA synthesis, or gamma-interferon, despite the presence of a functional gamma-interferon response. L929r1 constitutively produced TNF in the supernatant and expressed membrane-bound TNF, which was not bound to the TNF receptor. In fact, TNF receptors could not be demonstrated on L929r1 cells, not even after low pH treatment and/or incubation with antiserum to TNF. L929r1 exhibited a stable TNF-resistant phenotype in the absence of further TNF selection. No evidence could be obtained that TNF acted as an autocrine growth factor for these cells. L929r2, the second type of TNF-resistant L929 cells, became sensitive to TNF lysis in the presence of RNA or protein synthesis inhibitors, or in the presence of gamma-interferon. TNF induced the secretion of interleukin 6 in these cells, additionally showing that functional TNF signaling in these cells indeed takes place, but does not lead to cell lysis under normal conditions. L929r2 did not produce TNF, also not upon stimulation with exogenous TNF. The number and binding affinity of TNF receptors were not consistently different between L929s and L929r2 cells. In the absence of further TNF selection, L929r2 gradually reverted to TNF sensitivity. This sensitivity was not reversible to TNF resistance by the gene-regulatory agents 5-azacytidine or sodium butyrate. Treatment with these agents also did not affect the TNF sensitivity of L929s cells nor the TNF resistance of L929r1 and L929r2 cells. In summary, our results suggest the existence among cells of the same cell line of discrete mechanisms for acquisition of resistance to TNF-mediated cell lysis.

• Erikstein BK et al.
• Independent regulation of 55-kDa and 75-kDa tumor necrosis factor receptors during activation of human peripheral blood B lymphocytes.
• Eur J Immunol. 1991; 21: 1033-7
• Display abstract

• We have studied the expression of two different tumor necrosis factor receptors (TNFR; 55 kDa and 75 kDa) on resting and activated human peripheral blood B lymphocytes using specific monoclonal antibodies (mAb). Flow cytometric analysis revealed that most resting B cells expressed small amounts of the 75-kDa TNFR, and that the 75-kDa TNFR was markedly up-regulated upon stimulation with anti-mu or Staphylococcus aureus Cowan strain I (SAC). In contrast, the expression of the 55-kDa TNFR was low on resting as well as on activated cells. B cell activation was accompanied by an increased binding of biotinylated TNF-alpha, and this binding could be blocked by preincubation by utr-1 (anti-75-kDa TNRF), but not the htr (anti-55-kDa TNFR) antibodies. Notably, a number of cytokines tested (interleukin 1 to 8, interferon-gamma, TNF-alpha and -beta) did not influence the expression of either the 75-kDa or the 55-kDa TNFR when given to resting B cells. Moreover, phorbol 12-myristate 13-acetate led to an early, marked down-regulation of the 75-kDa TNFR expression, followed by a later modest increase after greater than 24 h. In contrast to other cell systems where htr mAb have been found either to mimic or to inhibit TNF action, htr mAb had insignificant effects in assays for restimulation of preactivated B cells. However, utr-1 markedly inhibited the TNF-beta but only partly inhibited the TNF-alpha-induced proliferation. Taken together, our data suggest that changes in 75-kDa protein expression is responsible for the increased TNFR expression on activated vs. resting peripheral blood B cells and that this protein also may play an important functional role.

• Cappuccini F et al.
• Identification of tumor necrosis factor and lymphotoxin blocking factor(s) in the ascites of patients with advanced and recurrent ovarian cancer.
• Lymphokine Cytokine Res. 1991; 10: 225-9
• Display abstract

• Ascites obtained from human ovarian cancer patients contains material(s) that inhibit the cytolytic activity of tumor necrosis factor (TNF) and lymphotoxin (LT) in vitro. These inhibitor(s) are found in ascites from ovarian cancer patients and are detected in very low amounts in the ascites from patients with nonmalignant hepatic disease. These ascites TNF/LT blocking factors are heat sensitive and heterogeneous with respect to molecular weight. Kinetic studies indicate these factors inhibited cytolysis at the stage of TNF/LT interaction with membrane receptors on L929 cells. Because TNF and LT are key cytokines in host cell-mediated antitumor mechanisms, factor(s) that inhibit these cytokines could have a profound effect on the tumor host interaction and their presence in the ascitic fluid, should be considered before designing clinical trials that employ intraperitoneal administration of TNF or LT for immunotherapy of ovarian cancer.

• Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino MA Jr, Goeddel DV
• The two different receptors for tumor necrosis factor mediate distinct cellular responses.
• Proc Natl Acad Sci U S A. 1991; 88: 9292-6
• Display abstract

• The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses.

• Smith CA et al.
• T2 open reading frame from the Shope fibroma virus encodes a soluble form of the TNF receptor.
• Biochem Biophys Res Commun. 1991; 176: 335-42
• Display abstract

• A transcriptionally active open reading frame (T2) from Shope Fibroma Virus was recently shown to have striking sequence homology with members of a new superfamily of cell surface proteins, including a receptor for human tumor necrosis factor. Here we report that recombinant T2 protein expressed in COS cells is a soluble, secreted glycoprotein which specifically binds human TNF alpha and beta, and inhibits binding of these cytokines to native TNF receptors on cells. T2 binding of TNF is not inhibited by nerve growth factor, although the nerve growth factor receptor is also a member of the same family, nor by nine other recombinant cytokines. Further, the repeating domain structure of T2 most closely resembles that of the type I TNF receptor (p75) and is significantly different from other family members, including the type II TNF receptor (p55). Since T2 possesses a leader sequence but lacks a transmembrane domain, these results confirm the original suggestion (1) that T2 represents a soluble form of the type I TNF receptor which is secreted from virally infected cells, and whose function is to immunosuppress the host by abrogating the potentially destructive effects of TNF. This is the first such virally-encoded soluble cytokine receptor to be identified, and may represent a more general mechanism by which viruses subvert the host immune system.

• Ashkenazi A et al.
• Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin.
• Proc Natl Acad Sci U S A. 1991; 88: 10535-9
• Display abstract

• Tumor necrosis factors (TNF) alpha and beta are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-alpha induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-alpha and TNF-beta and exhibits 6- to 8-fold higher affinity for TNF-alpha than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-alpha or TNF-beta on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-alpha antibodies (4-fold) in inhibiting TNF-alpha. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-alpha in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.

• Chouaib S, Branellec D, Buurman WA
• More insights into the complex physiology of TNF.
• Immunol Today. 1991; 12: 141-2
• Display abstract

• Tumor necrosis factor (TNF), a multifactorial cytokine involved in a plethora of cell regulatory and differentiative processes, was the subject of a recent conference. It is perhaps best known as a major player in the pathophysiology of numerous diseases but TNF clearly has many beneficial effects and is considered to be a promising therapeutic agent.

• Upton C, Macen JL, Schreiber M, McFadden G
• Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence.
• Virology. 1991; 184: 370-82
• Display abstract

• Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture. However, many of these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection. Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNF alpha and TNF beta. We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein. To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker (Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid. The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52-56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at early times after infection. Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated. The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered. These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF. We propose the term "viroceptor" to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.

• Miloszewska J, Janik P, Ostrowski J
• The effect of tumor necrosis factor (TNF-alpha) on calcium (Ca2+) level.
• Arch Immunol Ther Exp (Warsz). 1991; 39: 99-102
• Display abstract

• Following addition TNF-alpha to variety of cells the diversity of action has been observed. TNF-alpha induced rapid elevation of intercellular Ca2+ level in all studied cells. This suggests TNF receptor is coupled to phospholipase C.

• Chambaut-Guerin AM, Thomopoulos P
• Regulation by dexamethasone and 1,25-dihydroxyvitamin D3 of the 75 kD-type A tumor necrosis factor receptor surface expression and mRNA level in human U937 cells.
• Eur Cytokine Netw. 1991; 2: 355-60
• Display abstract

• 1. Dexamethasone selectively reduced the number of the type A (75 kD) receptors whereas 1,25-dihydroxyvitamin D3 reduced the number of both A (75 kD) and B (55 kD) receptors, without any change of their dissociation constants (3-4 x 10(-10) M and 1-2 x 10(-9) M respectively). The level of the type A (75 kD) TNF receptor mRNA was increased following treatment with either steroid. 2. Other agonists which either down-regulated (phorbol dibutyrate) or up-regulated (dibutyryl cAMP and recombinant GM-CSF) rTNF alpha binding also increased the level of this mRNA species. 3. The data suggest that these steroids might alter post-transcriptional event(s).

• Horton TM et al.
• Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis.
• J Virol. 1991; 65: 2629-39
• Display abstract

• A 14,700-kDa protein (14.7K) encoded by the E3 region of adenovirus has been shown to protect adenovirus-infected mouse C3HA cells from lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). These infected cells are sensitized to TNF by expression of the adenovirus E1A proteins (P. Duerksen-Hughes, W. S. M. Wold, and L. R. Gooding, J. Immunol. 143:4193-4200, 1989). In this study we show that 14.7K suppresses TNF cytolysis independently of adenovirus infection. Mouse C3HA and C127 cells were transfected with the 14.7K gene controlled by the mouse metallothionein promoter, and permanent 14.7K-expressing cell lines were tested for sensitivity to TNF cytolysis. Transfected cells which were sensitized to TNF either by inhibitors of protein synthesis, microfilament-destabilizing agents, or adenovirus infection were found to be resistant to TNF cytolysis. Two monoclonal antibodies were isolated and used to quantitate 14.7K in transfected and infected cells. Enzyme-linked immunosorbent assay (ELISA) analysis with these monoclonal antibodies and 14.7K immunoblots showed that 14.7K expression can be induced with cadmium in C3HA and C127 transfectants. The 14.7K induction correlated with a dose-dependent decrease in sensitivity to TNF cytotoxicity. The 14.7K protein does not substantially alter cell surface TNF receptor numbers or affinity on C3HA mouse fibroblasts, as determined by Scatchard analysis of 125I-TNF binding. The 14.7K protein also does not alter TNF signal transduction in general, because TNF induction of cell surface class I major histocompatibility complex molecules on 14.7K transfectants was unmodified. Our findings indicate that the adenovirus 14.7K protein functions as a specific inhibitor of TNF cytolysis in the absence of other adenovirus proteins and thus is a unique tool to study the mechanism of TNF cytotoxicity.

• Goh CR, Loh CS, Porter AG
• Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin).
• Protein Eng. 1991; 4: 785-91
• Display abstract

• Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer. When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits. Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta.

• Lewis M et al.
• Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific.
• Proc Natl Acad Sci U S A. 1991; 88: 2830-4
• Display abstract

• Complementary DNA clones encoding two distinct tumor necrosis factor receptors were isolated from a mouse macrophage cDNA library. The cDNA for murine tumor necrosis factor receptor type 1 (mTNF-R1) predicts a mature polypeptide of 425 amino acids that is 64% identical to its human counterpart, whereas the cDNA of murine tumor necrosis factor receptor type 2 (mTNF-R2) predicts a mature protein of 452 amino acids that is 62% identical to human tumor necrosis factor receptor type 2. The two murine tumor necrosis factor receptors have limited sequence homology (approximately 20% identity) in their extracellular regions but no apparent similarity in their cytoplasmic portions. Northern (RNA) analysis indicates a single 2.6-kilobase (kb) transcript for mTNF-R1; a 3.6-kb and a more predominant 4.5-kb transcript are observed for mTNF-R2. A human cell line transfected with either mTNF-R1 or mTNF-R2 expression vectors specifically bound 125I-labeled recombinant murine tumor necrosis factor alpha (TNF-alpha). Although mTNF-R1 had a similar affinity for both recombinant murine TNF-alpha and human TNF-alpha, mTNF-R2 showed strong specificity for recombinant murine TNF-alpha. This result suggests that the various activities of human tumor necrosis factor alpha reported in mice or in murine cell lines are probably mediated by mTNF-R1.

• Corkey BE, Geschwind JF, Deeney JT, Hale DE, Douglas SD, Kilpatrick L
• Ca2+ responses to interleukin 1 and tumor necrosis factor in cultured human skin fibroblasts. Possible implications for Reye syndrome.
• J Clin Invest. 1991; 87: 778-86
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• Elevated concentrations of cytokines were found in the plasma of patients acutely ill with Reye syndrome (RS) but not in control subjects or recovered RS patients. To determine whether this disorder involves a genetically determined abnormal response to cytokines, the effects of tumor necrosis factor (TNF) and IL-1 on intracellular free Ca2+ were compared in cultured skin fibroblasts from control subjects and patients with RS. IL-1 and TNF caused rapid, transient, and concentration-dependent increases in cytosolic free Ca2+. The peak cytosolic free Ca2+ was greater and occurred at higher concentrations of IL-1 and TNF in patient cells than in cells from age-matched controls. In control cells, the Ca2+ transient diminished sharply with increasing amounts of IL-1 or TNF above the maximum stimulatory concentration. In contrast, in patient fibroblast this bell-shaped curve of concentration dependency was much less apparent. Bradykinin-stimulated Ca2+ transients were similar in the two groups and did not exhibit the bell-shaped concentration dependency. Thus, plasma cytokine levels are elevated in RS patients and the Ca2+ response to cytokines is increased in cells derived from these patients. We propose that the increased response reflects a genetic defect in cytokine receptor-modulated signal transduction.

• Rothe JG, Brockhaus M, Gentz R, Lesslauer W
• Molecular cloning and expression of the mouse Tnf receptor type b.
• Immunogenetics. 1991; 34: 338-40

• Fruehauf JP, Mimnaugh EG, Sinha BK
• Doxorubicin-induced cross-resistance to tumor necrosis factor (TNF) related to differential TNF processing.
• J Immunother. 1991; 10: 165-73
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• To evaluate whether cells selected for doxorubicin resistance were cross-resistant to tumor necrosis factor, the effects of doxorubicin and recombinant human tumor necrosis factor-alpha (TNF) on doxorubicin-sensitive (WT) and 40-fold doxorubicin-resistant (40F) MCF-7 cell proliferation were assessed. The median dose (MD) for doxorubicin was 14.5 nM for WT cells and 474 nM for 40F cells. The MD for TNF was 0.18 nM for WT cells, while 40F cells were highly resistant to TNF concentrations up to 60 nM. Doxorubicin and TNF in combination were synergistic against WT cells, but not 40F cells. Glutathione depletion by buthionine sulfoxamine sensitized WT cells threefold to TNF, with no change in their response to doxorubicin, while 40F cells showed a twofold increase in doxorubicin sensitivity, with no apparent change in their resistance to TNF. No significant differences in TNF receptor number, Kd, or capacity for TNF internalization were noted between the two cell types. WT cells produced a single 15 kDa TNF degradation product, while the 40F cells produced three lower molecular weight degradation products. We conclude that cross-resistance to TNF in doxorubicin-resistant MCF-7 cells may be explained in part by altered TNF degradation.

• Camussi G, Albano E, Tetta C, Bussolino F
• The molecular action of tumor necrosis factor-alpha.
• Eur J Biochem. 1991; 202: 3-14
• Display abstract

• Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-alpha may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-alpha may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF-alpha are triggered by the binding to specific cell surface receptors. The formation of TNF-alpha-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-alpha. The full understanding of the molecular mechanisms of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.

• Lesslauer W et al.
• Recombinant soluble tumor necrosis factor receptor proteins protect mice from lipopolysaccharide-induced lethality.
• Eur J Immunol. 1991; 21: 2883-6
• Display abstract

• 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.

• Fiers W
• Tumor necrosis factor. Characterization at the molecular, cellular and in vivo level.
• FEBS Lett. 1991; 285: 199-212
• Display abstract

• TNF was originally characterized as an antitumor agent and a factor cytotoxic for many malignant cells. It is now clear that it plays an important role in the defense against viral, bacterial and parasitic infections, - and in (auto-)immune responses. Natural induction of TNF is protective, but its overproduction may be detrimental and even lethal to the host. The structure of TNF and its interaction with the two types of cellular receptor are becoming better understood. TNF elicits a variety of events in different cell types. It subverts the electron transport system or the mitochondria into production of oxygen radicals, which can kill the (malignant) cells when these do not contain or produce protective enzymes. Furthermore, TNF induces a set of genes and at least part of this transcriptional activation is mediated by NF kappa B. The prospects of TNF as an antitumor drug can be improved on the one hand by agents such as LI+, which synergizes, and on the other hand by inhibitors of the systemic toxicity which do not interfere with the antitumor efficacy. Also, in tumor-bearing animals which have been rendered tolerant by administration of small doses of TNF, an effective and complete elimination of the tumors can be obtained by the combined action of TNF plus interferon.

• Dinarello CA
• Inflammatory cytokines: interleukin-1 and tumor necrosis factor as effector molecules in autoimmune diseases.
• Curr Opin Immunol. 1991; 3: 941-8
• Display abstract

• The nature of the events that precipitate autoimmune diseases varies. Interleukin-1 and tumor necrosis factor do not precipitate autoimmune diseases but rather act as effector molecules. They induce eicosanoid and nitric oxide synthesis, stimulate collagenases and collagen synthesis, and trigger the genes for other cytokines, namely interleukin-2, interleukin-6 and interleukin-8. The ability to block interleukin-1 with the receptor antagonist, and tumor necrosis factor with soluble receptors, has given investigators specific tools to test the role of these two cytokines in the pathological processes of autoimmune disease.

• Schutze S, Berkovic D, Tomsing O, Unger C, Kronke M
• Tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C.
• J Exp Med. 1991; 174: 975-88
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• Tumor necrosis factor (TNF) is a proinflammatory polypeptide that is able to induce a great diversity of cellular responses via modulating the expression of a number of different genes. One major pathway by which TNF receptors communicate signals from the membrane to the cell nucleus involves protein kinase C (PKC). In the present study, we have addressed the molecular mechanism of TNF-induced PKC activation. To this, membrane lipids of the human histiocytic cell line U937 were labeled by incubation with various radioactive precursors, and TNF-induced changes in phospholipid, neutral lipid, and water-soluble metabolites were analyzed by thin layer chromatography. TNF treatment of U937 cells resulted in a rapid and transient increase of 1'2'diacylglycerol (DAG), a well-known activator of PKC. The increase in DAG was detectable as early as 15 s after TNF treatment and peaked at 60 s. DAG increments were most pronounced (approximately 360% of basal levels) when cells were preincubated with [14C]lysophosphatidylcholine, which was predominantly incorporated into the phosphatidylcholine (PC) pool of the plasma-membranes. Further extensive examination of changes in metabolically labeled phospholipids indicated that TNF-stimulated hydrolysis of PC is accompanied by the generation of phosphorylcholine and DAG. These results suggest the operation of a PC-specific phospholipase C. Since no changes in phosphatidic acid (PA) and choline were observed and the production of DAG by TNF could not be blocked by either propranolol or ethanol, a combined activation of phospholipase D and PA-phosphohydrolase in DAG production appears unlikely. TNF-stimulated DAG production as well as PKC activation could be blocked by the phospholipase inhibitor p-bromophenacylbromide (BPB). Since BPB did not inactivate PKC directly, these findings underscore that TNF activates PKC via formation of DAG. TNF stimulation of DAG production could be inhibited by preincubation of cells with a monoclonal anti-TNF receptor (p55-60) antibody, indicating that activation of a PC-specific phospholipase C is a TNF receptor-mediated event.

• Drapier JC, Wietzerbin J
• IFN-gamma reduces specific binding of tumor necrosis factor on murine macrophages.
• J Immunol. 1991; 146: 1198-203
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• Because IFN-gamma is the main cytokine activating macrophages and TNF cooperates in this activation, we assessed TNF binding capacity during the course of murine macrophage activation by IFN-gamma. TNF binding to elicited macrophages increased with time, was maximal by 8 h of culture, and required de novo protein synthesis. 125I-TNF bound to about 40,000 sites/cell with a Kd of 1 x 10(-9) M. Cross-linking experiments performed with a bifunctional cross-linking agent revealed a specific band with a m.w. of 94,000. Preincubation of macrophages with IFN-gamma prevented the binding of TNF to receptors. This effect was dose-dependent and maximal at 100 U/ml. IFN-gamma also reduced specific TNF binding to preexisting receptors (50% inhibition in 3 h), but IFN-gamma did not change the internalization rate of TNF. These studies showed that the number of TNF receptors increased on macrophages vs maturation in culture and was negatively controlled by IFN-gamma.

• Ryffel B, Brockhaus M, Durmuller U, Gudat F
• Tumor necrosis factor receptors in lymphoid tissues and lymphomas. Source and site of action of tumor necrosis factor alpha.
• Am J Pathol. 1991; 139: 7-15
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• Tumor necrosis factor alpha (TNF alpha), which is produced by germinal center dendritic reticulum cells (DRC) in lymphoid tissue, plays a regulatory role in a local immune response. However no information is available on the nature and location of cells responding to this cytokine. Thus TNF receptor distribution was investigated in situ by immunohistochemistry using monoclonal antibodies directed against the p75 and p55 receptor proteins. Receptor expression was unique and restricted to the lymphoreticular tissue. The p75 receptor was found on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal center DRCs, which are the main site of TNF alpha production. The two receptor proteins were expressed on distinct cell populations of the lymphoid system and no coexpression was observed. Preliminary results indicate that TNF receptor (TNFR) expression is regulated; Upregulation of TNFR proteins was found in reactive hyperplasia together with increased TNF alpha expression. In lymphoproliferative disorders, expression of the p75 receptor and TNF alpha was found mainly in high-grade malignant non-Hodgkin lymphomas. In summary, TNF alpha produced by germinal center DRCs might regulate an in vivo immune response through autocrine and paracrine pathways. Thus TNF alpha might signal, through the distinct TNFR proteins, the p55 and p75 receptor, which are expressed on different cell types in lymphoid tissue.

• Kolb E
• [Current knowledge of characteristics and effects of alpha and beta tumor necrosis factors (cachectins)]
• Z Gesamte Inn Med. 1991; 46: 139-44
• Display abstract

• The tumour necrosis factors (TNF) alpha and beta play a role in the activation of the immune system in bacterial and viral infections as well as in parasitoses and inflammatory processes, respectively. The TNF alpha is formed mainly by activated macrophages and mast cells, the TNF beta is formed by activated T- and B-lymphocytes. The evocation of necroses in certain forms of tumours is done by the furthering of the formation of activated O2-molecules (H2O2, peroxide anions, hydroxyl radicals). In tumour cells with a high activity of glutathione-peroxidase and of Mn peroxide-dismutase the TNF are without any effect. The TNF activate neutrophil and eosinophil granulocytes as well as macrophages. By induction of the formation of an oligoadenylate synthetase in virus-infected cells they inhibit the virus reproduction. In chronic infections and parasitoses they evoke a retrogression of the fatty tissue (cachexia). The division of fibroblasts is stimulated via the activation of the genes c-myc and c-fos. In the same way like interleukin 1 and interferon gamma they cause fever.

• Lentz SR, Tsiang M, Sadler JE
• Regulation of thrombomodulin by tumor necrosis factor-alpha: comparison of transcriptional and posttranscriptional mechanisms.
• Blood. 1991; 77: 542-50
• Display abstract

• The procoagulant properties of cultured vascular endothelial cells are enhanced in response to inflammatory cytokines such as tumor necrosis factor-alpha (TNF). A major component of this response is a reduction in expression of thrombomodulin, a cell surface cofactor for the activation of protein C. Regulation of thrombomodulin expression by TNF has been reported to occur through multiple mechanisms. To determine the relative roles of transcriptional and posttranscriptional regulation, the effect of TNF on the turnover of thrombomodulin protein and mRNA was examined in human and bovine endothelial cells. Quantitative nuclease S1 protection assays showed a 70% to 90% reduction in thrombomodulin mRNA within 4 hours of the addition of 1.0 nmol/L TNF to the culture medium. The decrease in thrombomodulin mRNA resulted from inhibition of transcription, followed by rapid degradation of thrombomodulin transcripts (t1/2 less than or equal to 3 hours). In pulse-chase incubations, thrombomodulin synthesis decreased parallel with mRNA, but the rate of degradation of radiolabeled thrombomodulin was not significantly altered by TNF. Human thrombomodulin was degraded with a t1/2 of 8.2 +/- 2.4 hours (SD) or 7.5 +/- 1.3 hours (SD) in the absence or presence of TNF, respectively. We conclude that TNF acts primarily to inhibit thrombomodulin transcription. The subsequent decrease in activity results from the inherent instability of thrombomodulin mRNA and protein in these cells, and not from the regulation of thrombomodulin degradation.

• Schoenfeld HJ et al.
• Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors.
• J Biol Chem. 1991; 266: 3863-9
• Display abstract

• A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

• Ohdama S, Takano S, Ohashi K, Miyake S, Aoki N
• Pentoxifylline prevents tumor necrosis factor-induced suppression of endothelial cell surface thrombomodulin.
• Thromb Res. 1991; 62: 745-55
• Display abstract

• Thrombomodulin (TM) expression has been reported to be down-regulated by cytokines (endotoxin, interleukin-1, and tumor necrosis factor). We report, in the present study, up-regulation of surface TM antigen of human umbilical vein endothelial cells (HUVECs) by pentoxifylline (PTX) which is one of the agents that can increase intracellular cyclic AMP in HUVECs at therapeutic concentrations. Surface TM antigen was measured by an enzyme immunoassay. PTX increased surface TM antigen and intracellular cAMP in HUVECs in a dose dependent manner. Upregulation of TM by PTX was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. PTX counterbalanced the TNF-induced suppression of TM expression. These results suggest that protein kinase A may be involved in cellular regulatory mechanism for TM expression and PTX may protect partially against TNF-induced endothelial cell injury and restore anticoagulant state of endothelium.

• Camerini D, Walz G, Loenen WA, Borst J, Seed B
• The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis factor receptor gene family.
• J Immunol. 1991; 147: 3165-9
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• CD27 is a dimeric membrane glycoprotein found on the surface of most human T lymphocytes. Activation of T cells by engagement of the Ag receptor increases CD27 surface expression, and anti-CD27 antibodies augment Ag receptor-mediated T cell proliferation. In this study a cDNA-encoding CD27 was isolated by expression and immunoselection in COS cells. The predicted polypeptide was found to belong to a recently characterized family of cysteine-rich receptors whose known ligands include nerve growth factor and TNF-alpha and -beta. Structural similarities suggest that CD27 belongs to a lymphocyte-specific subgroup of the family, comprised of the B cell Ag CD40, the rat T cell subset Ag OX40, and the mouse T cell activation Ag 4-1BB. Recent studies suggest some of these molecules may play a role in the survival of activated cells.

• Johnson DR, Pober JS
• Tumor necrosis factor regulation of major histocompatibility complex gene expression.
• Immunol Res. 1991; 10: 141-55

• Libert C, Van Bladel S, Brouckaert P, Fiers W
• The influence of modulating substances on tumor necrosis factor and interleukin-6 levels after injection of murine tumor necrosis factor or lipopolysaccharide in mice.
• J Immunother. 1991; 10: 227-35
• Display abstract

• In this study, we investigated the influence of D-galactosamine (GalN), indomethacin, and dexamethasone on the pharmacokinetics of injected or induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) after a bolus injection of murine TNF (mTNF) or lipopolysaccharide (LPS). It is well known that GalN treatment renders mice much more vulnerable to TNF or LPS lethality. Nevertheless, GalN had no influence on TNF clearance or IL-6 induction after mTNF injection; however, the induced TNF and IL-6 levels were considerably augmented by the GalN cotreatment when a high dose of LPS was injected (GalN was given as a single injection together with TNF or LPS). Indomethacin and dexamethasone, either of which shows a clear protection against TNF/LPS lethality in normal mice, did not change the clearance of injected mTNF, but both reduced the TNF-induced IL-6 levels. Indomethacin did not affect the level and clearance of LPS-induced TNF, whereas the induced IL-6 levels were significantly lower than in the control mice. The circulating TNF and IL-6 concentrations after LPS injection in mice pretreated with dexamethasone were very considerably reduced. Furthermore, neither agent had an influence on the number of TNF binding sites on hepatocytes. We conclude that the strongly enhanced sensitivity of GalN-treated mice towards mTNF-induced or LPS-induced lethality was not reflected in circulating TNF or IL-6 levels, and that dexamethasone and indomethacin both reduce circulating IL-6 concentrations in mice treated with TNF and LPS.

• Lau AS, Der SD, Read SE, Williams BR
• Regulation of tumor necrosis factor receptor expression by acid-labile interferon-alpha from AIDS sera.
• AIDS Res Hum Retroviruses. 1991; 7: 545-52
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• High levels of an unusual acid-labile interferon (IFN) alpha in sera of patients with human immunodeficiency virus (HIV) infection are associated with disease progression to acquired immunodeficiency syndrome (AIDS). Since IFNs have been shown to enhance the cytotoxic actions of tumor necrosis factor (TNF), a potent mediator of inflammation and cachexia, a study was undertaken to investigate whether the acid-labile IFN alpha produced in AIDS can regulate TNF receptor expression. The expression of TNF receptors was determined by studying the interaction of [125I]TNF with cellular receptors. The results show the acid-labile IFN alpha present in AIDS sera is capable of inducing the expression of cellular receptors for TNF. The extent of induction of TNF receptors depends on the concentration of the acid-labile IFN alpha in the AIDS sera. There is no significant induction of TNF receptors when the AIDS sera are preneutralized with polyclonal anti-IFN alpha antibodies. It is also shown that the synthesis of TNF by peripheral blood monocytes (PBM) from patients with HIV infection is enhanced during the progression of HIV infection in vivo. Thus, the TNF system is activated in patients with HIV infection. This activation may be a contributing factor to some of the physiological disturbances including the wasting syndrome observed in AIDS.

• Barrett K et al.
• Cloning, expression and cross-linking analysis of the murine p55 tumor necrosis factor receptor.
• Eur J Immunol. 1991; 21: 1649-56
• Display abstract

• Tumor necrosis factor (TNF) mediates its pleiotropic effects via high-affinity cell surface receptors. In man, molecular cloning has identified two distinct, independent TNF receptors (TNFR) of 55 and 75 kDa. It is unclear, however, whether the multiple effects of TNF are suggested between the receptor types. In the mouse, previous studies had shown functional heterogeneity of TNFR, since the WEHI 164 fibroblast line is sensitive to the cytotoxic effects of both murine and human TNF, whereas the murine T cell line, CT6, proliferates in response to murine but not human TNF. In this study, the cloning of a cDNA encoding the murine homologue of the p55 TNFR is reported. This receptor binds murine and human TNF with equal affinity and is expressed on WEHI 164 and a number of other cell lines, but only low levels of mRNA and no protein is detectable on CT6 cells. CT6 cells, however, express a second TNFR of approximately 75 kDa, identified by cross-linking analysis, which is also found on WEHI 164 cells, and binds only murine TNF. These studies establish that there are also two TNFR in the mouse, and suggests that there may be segregation of the cytotoxic and proliferative responses between different receptors, at least in these cell lines.

• Konig M, Wallach D, Resch K, Holtmann H
• Induction of hyporesponsiveness to an early post-binding effect of tumor necrosis factor by tumor necrosis factor itself and interleukin 1.
• Eur J Immunol. 1991; 21: 1741-5
• Display abstract

• Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV-80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV-80 cells respond to TNF also with enhanced phosphorylation of the small heat-shock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reversed already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.

• Dett CA et al.
• Enhancement of lymphokine-activated T killer cell tumor necrosis factor receptor mRNA transcription, tumor necrosis factor receptor membrane expression, and tumor necrosis factor/lymphotoxin release by IL-1 beta, IL-4, and IL-6 in vitro.
• J Immunol. 1991; 146: 1522-6
• Display abstract

• Co-culture with IL-2 can induce human T lymphocytes to proliferate and become nongenetically restricted, lymphokine-activated killer (LAK) cells in vitro. Our studies were conducted with long term cultured, human T-LAK cells from peripheral blood, which are 95 to 99% CD3+. We found that proliferating 7 to 10-day human T-LAK cells express TNFR, by using a 125I-TNF binding assay. Additional treatment of these cells with the cytokines IL-1 beta, IL-4, or IL-6 rapidly up-regulated 55-kDa TNFR mRNA transcription and doubled TNFR membrane expression. Further studies revealed that these cytokines also increased the release of TNF and lymphotoxin (LT). Antibody neutralization studies indicated that IL-1 induces release of both TNF and LT; however, IL-4 and IL-6 induce primarily LT release. These results further support the concept that these cytokines are involved in the regulation of TNF/LT release, TNFR synthesis, and TNFR membrane expression. It is apparent that cytokines and their membrane receptors are involved in the autocrine/paracrine control of T cell proliferation, differentiation, and expression of functional activity after IL-2 stimulation in vitro.

• Wallach D et al.
• Soluble and cell surface receptors for tumor necrosis factor.
• Agents Actions Suppl. 1991; 35: 51-7
• Display abstract

• Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptors have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine. The intracellular domains of the two receptors differ in structure, suggesting that they mediate different activities. Their extracellular domains, however, are structurally related. Both contain cysteine-rich repeats which are homologous to repeated structures found in the extracellular domains of the nerve growth factor receptor and the CDw40 protein. Truncated soluble forms of the two receptors, corresponding to these cysteine-rich repeated structures, have been detected in human urine and were later found to be present also in the serum. The serum levels of those soluble TNF receptors increase dramatically in certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect on TNF function, and may thus act as physiological attenuators of its activity.

• Howard ST, Chan YS, Smith GL
• Vaccinia virus homologues of the Shope fibroma virus inverted terminal repeat proteins and a discontinuous ORF related to the tumor necrosis factor receptor family.
• Virology. 1991; 180: 633-47
• Display abstract

• Nucleotide sequencing data from a region extending 35 kb inward from the right inverted terminal repeat (ITR) of the vaccinia virus (VV) genome established the presence of VV homologues of the Shope fibroma virus (SFV) ITR proteins. The nucleotide sequences, comprising a total of 8.6 kb, and the amino acid translations for nine predicted open reading frames (ORFs) (designated SalF4L, SalF 19R, SalF21R, B4R, B8R, B9R, B10R, and B14R) are presented. Eight of the nine VV genes and all the SFV ORFs are transcribed towards their genomic termini. However, the relative positions of the VV genes (genus Orthopoxvirus) are different than those of the corresponding ORFs in SFV (genus Leporipoxvirus), indicating complex rearrangements of DNA in the genome of one or both of these viruses subsequent to their divergence from a common ancestor. Several other features of the VV ORFs were noted. SalF4L, B7R, B8R, and B9R have hydrophobic amino-terminal signal sequences but lack discernible membrane anchor domains suggesting that the proteins may be secreted. VV ORF SalF19R has a single cysteine-rich region homologous to the multiple domains of nerve growth factor receptor (NGFR), CD40, OX40 (a glycoprotein from the surface of activated murine T lymphocytes), and the recently described tumor necrosis factor receptors. Just downstream of the ORF SalF19R and in a different reading frame, there are another two related cysteine-rich domains, indicating that SalF19R was once a larger gene. B4R has homology to the host range gene of cowpox virus and to related genes near the opposite end of the vaccinia virus genome, and contains regions homologous to the repeat domains of erythrocyte ankyrin. In addition, several of the VV ORFs have homology to ORFs from near the opposite end of the VV genome, thus increasing the number of known VV gene families.

• Nakamura S et al.
• A novel recombinant tumor necrosis factor-alpha mutant with increased anti-tumor activity and lower toxicity.
• Int J Cancer. 1991; 48: 744-8
• Display abstract

• We prepared a novel recombinant tumor necrosis factor-alpha (TNF) mutant (mutant 471), in which 7 N-terminal amino-acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, and compared its biological activity with that of wild-type recombinant TNF. Mutant 471 had a 7-fold higher anti-tumor activity against murine L-M cells in vitro, and a higher binding activity to TNF receptors on L-M cells, than wild-type TNF. Furthermore, mutant 471 showed a higher anti-tumor effect on murine Meth A-HM tumors transplanted into BALB/c mice, with complete regression of the tumors being observed in the animals. The possible cachectin activity of mutant 471 was almost the same as that of wild-type TNF. The acute lethal toxicity of mutant 471 in beta-D-galactosamine-sensitized C3H/HeJ mice was 18 times lower than that of wild-type TNF. These results suggest that mutant 471 might be a more promising anti-cancer agent than wild-type TNF.

• Loetscher H et al.
• Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.
• J Biol Chem. 1991; 266: 18324-9
• Display abstract

• 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.

• Lau JY, Sheron N, Nouri-Aria KT, Alexander GJ, Williams R
• Increased tumor necrosis factor-alpha receptor number in chronic hepatitis B virus infection.
• Hepatology. 1991; 14: 44-50
• Display abstract

• Production of the antiviral cytokine, tumor necrosis factor-alpha is increased in chronic hepatitis B virus infection, and clinical studies of tumor necrosis factor-alpha have indicated a proviral effect at higher doses. To determine whether this might be related to abnormal cell surface tumor necrosis factor-alpha receptor expression, binding characteristics of cell surface tumor necrosis factor-alpha receptor on peripheral blood mononuclear cells in chronic hepatitis B virus carriers were studied using radioiodinated recombinant tumor necrosis factor-alpha. The specific binding curves generated were analyzed according to the method of Scatchard to determine cell surface receptor numbers and dissociation constants. A single class of cell surface tumor necrosis factor-alpha receptor was demonstrated on peripheral blood mononuclear cells and mononuclear subsets. The median number (range) of cell surface tumor necrosis factor-alpha receptors on peripheral blood mononuclear cells from controls (n = 11), chronic hepatitis B virus patients seropositive for hepatitis B virus DNA (n = 8) and seronegative for hepatitis B virus DNA (n = 8) were 2,329 (range = 1,538 to 3,133), 3,375 (range = 2,300 to 6,718) (p less than 0.01) and 3,113 (range = 2,229 to 5,246) (p less than 0.05) sites/cell, respectively. They all had similar dissociation constants of 8.4 x 10(-10) mol/L (range = 4.1 to 16.9), respectively. Further dissection of the peripheral blood mononuclear cells showed that this increase in cell surface receptor number was confined to the monocyte fraction (p less than 0.01). Plasma tumor necrosis factor-alpha levels in five patients with increased monocyte cell surface tumor necrosis factor-alpha receptor numbers were also elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

• Derre J, Kemper O, Cherif D, Nophar Y, Berger R, Wallach D
• The gene for the type 1 tumor necrosis factor receptor (TNF-R1) is localized on band 12p13.
• Hum Genet. 1991; 87: 231-3

• Porteu F, Brockhaus M, Wallach D, Engelmann H, Nathan CF
• Human neutrophil elastase releases a ligand-binding fragment from the 75-kDa tumor necrosis factor (TNF) receptor. Comparison with the proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils.
• J Biol Chem. 1991; 266: 18846-53
• Display abstract

• To localize the protease(s) involved in shedding of tumor necrosis factor receptors (TNF-R) from activated neutrophils (PMN) (Porteu, F., and C. Nathan (1990) J. Exp. Med. 172, 599-607), we tested subcellular fractions from PMN for their ability to cause loss of TNF-R from intact cells. Exposure of PMN to sonicated azurophil granules at 37 degrees C resulted in inhibition of 125I-TNF binding; 50% inhibition ensued when PMN were treated for approximately 1 min with azurophil granules equivalent to 2-3 PMN per indicator cell. The TNF-R-degrading activity in azurophil granules were identified as elastase by its sensitivity to diisopropyl fluorophosphate (DFP), alpha 1-antitrypsin and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MSAAPV-CK), and by the ability of purified elastase to reproduce the effect of azurophil granules. Elastase preferentially acted on the 75-kDa TNF-R, reducing by 85-96% the binding of 125I-TNF to mononuclear cells expressing predominantly this receptor, while having no effect on endothelial cells expressing almost exclusively the 55-kDa TNF-R. Elastase-treated PMN released a 32-kDa soluble fragment of p75 TNF-R that bound TNF and reacted with anti-TNF-R monoclonal antibodies. In contrast, fMet-Leu-Phe-activated PMN shed a 42-kDa fragment from p75 TNF-R, along with similar amounts of a 28-kDa fragment from p55 TNF-R. Shedding of both TNF-Rs by intact activated PMN was more extensive than shedding caused by elastase and was completely resistant to DFP and MSAAPV-CK. Thus, the TNF-R-releasing activity of azurophil granules is distinct from that operative in intact stimulated PMN and could provide an additional mechanism for the control of cellular responses to TNF at sites of inflammation.

• Kemper O, Derre J, Cherif D, Engelmann H, Wallach D, Berger R
• The gene for the type II (p75) tumor necrosis factor receptor (TNF-RII) is localized on band 1p36.2-p36.3.
• Hum Genet. 1991; 87: 623-4
• Display abstract

• The gene encoding the type II (p75) tumor necrosis factor receptor (TNF-RII) has been localized on human chromosome 1, band 1p36.2 by nonradioactive in situ hybridization. The gene encoding the type I (p55) TNF-R, which is structurally homologous to the type II (p75) TNF-R, has been previously localized on chromosome 12 band 12p13. Thus, despite their probable common ancestry, the genes for the two TNF-Rs are localized on different chromosomes.

• Williams GT
• Programmed cell death: apoptosis and oncogenesis.
• Cell. 1991; 65: 1097-8

• Loetscher H, Steinmetz M, Lesslauer W
• Tumor necrosis factor: receptors and inhibitors.
• Cancer Cells. 1991; 3: 221-6
• Display abstract

• Tumor necrosis factor (TNF) is a highly potent pleiotropic response modifier in inflammatory and immunologic host defense reactions. It can also be toxic to cells and elicit toxic systemic reactions, as evinced by certain pathophysiologic conditions that are initiated or aggravated by an excess of TNF. The cellular mechanisms for transducing TNF signals are complex. There are two forms of TNF, alpha and beta, and two distinct TNF receptors. Many cells express both receptor types simultaneously, even though neither membrane receptor can distinguish between TNF-alpha and TNF-beta. The effects of TNF are inhibited by binding proteins that are truncated fragments of the extracellular domains of the TNF receptors. The mechanisms by which these components of the TNF signal transmission pathways interact to mediate the pleiotropic effects of TNF remain unclear.

• Peck R, Brockhaus M, Frey JR
• The principal tumor necrosis factor receptor in monocyte cytotoxicity is on the effector cell, not on the target cell.
• Cell Immunol. 1991; 132: 308-18
• Display abstract

• Several tumor target cell lines, prototypically K562 cells, are resistant to lysis by recombinant tumor necrosis factor (TNF alpha) but are killed by monocytes expressing membrane-associated TNF, suggesting that membrane TNF could account for monocyte-mediated cytotoxicity. Formaldehyde-fixed monocytes or extracted monocyte membrane fragments are cytotoxic to K562 target cells. Treatment of monocytes with interferon-gamma (IFN-gamma) increases cytotoxicity by live and fixed cells or by extracted monocyte membranes. Both TNF and TNF receptors are detectable on monocyte membranes by FACS analysis, and the levels of each are modulated by treatment with IFN-gamma. Cytotoxicity can be inhibited by either anti-TNF or anti-TNF receptor antibodies. Incubation of effector cells with exogenous soluble TNF prior to fixation or membrane preparation increases their cytotoxicity. In contrast, incubation of the target cells with exogenous TNF neither increases nor decreases killing by effector cell membrane fragments or intact effector cells. The data suggest that the TNF receptors on the effector cell, but not on the target cell, play a crucial role in TNF-mediated cytotoxicity.

• Engelberts I, Stephens S, Francot GJ, van der Linden CJ, Buurman WA
• Evidence for different effects of soluble TNF-receptors on various TNF measurements in human biological fluids.
• Lancet. 1991; 338: 515-6

• Holtmann H et al.
• Mechanisms controlling the level of receptors for tumor necrosis factor.
• Agents Actions. 1991; 32: 106-8

• Kinouchi K, Brown G, Pasternak G, Donner DB
• Identification and characterization of receptors for tumor necrosis factor-alpha in the brain.
• Biochem Biophys Res Commun. 1991; 181: 1532-8
• Display abstract

• Specific receptors for murine TNF have been identified in homogenates of rodent brain. These receptors are saturable and bind TNF with sufficient affinity to ensure occupancy by cytokine elaborated during infection. 125I-mTNF was detected in four specific complexes of Mr 130,000, 90,000, 66,000 and 60,000 after affinity labeling. Solubilization of brain membranes into detergent increased binding capacity 4-fold which indicates the presence of latent receptors for mTNF in the brain. Specific binding was greatest in the brainstem, least in the cerebellum and was also detected in the cortex, thalamus and basal ganglia.

• Thoma B, Grell M, Pfizenmaier K, Scheurich P
• Identification of a 60-kD tumor necrosis factor (TNF) receptor as the major signal transducing component in TNF responses.
• J Exp Med. 1990; 172: 1019-23
• Display abstract

• We describe here a monoclonal antibody (H398) that immunoprecipitates a human 60-kD tumor necrosis factor (TNF) membrane receptor (p60) and competes with TNF binding to p60 but not to p85 TNF receptors. Despite partial inhibition of TNF binding capacity of cells coexpressing both TNF receptor molecules, H398 uniformly and completely inhibits very distinct TNF responses on a variety of cell lines. These data suggest a limited structural heterogeneity in those components actually contributing to TNF responsiveness and identify p60 as a common receptor molecule essential for TNF signal transduction. As H398 is a highly effective TNF antagonist in vitro, it might be useful as a therapeutic agent in the treatment of TNF-mediated acute toxicity.

• Dunne SL, Bajzer Z, Vuk-Pavlovic S
• Kinetics of receptor-mediated uptake and processing of interferon-alpha 2a and tumor necrosis factor-alpha by human tumor cells.
• Growth Factors. 1990; 2: 167-77
• Display abstract

• The kinetics of uptake and processing of recombinant human interferon-alpha 2a (IFN) and recombinant human tumor necrosis factor-alpha (TNF) were studied in human epithelial tumor cell lines differing in sensitivity to growth inhibition by IFN and TNF. Concentrations of [125I]IFN or [125I]TNF at the cell surface and internalized by confluent cell monolayers incubated at 37 degrees C were measured as a function of time. Cells incubated with [125I]IFN exhibited transient maxima of surface-associated and internalized [125I]IFN after 1-2 hr of incubation followed by a steady-state attained after approximately 6 hr of exposure to [125I]IFN. Concentration of [125I]TNF associated with the plasma membrane displayed a maximum within 20 min of incubation. Internalized radioactivity increased with time and did not achieve a steady state. We analyzed the time-dependent concentrations of membrane-associated and internalized cytokines by use of a compartmental model. This model describes changes in concentrations of free surface receptors, of ligand-receptor complex at the membrane and of internalized ligand and contains a term for receptor recycling. The rate constants for concentration changes were evaluated by fitting model functions to data. The best fits for IFN were obtained without receptor recycling. The best-fit values for the endocytotic rate constant (ke,IFN) varied among cell lines from 2.4 x 10(-4) to 7.8 x 10(-4) sec-1. To obtain fits to time-dependent concentrations of surface-associated and internalized [125I]TNF, introduction of a term for receptor recycling was required. Best-fit values for ke,TNF ranged among cell lines from 8.4 x 10(-4) to 2.5 x 10(-3) sec-1. For every cell line, the value of ke,TNF was larger than the value of ke,IFN. We tested the significance of these differences by substituting ke,IFN for ke,TNF as fixed parameters in fits to data for TNF and v.v., respectively. Under these conditions, fits were significantly worse. Our data indicate that recycling is insignificant in kinetics of Type-I IFN receptor; recycling is necessary to explain the kinetics of TNF receptor. Upon binding, TNF is taken up by cells faster than IFN and is eliminated from cells more slowly than IFN.

• Kohno T et al.
• A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor.
• Proc Natl Acad Sci U S A. 1990; 87: 8331-5
• Display abstract

• An inhibitor of tumor necrosis factor (TNF) has been isolated from the human histiocytic lymphoma cell line U-937 that is capable of inhibiting both TNF-alpha and TNF-beta. Protein sequencing has verified that it is distinct from a previously described TNF inhibitor that is a soluble fragment of a TNF receptor molecule (TNFrI). The cDNA sequence of this second TNF inhibitor clone suggests that it is also a soluble fragment of a TNF receptor. Expression of this cDNA sequence in COS-7 cells verified that it encodes a receptor for TNF-alpha (TNFrII) that can give rise to a soluble inhibitor of TNF-alpha, presumably through proteolytic cleavage. The extracellular domain of TNFrII has significant homology with that of TNFrI and these two receptors share a striking conservation of cysteine residue alignment with the extracellular domain of the nerve growth factor receptor. These three receptor molecules are therefore members of a family of polypeptide hormone receptors.

• Pirisi M, Vasiliadis D, Harrison LC
• Tumor necrosis factor secreted by transformed human B lymphocytes: lack of an autocrine growth effect.
• Lymphokine Res. 1990; 9: 27-33
• Display abstract

• There is evidence that tumor necrosis factor (TNF) may be a proliferation and differentiation factor for B lymphocytes. We found that three of four lymphoblastoid cell lines (ADD, IM-9, W1) secreted TNF-beta and expressed TNF receptors, whereas one pre-B cell leukemia and six Burkitt's lymphoma cell lines had no detectable TNF secretion and, except for one Burkitt's cell line (LOU), very low expression of TNF receptors. When IM-9, W1 or LOU cells were cultured over seven days in the presence of either TNF-alpha or antiserum to TNF-beta there was no difference between their growth rate, endogenous TNF-beta secretion or immunoglobulin secretion compared to untreated cells. These findings indicate that TNF does not have a universal role as an autocrine growth factor in transformed B lymphocytes.

• Ohmann HB, Campos M, McDougall L, Lawman MJ, Babiuk LA
• Expression of tumor necrosis factor-alpha receptors on bovine macrophages, lymphocytes and polymorphonuclear leukocytes, internalization of receptor-bound ligand, and some functional effects.
• Lymphokine Res. 1990; 9: 43-58
• Display abstract

• Tumor necrosis factor-alpha (TNF) is a pluripotent protein produced by cells of the monocyte-macrophage lineage. It has important pro-inflammatory functions and is thought to be involved in the pathogenesis of viral-bacterial lung infections of cattle. Binding and internalization of gold-labeled TNF (TNF-G10) by bovine alveolar macrophages as well as by peripheral blood mononuclear leukocytes (PBML) and polymorphonuclear granulocytes (PMN) have therefore been investigated in conjunction with studies of the effect of TNF on some leukocyte functions. TNF-G10 bound to all leukocyte types to varying extents, which however, did not correlate with the influence of TNF on cell functions. Thus, PMN appeared to possess the lowest number of TNF-receptors, but were nevertheless the most sensitive to functional modulation by TNF. The expression of TNF-receptors appeared to be regulated by other cytokines such as IFN-gamma and IL-2, with IFN-gamma down-regulating receptor expression on all cell types, and IL-2 up-regulating receptor expression on lymphocytes. In macrophages internalization of TNF-G10 occurred via clathrin-coated structures, whereas in lymphocytes and PMN the endosomes appeared to lack distinct coating.

• Giaccone G, Kadoyama C, Maneckjee R, Venzon D, Alexander RB, Gazdar AF
• Effects of tumor necrosis factor, alone or in combination with topoisomerase-II-targeted drugs, on human lung cancer cell lines.
• Int J Cancer. 1990; 46: 326-9
• Display abstract

• Tumor necrosis factor alpha (TNF) exhibits cytotoxic activity on some solid tumors and has been reported to be synergistic with topoisomerase-II-targeted antineoplastic agents. A wide range of TNF concentrations (from 10 to 10,000 U/ml) was tested in 9 human lung cancer cell lines (5 small-cell and 4 non-small-cell carcinomas) using a semi-automated MTT assay. TNF was not cytotoxic in 8 cell lines, while an adenocarcinoma cell line was marginally sensitive to the cytokine. Using 125I-TNF we were able to show the presence of specific binding sites for TNF in 4/9 human lung cancer cell lines. Scatchard analysis of the marginally sensitive cell line showed high-affinity, saturable binding. With 5 cell lines we also tested whether TNF affected the cytotoxicity of doxorubicin and etoposide, 2 topoisomerase II-targeted drugs which are widely used in the therapy of lung cancer. No significant increase in cytotoxicity was seen when TNF was added to the 2 anti-neoplastic agents. In contrast to certain other human and mouse lines, human lung cancer cell lines appear to be resistant to TNF, despite the presence of the receptor in some of them; moreover, no synergistic effect of TNF and 2 topoisomerase-II-targeted drugs was evident in these human cell lines.

• Gatanaga T et al.
• Purification and characterization of an inhibitor (soluble tumor necrosis factor receptor) for tumor necrosis factor and lymphotoxin obtained from the serum ultrafiltrates of human cancer patients.
• Proc Natl Acad Sci U S A. 1990; 87: 8781-4
• Display abstract

• Serum ultrafiltrates (SUF) from human patients with different types of cancer contain a blocking factor (BF) that inhibits the cytolytic activity of human tumor necrosis factor alpha (TNF-alpha) in vitro. BF is a protein with a molecular mass of 28 kDa on reducing sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The active material was purified to homogeneity by a combination of affinity chromatography, PAGE, and high-pressure liquid chromatography. Amino acid sequence analysis revealed that BF is derived from the membrane TNF receptor. Purified BF blocks the lytic activity of recombinant human and mouse TNF-alpha and recombinant human lymphotoxin on murine L929 cells in vitro. However, BF inhibits the lytic activity of TNF-alpha more effectively than it does that of lymphotoxin. The BF also inhibits the necrotizing activity of recombinant human TNF-alpha when coinjected into established cutaneous Meth A tumors in BALB/c mice. The BF may have an important role in (i) the regulation and control of TNF-alpha and lymphotoxin activity in cancer patients, (ii) interaction between the tumor and the host antitumor mechanisms, and (iii) use of systemically administered TNF-alpha in clinical trials with human cancer patients.

• Shanahan F, Niederlehner A, Carramanzana N, Anton P
• Sulfasalazine inhibits the binding of TNF alpha to its receptor.
• Immunopharmacology. 1990; 20: 217-24
• Display abstract

• Sulfasalazine was found to exhibit a dose-dependent inhibition of human mucosal and peripheral blood cytotoxic T-cell function. The drug also inhibited the cytotoxic activity of supernatants from anti-CD3-triggered T-cells against murine L929 fibroblasts. TNF alpha has previously been shown to be primarily responsible for the lytic activity of such supernatants and this was confirmed. Sulfasalazine also inhibited the lytic activity of recombinant TNF alpha. When tested under conditions where TNF alpha was allowed to bind to but not lyse the target cells, the results suggested that the drug inhibits the action of this cytokine by inhibiting its binding to the cell membrane receptor. Additional evidence for an inhibitory effect of sulfasalazine on the membrane binding of TNF alpha was obtained by demonstrating a dose-dependent displacement of 125I-TNF alpha from HL60 cells. Although sulfasalazine is often considered to be a pro-drug for site-specific delivery of its component fragments 5-ASA and sulfapyridine, the results demonstrate an immunopharmacological property of the parent compound that is not shared with its component molecules.

• Kronke M et al.
• Tumour necrosis factor signal transduction.
• Cell Signal. 1990; 2: 1-8

• Shalaby MR, Sundan A, Loetscher H, Brockhaus M, Lesslauer W, Espevik T
• Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors.
• J Exp Med. 1990; 172: 1517-20
• Display abstract

• The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions.

• Lindvall L, Lantz M, Gullberg U, Olsson I
• Modulation of the constitutive gene expression of the 55 kD tumor necrosis factor receptor in hematopoietic cells.
• Biochem Biophys Res Commun. 1990; 172: 557-63
• Display abstract

• The expression of the 55 kD human tumor necrosis factor (TNF) receptor gene was investigated. By use of a 1.2 kb cDNA we demonstrated a constitutive expression of a single 2.3 kb transcript in cell lines and fresh blood cells. The TNF receptor gene expression was not affected by phorbol esters, dibutyryl cAMP (dbcAMP), interleukin-1 (IL-1), interferon-gamma (IFN-gamma) or by TNF, agents known to modulate functional TNF receptors. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) showed a dose dependent inhibition of the TNF receptor gene expression. This inhibition is not mediated by cAMP, since neither dbcAMP nor forskolin had any effects on the expression of the 55 kD receptor gene. The results suggest that the effects of phorbol diesters, IL-1, IFN-gamma, TNF and dbcAMP previously observed on binding of TNF to cells are limited to posttranscriptional regulation of the 55 kD TNF receptor.

• Johnson DR, Pober JS
• Tumor necrosis factor and immune interferon synergistically increase transcription of HLA class I heavy- and light-chain genes in vascular endothelium.
• Proc Natl Acad Sci U S A. 1990; 87: 5183-7
• Display abstract

• Tumor necrosis factor and immune interferon synergistically increase cell-surface expression of class I major histocompatibility complex molecules in cultured human endothelial cells. We report that tumor necrosis factor and interferon gamma each independently increase mRNA levels and together cause a greater-than-additive (i.e., synergistic) increase in steady-state mRNA levels and transcriptional rates of the class I heavy- and light-chain genes. HLA heavy-chain mRNA is equally stable in cytokine-treated and -untreated endothelial cells. Interferon gamma does not increase tumor necrosis factor receptor number or affinity on human endothelial cells. We conclude that the synergistic increase in class I major histocompatibility complex cell-surface expression results principally from the synergistic increase in transcriptional rates. We propose that this increase is caused by the cooperative binding of independently activated transcription factors to the promoter/enhancer sequences of class I genes.

• Seckinger P, Zhang JH, Hauptmann B, Dayer JM
• Characterization of a tumor necrosis factor alpha (TNF-alpha) inhibitor: evidence of immunological cross-reactivity with the TNF receptor.
• Proc Natl Acad Sci U S A. 1990; 87: 5188-92
• Display abstract

• Previous studies have shown that urine of febrile patients contains a tumor necrosis factor alpha inhibiting activity (TNF-alpha Inh) when tested in a cytotoxicity assay using the tumor necrosis factor alpha (TNF-alpha)-susceptible cell line L929. In the present study, we investigated the relationship between the TNF-alpha Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-alpha activities by binding to the ligand. We demonstrate that human TNF-alpha is affected to a greater extent than is murine TNF-alpha. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-alpha Inh that neutralizes its activity and does not recognize TNF-alpha. Solubilized cross-linked 125I-labeled TNF-alpha receptor complex could be immunoprecipitated by using either anti-TNF-alpha or anti-TNF-alpha Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-alpha, it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-alpha receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-alpha Inh antibody revealed with a fluorescein isothiocyanate-coupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-alpha Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-alpha Inh originally described might be a soluble form of the TNF receptor itself.

• Brockhaus M, Schoenfeld HJ, Schlaeger EJ, Hunziker W, Lesslauer W, Loetscher H
• Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies.
• Proc Natl Acad Sci U S A. 1990; 87: 3127-31
• Display abstract

• The pleiotropic cyto/lymphokine tumor necrosis factor (TNF) exerts its functions by binding to specific cell-surface receptors. We have prepared two sets of monoclonal antibodies (mAbs) against TNF-binding proteins from the HL-60 (htr-mAb series) and U-937 (utr-mAb series) cell lines. The htr antibodies inhibit the binding of 125I-labeled TNF-alpha to HL-60 cells only partially, whereas they block the TNF-alpha binding to several adenocarcinoma cell lines (HEp-2, HeLa, and MCF7) almost completely. In contrast, the utr antibodies have no effect on TNF-alpha binding to the adenocarcinoma cell lines but partially inhibit TNF-alpha binding to HL-60 and U-937 cells. However, htr-9 and utr-1 antibodies in combination fully inhibit the TNF-alpha binding to HL-60 and U-937 cells. The binding of TNF-beta to HEp-2 and U-937 cells is also inhibited by htr and utr antibodies. Neither htr nor utr mAb has an effect on the TNF-sensitive murine cell lines L929 and WEHI 164. Flow cytometry studies show that mAbs htr-9 and utr-1 detect two distinct TNF-binding sites on human cell lines. Immunologic blot and immunoprecipitation analyses indicate that mAbs htr-9 and utr-1 recognize proteins of approximately 55 kDa and 75 kDa, respectively. These data provide evidence for the existence of two distinct TNF receptor molecules that contribute to varying extent to the TNF binding by different human cells.

• Crump WL 3rd, Owen-Schaub LB, Grimm EA
• Molecular identification of the human tumor necrosis factor receptor on interleukin-2-stimulated peripheral blood lymphocytes.
• Cell Immunol. 1990; 131: 150-8
• Display abstract

• The human tumor necrosis factor (TNF) receptor on interleukin (IL)-2-stimulated lymphocytes was characterized by binding and crosslinking techniques. The TNF receptor on IL-2-activated lymphocytes has an affinity of approximately 50 pM. Conventional crosslinking studies with the DSS analog bis(sulfosuccinimidyl) suberate demonstrated a ligand-receptor complex molecular weight of 106-108 kDa. Lectin precipitation experiments indicated that the receptor is a glycoprotein with an affinity for lectin isolated from Ricinus communis. Affinity crosslinking studies with the iodinateable, cleavable crosslinker sulfosuccinimidyl 2-(p-azido-salicylamido) ethyl 1,3'-dithiopropionate demonstrated that the TNF receptor, by itself, in the absence of bound ligand, has a molecular weight of approximately 90 kDa. Furthermore, these results indicate that the crosslinked TNF:TNF-receptor complexes observed at 104-108 kDa are composed of receptor and monomeric TNF.

• Engelmann H, Novick D, Wallach D
• Two tumor necrosis factor-binding proteins purified from human urine. Evidence for immunological cross-reactivity with cell surface tumor necrosis factor receptors.
• J Biol Chem. 1990; 265: 1531-6
• Display abstract

• 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.

• Nakaya K, Deguchi Y, Arata S, Kasai N, Nakamura Y
• Tumor necrosis factor changes sensitivity of differentiation of mouse leukemia M1 cells by lipopolysaccharide.
• Jpn J Cancer Res. 1990; 81: 396-402
• Display abstract

• A clone of mouse leukemia M1 cells was induced to differentiate by lipopolysaccharide (LPS) (LPS-sensitive clone) while another clone of the same cells was resistant (LPS-resistant clone). LPS and lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis were as active as Escherichia coli LPS in the induction of differentiation of the LPS-sensitive clone. Synthetic lipid A precursor Ia (compound 406), which has no interleukin 1 (IL-1)-inducing activity toward monocytes, had strong differentiation-inducing activity toward the LPS-sensitive clone. The combined treatment of the LPS-sensitive clone with LPS and recombinant tumor necrosis factor (rTNF) did not further increase the degree of differentiation induced by LPS alone. By contrast, the LPS-resistant clone was markedly induced to differentiate by LPS in the presence of rTNF. Combined treatment of the LPS-resistant clone with LPS and other cytokines such as recombinant IL-1 alpha, recombinant granulocyte colony-stimulating factor, and interferon-gamma was not effective in inducing marked synergistic differentiation. These results raise the possibility that rTNF changes the sensitivity of M1 cells to induction of differentiation by LPS.

• Schleiffenbaum B, Fehr J
• The tumor necrosis factor receptor and human neutrophil function. Deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation.
• J Clin Invest. 1990; 86: 184-95
• Display abstract

• Despite numerous reports, the role of tumor necrosis factor (TNF) in polymorphonuclear leukocyte (PMN) function remains controversial. We found TNF to be a potent, pertussis toxin-independent stimulator of PMN adhesion (ED50 2.6 pM). TNF-stimulated PMN under adherent conditions released up to 65% of their transcobalamine content (ED50 3.9 pM) and increased their burst activity 10-fold (ED50 3.2 pM) as measured by the hexose monophosphate shunt, whereas PMN held in suspension hardly degranulated at all and only little burst activity was demonstrable. However, preincubation of PMN with TNF in suspension led to a decrease in cellular adhesiveness, degranulation, and burst activity in response to a secondary stimulus of TNF under adherent conditions, although cells remained fully responsive toward phorbol myristate acetate. A concomitant dose-dependent decline of TNF receptor numbers that correlated well with the inhibition of PMN function (r = 0.91) suggests receptor down-regulation as the mechanism of functional PMN deactivation. Remarkably, preincubation with other PMN stimuli such as N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor, leukotriene B4, complement component fragment 5a (C5a)/C5a (desarginated), and endotoxin also led to a reduction of TNF-specific PMN responses (cross-deactivation) from 35% (LTB4) to 90% (endotoxin), corresponding with the down-regulation of TNF receptors. Deactivation and receptor down-regulation are independent of pertussis toxin-sensitive G proteins and protein kinase C but seemed to depend on changes in calcium metabolism. Granulocyte hyporesponsiveness towards TNF in sepsis (with elevated blood levels of endotoxin and TNF) might be a mechanism of self-protection or, to the contrary, might impair a possibly central mode of host defense.

• Heller RA, Song K, Onasch MA, Fischer WH, Chang D, Ringold GM
• Complementary DNA cloning of a receptor for tumor necrosis factor and demonstration of a shed form of the receptor.
• Proc Natl Acad Sci U S A. 1990; 87: 6151-5
• Display abstract

• Tumor necrosis factor (TNF) receptor (TNFR) was isolated as a 68-kDa glycoprotein from UC/HeLa 2-5 cells developed from a parental B-cell line (UC cells) to overexpress the receptor. Tryptic digests of two separate TNFR preparations provided amino acid sequences of four different peptides. Amino-terminal analysis indicated the presence of the amino-acid sequence Val-Ala-Phe-Thr-Pro, reported to be the amino-terminal sequence of a 30-kDa urinary TNF-binding protein II. Examination of the cultured medium of UC/HeLa 2-5 cells showed an abundance of a 40-kDa TNF-binding protein, indicating that the previously cited 30-kDa TNF-binding protein II is likely to be a shed form of the TNFR. Based on the peptide sequences, oligonucleotides were synthesized, and two of these were used as primers in the polymerase chain reaction to amplify cDNA sequences from poly(A)+ RNA of UC/HeLa 2-5 cells. These PCR fragments were radiolabeled and used to screen a cDNA library made from UC/HeLa 2-5 mRNA. Further analysis identified cDNA sequences that encoded the amino acid sequences of all four TNFR peptides. RNA blot-hybridization analysis of UC/HeLa 2-5 mRNA revealed a 3.8-kilobase transcript of the same size as the mRNA in the parental UC cells. Genomic Southern blots indicated the presence of a single gene in parental cells and a second, amplified gene in TNFR-overexpressing cells, suggesting amplification of the transfected gene as a possible mechanism for the increase in TNFR numbers in UC/HeLa 2-5 cells.

• Ding AH, Porteu F, Sanchez E, Nathan CF
• Shared actions of endotoxin and taxol on TNF receptors and TNF release.
• Science. 1990; 248: 370-2
• Display abstract

• Bacterial lipopolysaccharide (LPS) exerts profound effects on mammalian hosts in part by inducing macrophages to release tumor necrosis factor-alpha (TNF-alpha); the mechanisms involved are unresolved. The microtubule stabilizer taxol shared two actions of LPS on macrophages: it rapidly decreased TNF-alpha receptors and triggered TNF-alpha release. Both actions of taxol were absent in LPS-hyporesponsive C3H/HeJ mice. In recombinant inbred mice, the genes controlling responses to LPS and to taxol were closely linked. Dexamethasone blocked release of TNF-alpha by both stimuli but did not block the decrease in TNF-alpha receptors. Thus, a protein associated with microtubules may be a cellular target of LPS.

• Bethea JR, Gillespie GY, Chung IY, Benveniste EN
• Tumor necrosis factor production and receptor expression by a human malignant glioma cell line, D54-MG.
• J Neuroimmunol. 1990; 30: 1-13
• Display abstract

• Human malignant gliomas possess some of the same immune-related functions as astrocytes do. For instance, they are capable of secreting various immunoregulatory molecules and expressing HLA-DR antigens on their surface. The human malignant glioma cell line, D54-MG, was used to investigate the proliferative effects of tumor necrosis factor-alpha (TNF-alpha) and the expression of specific surface receptors for TNF-alpha. Additionally, we were interested in examining whether D54-MG cells are capable of synthesizing and secreting biologically active TNF-alpha. D54-MG cells responded in a mitogenic fashion upon incubation with TNF-alpha for 48 h under serum-free conditions. 125I-labeled TNF-alpha was used in this study to investigate the expression of receptors specific for TNF-alpha on D54-MG cells. Scatchard analysis of our receptor binding data produced curvilinear plots indicating there are two distinct receptor sites for TNF-alpha. From these data, we calculated that there are approximately 3500 high affinity and 24,666 low affinity binding sites per cell. Pretreating these cells with interferon-gamma (IFN-gamma) resulted in a 2-fold increase in the number of high affinity binding sites and a moderate increase in the number of low affinity binding sites, with no appreciable change in binding affinity (Kd) of either site. D54-MG cells were unable to constitutively secrete TNF-alpha; however, upon stimulation, these cells synthesize and secrete biologically active TNF-alpha. Polyclonal antisera reactive with human macrophage-derived TNF-alpha neutralized the cytotoxicity of D54-MG-derived TNF-alpha, demonstrating that the cytotoxic activity was in fact due to TNF-alpha. Our observations indicate that TNF-alpha could act in an autocrine fashion to induce the proliferation of this malignant glioma cell line and that TNF-alpha exerts its effect by binding to specific TNF-alpha receptors whose expression was enhanced by IFN-gamma.

• Espevik T, Brockhaus M, Loetscher H, Nonstad U, Shalaby R
• Characterization of binding and biological effects of monoclonal antibodies against a human tumor necrosis factor receptor.
• J Exp Med. 1990; 171: 415-26
• Display abstract

• Three different antibodies against a human TNF receptor (htr-1, htr-5, and htr-9) have been examined for their binding pattern to U937 cells and ability to mimic TNF-alpha activity in U937 cells, Fs4 fibroblasts, and human endothelial cells. Flow cytometric analysis revealed that htr-5 and htr-9 bound specifically to a TNF receptor on U937 cells that could be blocked by pretreatment with rTNF-alpha. Pretreatment of U937 cells with rTNF-beta blocked the binding of htr-9, but to a lesser extent htr-5 binding. Pretreatment with htr-5 inhibited the binding of htr-9 to U937 cells while pretreatment with htr-9 did not inhibit htr-5 binding. These results indicate that htr-5 and htr-9 recognize distinct but overlapping epitopes of a human TNF receptor on U937 cells and that htr-5 may be close to a TNF-alpha-specific domain of the binding site. Pretreatment with htr-5 or htr-9 only minimally reduced binding of BrTNF-alpha to U937 cells; however, these antibodies were much more effective in inhibiting BrTNF-alpha binding to HL-60 cells. Furthermore, it was found that htr-1 and htr-9, but not htr-5, had TNF-alpha activity on U937 cells, Fs4 fibroblasts, and endothelial cells and that the TNF-alpha activity induced by htr-9 was completely inhibited by htr-5. However, the cytotoxic activity of TNF-alpha was only partially inhibited by htr-5 on U937 cells while htr-5 had no effect on TNF-alpha activity on Fs4 cells. The data suggest that a common epitope is involved in inducing TNF-alpha activity in three different cell systems.

• Loetscher H et al.
• Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor.
• Cell. 1990; 61: 351-9
• Display abstract

• Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.

• Porteu F, Nathan C
• Shedding of tumor necrosis factor receptors by activated human neutrophils.
• J Exp Med. 1990; 172: 599-607
• Display abstract

• The capacity of human neutrophils (PMN) to bind tumor necrosis factor (TNF) was rapidly lost when the cells were incubated in suspension with agents that can stimulate their migratory and secretory responses. Both physiological (poly)peptides (FMLP, C5a, CSF-GM) and pharmacologic agonists (PMN, calcium ionophore A23187) induced the loss of TNF receptors (TNF-R) from the cell surface. Half-maximal loss in TNF-R ensued after only approximately 2 min with 10(-7) M FMLP at 37 degrees C, and required only 10(-9) M FMLP during a 30-min exposure. However, there were no such changes even with prolonged exposure of PMN to FMLP at 4 degrees or 16 degrees C. Scatchard analysis revealed loss of TNF-binding sites without change in their affinity (Kd approximately 0.4 nM) as measured at incompletely modulating concentrations of FMLP, C5a, PMA, or A23187. The binding of anti-TNF-R mAbs to PMN decreased in parallel, providing independent evidence for the loss of TNF-R from the cell surface. At the same time, soluble TNF-R appeared in the medium of stimulated PMN. This inference was based on the PMN- and FMLP-dependent generation of a nonsedimentable activity that could inhibit the binding of TNF to fresh human PMN or to mouse macrophages, and the ability of mAbs specific for human TNF-R to abolish inhibition by PMN-conditioned medium of binding of TNF to mouse macrophages. Soluble TNF-R activity was associated with a protein of Mr approximately 28,000 by ligand blot analysis of cell-free supernatants of FMLP-treated PMN. Thus, some portion of the FMLP-induced loss of TNF-R from human PMN is due to shedding of TNF-R. Shedding was unaffected by inhibitors of serine and thiol proteases and could not be induced with phosphatidylinositol-specific phospholipase C. Loss of TNF-R from PMN first stimulated by other agents may decrease their responsiveness to TNF. TNF-R shed by PMN may be one source of the TNF-binding proteins found in body fluids, and may blunt the actions of the cytokine on other cells.

• Himmler A et al.
• Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.
• DNA Cell Biol. 1990; 9: 705-15
• Display abstract

• Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor. The calculated Mr of the mature human and rat TNF receptor chains is 47,526 and 48,072, respectively. The extracellular ligand binding domain represents the soluble TNF-BP which is released by proteolytic cleavage. TNF-BP contains 24 cysteine residues and three potential N-glycosylation sites and shows sequence homology to the extracellular portions of TNF-R p80 chain and nerve growth factor receptor. Transfection of the human TNF receptor cDNA into mammalian cells resulted in increased binding capacity for TNF-alpha and increased reactivity with a monoclonal antibody directed against the human TNF receptor chain p60. When a stop codon was introduced into the cDNA at the site corresponding to the carboxyl terminus of TNF-BP, transfected cells secreted a protein that reacted with antibodies raised against natural TNF-BP.

• Engelmann H et al.
• Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.
• J Biol Chem. 1990; 265: 14497-504
• Display abstract

• 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.

• Hohmann HP, Brockhaus M, Baeuerle PA, Remy R, Kolbeck R, van Loon AP
• Expression of the types A and B tumor necrosis factor (TNF) receptors is independently regulated, and both receptors mediate activation of the transcription factor NF-kappa B. TNF alpha is not needed for induction of a biological effect via TNF receptors.
• J Biol Chem. 1990; 265: 22409-17
• Display abstract

• The expression and biological function of the types A and B tumor necrosis factor (TNF) receptors were studied using three cell types. SW480T, HEp2, and HL60 cells had, respectively, mainly the type A, only the type B, and roughly similar amounts of both receptors. Dibutyric cAMP treatment induced a 3-6-fold increase in the amount of the type A receptor in HL60 cells without affecting the amount of the type B receptor. Expression of both receptors can thus be regulated independently. HEp2 and human umbilical vein endothelial cells only showed the type B receptor, and expression of the type A receptor could not be induced in these cells. HL60 cells showed, upon Scatchard analysis, a single binding site for TNF alpha, and its Kd may correspond to that of the type A receptor. The approximately 7-fold lower affinity of TNF alpha binding to the type B receptor of HL60 cells was only detected after blocking all TNF alpha binding to the type A receptor. Both the types A and B receptors mediated TNF alpha-induced activation of the transcription factor NF-kappa B. The agonistic antibody htr9 to the type B receptor also activated NF-kappa B. Thus, signal transduction via the type B receptor may only require interaction with the receptor's extracellular domain.

• Heller RA et al.
• Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries.
• J Biol Chem. 1990; 265: 5708-17
• Display abstract

• As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.

• Earl CQ, Stadel JM, Anzano MA
• Tumor necrosis factor-mediated biological activities involve a G-protein-dependent mechanism.
• J Biol Response Mod. 1990; 9: 361-7
• Display abstract

• The guanine nucleotide binding protein (G-protein) dependency of several of the activities of tumor necrosis factor (TNF), including cytotoxicity, inhibition of lipoprotein lipase activity, blockade of 3T3-L1 differentiation, and receptor binding were examined. TNF induced killing of the TNF-sensitive cell line L929S (ED50 = 30 pM), but had little to no effect on the TNF-resistant cell line L929R (ED50 = 5,300 pM). TNF-induced cytotoxicity in L929S was antagonized in a dose-dependent manner by pertussis toxin (sevenfold increase in ED50). However, TNF-induced cytotoxicity in L929R cells was only minimally affected by pretreatment with a high dose (50 ng/ml) of pertussis toxin (1.5-fold increase in ED50). Parallel biochemical investigations revealed that inhibition was accompanied by toxin-induced ADP ribosylation of a Gi alpha-like subunit in L929 and 3T3-L1 cell membranes. Pertussis toxin also significantly reduced TNF-induced inhibition of lipoprotein lipase activity in 3T3-L1 adipocytes and TNF blockade of 3T3-L1 preadipocyte differentiation. However, pertussis toxin pretreatment of L929S, L929R, and 3T3-L1 cell cultures had little to no effect on TNF receptor binding. These data indicate that several TNF-induced biological activities in the L929 and 3T3-L1 cell lines are partially dependent upon a pertussis toxin-sensitive G-protein.

• Turetskaia RL, Kuprash DV, Udalova IA, Azizov MM, Nedospasov SA
• [The molecular cloning and characteristics of the chromosomal copy of the human gene coding for the tumor-necrosis factor receptor]
• Dokl Akad Nauk SSSR. 1990; 315: 1475-8

• Jones EY, Stuart DI, Walker NP
• The structure of tumour necrosis factor--implications for biological function.
• J Cell Sci Suppl. 1990; 13: 11-8
• Display abstract

• The three-dimensional structure of TNF has been determined at 0.29 nm using the technique of X-ray crystallography. Published data on site-directed mutagenesis and antibody binding may now be assessed in the light of the structure, thus the links between structure and function for TNF may be addressed. TNF is a compact trimer composed of three identical subunits of 157 amino acids. The main-chain topology for a single subunit is essentially a beta-sandwich structure formed by two anti-parallel beta-pleated sheets. This mainchain fold corresponds to the 'jelly roll' motif observed in viral coat proteins such as VP1, VP2 and VP3 of rhinovirus, or the hemagglutinin molecule of influenza. TNF is the first non-viral protein to contain this motif. The subunits associate tightly about a threefold axis interacting through a simple edge-to-face packing of the beta-sandwich to form the solid, conical shaped trimer. A large number of the residues conserved between the amino acid sequences of TNF and lymphotoxin lie within the beta-sandwich or at the threefold axis of the trimer. This implies the presence of the same beta-sandwich motif in the lymphotoxin monomer and preservation of the edge-to-face mode of trimeric association. The detailed three dimensional structure for TNF explains a wide range of observations, including data on antibody binding and site directed mutagenesis. The currently available evidence points to a region of biological importance situated at the interface between two subunits on the lower half of the trimer.

• Aggarwal BB
• Lymphotoxin and tumor necrosis factor: qualitative and quantitative differences in their receptors and signal transduction in various cell types.
• Prog Clin Biol Res. 1990; 349: 375-84

• Hohmann HP, Remy R, Poschl B, van Loon AP
• Tumor necrosis factors-alpha and -beta bind to the same two types of tumor necrosis factor receptors and maximally activate the transcription factor NF-kappa B at low receptor occupancy and within minutes after receptor binding.
• J Biol Chem. 1990; 265: 15183-8
• Display abstract

• HL60 cells have types A and B tumor necrosis factor (TNF) receptors whereas HEp2 cells have only the type B receptor (Hohmann, H.-P., Remy, R., Brockhaus, M., and van Loon, A.P.G.M. (1989) J. Biol. Chem. 264, 14927-14934). TNF-beta can be cross-linked to each of these receptors and competes with TNF-alpha for binding to both receptors. TNF-alpha and TNF-beta activate the transcription factor NF-kappa B in HL60 and HEp2 cells. Maximal activation of NF-kappa B required binding of TNF-alpha or TNF-beta to 20-25% of the total number of TNF receptors and was achieved within minutes after the addition of TNF-alpha to HL60 cells. Both TNF-alpha and TNF-beta activate NF-kappa B at 5-10-fold lower concentrations in HL60 cells compared with HEp2 cells, and this correlates well with their different affinities for binding to these cells. Thus, TNF-alpha and TNF-beta are indistinguishable with respect to the correlation between degrees of receptor binding and activation of NF-kappa B.

• Vlassara H
• Advanced nonenzymatic tissue glycosylation: cell-mediated interactions implicated in the complications associated with diabetes and aging.
• Blood Purif. 1990; 8: 223-32
• Display abstract

• Tissue and cell surface proteins modified nonenzymatically by glucose are shown to be highly active in protein cross-linking and have been implicated in tissue damage. The production of such protein-glucose interactions called advanced glycosylation endproducts (AGE) are recently shown to be processed by macrophages through a recently characterized high-affinity receptor. Coupling of AGE proteins to their AGE receptor results in TNF and IL-1 synthesis and secretion. This suggests that AGE may act as a signal for growth-promoting factor secretion in a coordinated replacement process during tissue remodeling. A disturbance of this balance may lead to pathologic proliferative response such as in the vasculopathy of diabetes and aging. Since peritoneal surface proteins can be modified by AGE after exposure to high-glucose, a similar pathogenetic process may be involved in the peritoneal fibrosis associated with chronic peritoneal dialysis.

• Ding AH, Porteu F, Sanchez E, Nathan CF
• Downregulation of tumor necrosis factor receptors on macrophages and endothelial cells by microtubule depolymerizing agents.
• J Exp Med. 1990; 171: 715-27
• Display abstract

• Exposure of murine and human macrophages and human umbilical vein endothelial cells to micromolar concentrations of five microtubule (MT)-depolymerizing agents (colchicine, nocodazole, podophyllotoxin, vincristine, and vinblastine) resulted in a loss of binding sites for iodinated TNF-alpha. The reduction amounted to 40-60% by 1 h and approximately 75% by 2-4 h. In 1 h, specific binding was reduced 50% by 0.1-5 microM of these drugs at 37 degrees C, but not at 4 degrees C. Inactive isomers of colchicine were ineffective, as were microfilament-destabilizing cytochalasins. The active agents did not compete with TNF-alpha R for binding. Antiserum against TNF-alpha did not neutralize the effect of colchicine and nocodazole. PGE1 and dibutyryl-cAMP could not mimic, and cyclooxygenase inhibitors could not prevent the drug effects. All the binding sites were regenerated within 3 h after removal of nocodazole, which binds tubulin reversibly, whereas little recovery was found even 18 h after the removal of colchicine, which binds tubulin irreversibly. These findings suggested that MT disassembly was responsible for the observed downregulation of TNF-alpha R. The protein synthesis inhibitor cycloheximide inhibited binding of TNF-alpha to a similar extent and with a similar time course as colchicine in the absence of added ligand. Neither drug affected binding of IFN-gamma to macrophages, nor binding of TNF-alpha to human polymorphonuclear leukocytes. Thus, an intact MT network appears to be important in maintenance of the steady state of TNF-alpha R on those cells in which TNF-alpha R turns over rapidly in the absence of ligand. The antiinflammatory actions of MT-depolymerizing agents may result in part from their interference with the ability of such cells to respond to TNF-alpha.

• Aiyer RA, Aggarwal BB
• Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes.
• Lymphokine Res. 1990; 9: 333-44
• Display abstract

• High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-alpha iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-alpha receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-alpha to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37 degrees C, 24 degrees C and 4 degrees C, respectively. However, the maximum binding obtainable at 4 degrees C was only 40% of that at 37 degrees C. The binding 125I-rhTNF-alpha to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-alpha, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-beta; previously called lymphotoxin). This is similar to the behavior of TNF-alpha receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-alpha and rhTNF-beta bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,000 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-alpha are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-beta.

• Klostergaard J, Stoltje PA, Kull FC Jr
• Tumoricidal effector mechanisms of murine BCG-activated macrophages: role of TNF in conjugation-dependent and conjugation-independent pathways.
• J Leukoc Biol. 1990; 48: 220-8
• Display abstract

• The roles of secreted and membrane-associated TNFs were investigated in activated macrophage cytolysis of L929, EMT-6, and P815 targets. While all three targets were susceptible to cytolysis in coculture, an anti-TNF antiserum blocked lysis of L929 and EMT-6 but not of the P815 targets. Of the three targets, recombinant human or mouse TNF could only lyse the L929 target; despite the fact that a role for TNF was invoked in lysis of EMT-6 targets in coculture, the latter was strongly resistant to soluble rTNF, even at concentrations 30-40-fold higher than the Ka for its TNF-receptor. Cytolysis of the L929 target occurred when it was cocultured with BCG-activated macrophages even when these effector cells did not secrete TNF, either due to prior chemical crosslinking or to lack of exposure to a triggering level of lipopolysaccharide. Furthermore, by introduction of the anti-TNF antiserum over a dose-range, it was shown that macrophage cytolysis both of L929 and EMT-6 targets occurred in the absence of bioavailable, fluid-phase TNF. Thus, even for targets susceptible to fluid-phase TNF, TNF-dependent, direct macrophage-mediated cytolysis appears to be a function independent of secreted TNF and one that utilizes effector-target contact to express the action of a membrane form of the molecule.

• Dower SK, Smith CA, Park LS
• Human cytokine receptors.
• J Clin Immunol. 1990; 10: 289-99

• Smith MR, Munger WE, Kung HF, Takacs L, Durum SK
• Direct evidence for an intracellular role for tumor necrosis factor-alpha 1. Microinjection of tumor necrosis factor kills target cells.
• J Immunol. 1990; 144: 162-9
• Display abstract

• TNF-alpha is a small peptide cytokine produced primarily by activated macrophages. One of the many biologic activities of TNF is the killing of diverse types of tumor cells. We considered the possibility that killing was mediated by TNF itself at an intracellular site, subsequent to receptor-mediated endocytosis. To test this hypothesis, we microinjected TNF into various murine normal cells and cell lines, some of which were killed by TNF given by the usual extracellular route, and others that were not. Cytotoxic effects of microinjected TNF were observed in several cell types 2 to 4 h after injection. L929 fibroblasts were killed by either extracellular or intracellular TNF. A TNF-resistant subline of L929 was insensitive to either extracellular or intracellular TNF. L6 fibroblasts were found to be resistant to high doses of TNF given either extracellularly or microinjected. Normal macrophages and the J774 macrophage-like cell line were not killed by extracellular TNF, but were rapidly killed by microinjected TNF. Thus, TNF, an extracellular peptide ligand, has an intracellular activity, suggesting that internalization of this ligand may have important intracellular biochemical roles.

• Dinarello CA
• Cytokines and biocompatibility.
• Blood Purif. 1990; 8: 208-13
• Display abstract

• Issues in biocompatibility have traditionally focused on the interaction of membranes or materials with the activation of complement, red blood cell destruction, clotting dysfunctions, and platelet activation. Other studies are concerned with the replacement fluid formulations and host responses. Recent attention has examined the interaction of the blood monocyte with either complement or membrane surfaces. Activation of monocytes leads to the synthesis of cytokines, namely interleukin 1, tumor necrosis factor, and interleukin 6. These potent polypeptide 'cytokines' affect biological functions in nearly every tissue. Understanding the mechanisms by which gene expression of these cytokines is triggered will help in the better design of membrane material or substitution fluids. These are discussed in the context of increased cytokine synthesis during hemodialysis and continuous ambulatory peritoneal dialysis.

• Billard C, Sigaux F, Wietzerbin J
• IFN-alpha in vivo enhances tumor necrosis factor receptor levels on hairy cells.
• J Immunol. 1990; 145: 1713-8
• Display abstract

• Hairy cell leukemia (HCL) is a preplasma B cell neoplasia that is characterized by monocytopenia and responds to IFN-alpha therapy. We investigated the expression of receptors for TNF-alpha, a monocyte-derived cytokine, on the surface of hairy cells from seven HCL patients before and after treating them with IFN-alpha. Iodinated TNF-alpha binding experiments showed the presence of high affinity TNF-alpha receptors in six patients, but no specific binding was detected in the seventh. Scatchard's analysis revealed the presence of a single class of receptors, with 130 to 1200 sites/cell (mean 420) and Kd values of 0.37 to 0.89 nM. The TNF-alpha-R complex was identified by cross-linking as a single band of 94 kDa. Treatment of the patients with 3 x 10(6) U of IFN-alpha 2a markedly increased the number of TNF-alpha-R at 24 or 48 h, without changing the Kd. In the patient who lacked receptor expression on his tumor cells, high affinity TNF-alpha-R were detected at 48 h after IFN injection. These results suggest that in hairy cells, IFN-alpha can interact in vivo with TNF-alpha, through the modulation of TNF-R.

• Smith CA et al.
• A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins.
• Science. 1990; 248: 1019-23
• Display abstract

• Tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) bind surface receptors on a variety of cell types to mediate a wide range of immunological responses, inflammatory reactions, and anti-tumor effects. A cDNA clone encoding an integral membrane protein of 461 amino acids was isolated from a human lung fibroblast library by direct expression screening with radiolabeled TNF-alpha. The encoded receptor was also able to bind TNF-beta. The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.

• Larrick JW, Wright SC
• Cytotoxic mechanism of tumor necrosis factor-alpha.
• FASEB J. 1990; 4: 3215-23
• Display abstract

• Many intracellular pathways are set in motion by the binding of tumor necrosis factor (TNF) to its cell surface receptor. Major steps in the TNF-mediated cytotoxicity cascade include G protein-coupled activation of phospholipases, generation of free radicals, and damage to nuclear DNA by endonucleases. Ultimately the cells undergo apoptosis and die. Understanding how TNF initiates these pathways will facilitate the rational design of pharmaceuticals that can attenuate or potentiate the action of this important cytokine.

• Buck C et al.
• Tumor necrosis factor-alpha, but not lymphotoxin, stimulates growth of tumor cells in hairy cell leukemia.
• Leukemia. 1990; 4: 431-4
• Display abstract

• We investigated the effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) and recombinant lymphotoxin (rLT) in the growth modulation of purified hairy cell leukemia (HCL) cells. In response to rTNF-alpha, HCL cells from five of eight patients showed a 3 to 23-fold thymidine incorporation above their unstimulated controls. The effect was time and dose dependent with a maximum between 10 and 25 ng/ml rTNF-alpha after 120-hr incubation. rLT (1-50 ng/ml), however, could not enhance DNA synthesis in six of six cases. Cell number of rTNF-alpha stimulated cells ranged from 2-3 x 10(6)/ml from days 0-50 whereas cell number of unstimulated controls decreased from 3 x 10(6)/ml at day 0 to 0.01-0.02 x 10(6)/ml after 50 days in culture. rTNF-alpha induced proliferation could be suppressed in all HCL cell populations by 0.3 ng/ml recombinant interferon alpha (100 U/ml rIFN-alpha). TNF binding studies in two patients revealed that both TNF-sensitive HCL cells (1,990 +/- 148 receptors/cell) as well as TNF-insensitive HCL cells (1,261 +/- 101 receptors/cell) express specific receptors for TNF-alpha. These data show that rTNF-alpha and rLT have different effects on the growth of HCL cells. In addition there is a subgroup of patients who show no response to rLT or rTNF-alpha.

• Bermudez LE, Wu M, Young LS
• Effect of stress-related hormones on macrophage receptors and response to tumor necrosis factor.
• Lymphokine Res. 1990; 9: 137-45
• Display abstract

• Tumor Necrosis Factor (TNF) has been implicated in the early metabolic events following acute tissue injury or sepsis; it increases blood levels of glucocorticoids and glucagon or the cellular responses to the hormones. To examine whether stress-related hormones have any effect on macrophage activation by TNF, human monocyte-derived macrophages were exposed to somatostatin (S), ACTH, angiotensin (An), insulin (I), epinephrine (E), and glucagon (G) at physiologic concentrations. 125I-TNF binding as well as the ability of TNF to activate macrophages to kill an intracellular pathogen (Mycobacterium avium) were measured. While treatment with recombinant interferon gamma increased the number of TNF receptors by 53 +/- 8%, E, I, G, S, ACTH and An decreased the number of receptors by 81 +/- 6%, 83 +/- 6%, 15 +/- 5%, 83 +/- 4%, 17 +/- 4% and 21 +/- 4%, respectively. Treatment with I, E, and S also decreased the ability of macrophages to kill M. avium by 30 +/- 1%, 20 +/- 6%, and 51 +/- 2%, respectively. These in vitro results suggest that stress hormones influence TNF-mediated activation of macrophages.

• Han HM, Kolhatkar AA, Marino MW, Manchester KM, Donner DB
• Identification, characterization, and homologous up-regulation of latent (cryptic) receptors for tumor necrosis factor-alpha in rat liver plasma membranes.
• J Biol Chem. 1990; 265: 18590-4
• Display abstract

• A population of latent (cryptic) receptors for tumor necrosis factor-alpha (TNF) has been characterized in the rat liver plasma membrane (PM). 125I-TNF bound to high (Kd = 1.51 +/- 0.35 nM) and low (Kd = 13.58 +/- 1.45 nM) affinity receptors in PM. Solubilization of PM with 1% Triton X-100 prior to incubation with 125I-TNF increased both high affinity (from 0.33 +/- 0.04 to 1.67 +/- 0.05 pmol/mg of protein) and low affinity (from 1.92 +/- 0.16 to 7.57 +/- 0.50 pmol/mg of protein) TNF binding without affecting the affinities for TNF. Digestion of intact PM with chymotrypsin abolished most of the TNF binding capacity of PM. However, substantial binding activity was recovered by solubilization of chymotrypsin-treated PM with 1% Triton X-100, suggesting the presence of a large latent pool of TNF receptors. The affinities of the high and low affinity sites recovered from chymotrypsin-treated membranes were similar to those of intact PM. Affinity labeling of receptors whether from PM, solubilized PM, or membranes digested with chymotrypsin and then solubilized resulted in cross-linking of 125I-TNF into Mr 130,000, 90,000, and 66,000 complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, 125I-TNF binding to control and TNF-pretreated membranes was assayed. Specific binding was increased by pretreatment with TNF (p less than 0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF.

• Neale ML, Fiera RA, Matthews N
• Tumour cells which develop resistance to cytolysis by tumour necrosis factor have a different glycoform of a 105-kDa glycoprotein and lose the capacity to invade and metastasize.
• Int J Cancer. 1990; 45: 203-8
• Display abstract

• A plastic-adherent variant of human myelomonocytic leukaemia cells (U937) is highly susceptible to direct TNF cytolysis in vitro. Previously, we found that a subline selected for resistance to TNF cytolysis (U937/R) was much less motile and more plastic-adherent than the parental line. In the present study we show that U937 and U937/R cells have different glycoforms of a 105-kDa cell-surface glycoprotein. This protein is predominantly N-glycosylated and has the physicochemical properties of the LAMP-I glycoprotein. In nude mice, U937 cells are highly malignant whereas U937/R cells form a benign, encapsulated tumour. Therefore, possession of a different glycoform of the 105-kDa glycoprotein by U937/R cells correlates not only with loss of TNF susceptibility but also with reduced invasiveness and metastasis.

• Lantz M, Gullberg U, Nilsson E, Olsson I
• Characterization in vitro of a human tumor necrosis factor-binding protein. A soluble form of a tumor necrosis factor receptor.
• J Clin Invest. 1990; 86: 1396-42
• Display abstract

• Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammatory responses. A cysteine-rich, highly glycosylated 30-kD TNF-binding protein (TNF-BP) purified from urine may have a role in regulation because it protects in vitro against the biological effects of TNF. The cytotoxic effect of TNF on the fibrosarcoma cell line WEHI 164 was inhibited by 50% at a 10-fold excess of TNF-BP. The binding of TNF to the receptor was partially reversed after the addition of TNF-BP. Results from biosynthetic labeling of cells with 35S-cysteine followed by immunoprecipitation with anti-TNF-BP indicated that TNF-BP is formed and released at the cell surface by cleavage because no corresponding cellular polypeptide was observed. A cellular 60-kD polypeptide, which was immunoprecipitated with anti-TNF-BP, may correspond to the transmembrane TNF-receptor molecule and be the precursor of TNF-BP. Thus, TNF-BP appears to be a soluble form of a transmembrane TNF-receptor. Moreover our results demonstrate that the production of TNF-BP is increased when the TNF receptor is downregulated in cells by treatment with TNF or by activation of protein kinase C with phorbol esters. TNF-BP may be an important agent that blocks harmful effects of TNF, and, therefore, useful in clinical applications.

• Farrar WL, Garcia Garcia G, Evans G, Michiel D, Linnekin D
• Cytokine regulation of protein phosphorylation.
• Cytokine. 1990; 2: 77-91

• Kusher DI, Ware CF, Gooding LR
• Induction of the heat shock response protects cells from lysis by tumor necrosis factor.
• J Immunol. 1990; 145: 2925-31
• Display abstract

• A minority of transformed cell lines are directly susceptible to lysis by TNF, whereas many cells can be made sensitive to TNF by treatment with inhibitors of protein synthesis. Other groups have shown that exposure to TNF induces in many cells a transcription/translation dependent response that protects the cell from TNF lysis. Heat shock proteins are involved in protecting cells from the lethal affects of heat and other metabolic poisons. In this report, we test the possibility that heat shock proteins are also involved in protecting cells from lysis by TNF. We find that after induction of the cellular heat shock response by either heat or arsenite treatment, both spontaneously TNF-sensitive cells and those cells made sensitive by inhibition of protein synthesis are nearly completely protected from TNF cytolysis. The heat-treated cells retained most of their capacity to bind TNF, suggesting that heat shock functions at a postreceptor binding phase of the lytic process. Mouse C3HA fibroblasts are also made sensitive to TNF lysis by treatment with cytochalasin E. We have previously found that elicitation of the cell's TNF-protective response by exposure to TNF suppresses killing of C3HA by subsequent treatment with TNF plus cytochalasin E. In contrast, we report here that induction of the heat shock response did not provide significant protection to C3HA from killing by TNF in the presence of cytochalasin E. Thus, although induction of heat shock proteins does protect cells from TNF, they appear to act by a mechanism distinct from that elicited by TNF itself.

• Digel W et al.
• Receptors for tumor necrosis factor on neoplastic B cells from chronic lymphocytic leukemia are expressed in vitro but not in vivo.
• Blood. 1990; 76: 1607-13
• Display abstract

• Recombinant tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces proliferation of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms involved in regulating TNF responsiveness, we have examined TNF receptor expression on neoplastic B-CLL cells. We have demonstrated that freshly isolated neoplastic B cells from patients with CLL did not express TNF receptors. After 1 day of incubation in culture medium, TNF receptors were detectable in the range of 540 to 1,500/cell. Kinetic experiments revealed that receptor expression was half-maximal after 3 hours of culturing and required de novo protein synthesis. The Scatchard plots of TNF-alpha binding indicated a single set of high-affinity TNF receptors with a dissociation constant of 70 pmol/L. TNF receptor expression in vitro was found in all examined cases. All cytokines tested, with the exception of IL-2, did not influence the expression of TNF receptors. The TNF receptor expression is enhanced in B-CLL cells cultured in the presence of interleukin-2 when compared with the receptor expression of cells cultured in medium alone. Our data suggest that neoplastic B-CLL cells in patients with stable disease do not express TNF receptors in vivo and that an unknown mechanism suppressing TNF receptor expression in vivo may play a role in growth regulation of neoplastic B cells.

• Nophar Y et al.
• Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor.
• EMBO J. 1990; 9: 3269-78
• Display abstract

• Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross-react immunologically with two species of cell surface TNF receptors (TNF-R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine-rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF-R closely resemble those of TBPI. The COOH-terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF-R. CHO cells transfected with type I TNF-R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF-Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.

• Brown AR, Fishman M
• Tumor necrosis factor-alpha analyzed within individual macrophages by combined immunocytochemistry and computer-aided image analysis.
• Cell Immunol. 1990; 130: 352-63
• Display abstract

• Immunocytochemical staining procedures were combined with computer-aided image analysis to quantitate the relative intracellular production of tumor necrosis factor-alpha (TNF) within individual macrophages. Optimal conditions for time and methods for the activation of TNF production, fixation of cells for optimal immunocytochemical staining, and image analysis methods were determined. Thioglycolate elicited peritoneal macrophages were readily activated to significantly increased levels of intracellular TNF, as early as 1 hr after activation with lypopolysaccharide (LPS) + interferon-gamma: maximum intracellular TNF was evident after 2-3 hr. Both LPS and interferon-gamma was necessary to increase intracellular TNF. Normal alveolar macrophages also readily produced increased intracellular TNF, but normal peritoneal and splenic macrophages were poorly activated to TNF production. Acid stripping of receptor bound TNF allowed discrimination between intracellular TNF/integral membrane TNF, and TNF-receptor-bound TNF. Results stress the importance of studying these TNF forms early after activation. Applications for TNF quantitation by these means are discussed.

• Gray PW, Barrett K, Chantry D, Turner M, Feldmann M
• Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein.
• Proc Natl Acad Sci U S A. 1990; 87: 7380-4
• Display abstract

• The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNF alpha with an affinity of 2.5 x 10(-9) M. This binding can be competitively inhibited with unlabeled TNF alpha or lymphotoxin (TNF beta).

• Andrews JS, Berger AE, Ware CF
• Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes. TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4+ T cell hybridoma.
• J Immunol. 1990; 144: 2582-91
• Display abstract

• TNF-alpha and lymphotoxin (LT or TNF-beta) are structurally related cytokines that share several proinflammatory and immunomodulatory activities. The shared biologic activities of TNF and LT have been attributed to their binding to a common cell surface receptor(s). We observed that rTNF enhanced the expression of MHC class I proteins on the human T cell hybridoma, II-23.D7, however LT was largely unable to regulate MHC expression. To determine the molecular basis of this disparity between LT and TNF the receptor binding characteristics of rTNF and rLT were investigated by direct and competitive radioligand assays on the II-23.D7 T hybridoma, and for comparison, anti-CD3 activated human T lymphocytes. Specific 125I-rTNF binding to the II-23.D7 line revealed a single class of sites with a Kd = 175 pM and 3000 sites/cell; anti-CD3 activated T cells exhibited specific TNF binding with similar properties. The relationship of receptor occupancy to the induction of MHC class I Ag yielded a hyperbolic curve indicating a complex relationship between rTNF binding and biologic response. LT appeared to function like a partial agonist in that rLT was 10- to 20-fold less effective than rTNF in competitively inhibiting 125I-rTNF binding on the II-23.D7 line. Scatchard type analysis revealed a single class of low affinity binding sites for 125I-rLT. No differences in the competitive binding activity of rTNF and rLT were observed on the anti-CD3-activated T cells. Receptors for rTNF and rLT were immunoprecipitated from the II-23.D7 and activated T cells with anticytokine antibodies after cross-linking of radioiodinated rTNF or rLT to intact cells by using chemical cross-linking reagents. Analysis of the cross-linked adducts by SDS-PAGE and autoradiography indicated a major adduct of 92 kDa for rTNF and 104 kDa for rLT. Enzymatic digestion with neuraminidase or V8 protease revealed a unique structure to these adducts consistent with the cross-linking of a single chain of cytokine to a cell surface glycoprotein. rTNF inhibited the formation of the 104-kDa adduct formed with 125I-rLT on the II-23.D7 line, indicating these two cytokines bind to the same receptor of approximately 80 kDa. These results suggest that the disparate activities of LT and TNF to induce MHC class I proteins on the II-23.D7 cells are, in part, associated with a modified state of a common receptor.

• Dembic Z et al.
• Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences.
• Cytokine. 1990; 2: 231-7
• Display abstract

• Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

• Loetscher H, Schlaeger EJ, Lahm HW, Pan YC, Lesslauer W, Brockhaus M
• Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells.
• J Biol Chem. 1990; 265: 20131-8
• Display abstract

• Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.

• Ranges GE, Bombara MP, Aiyer RA, Rice GG, Palladino MA Jr
• Tumor necrosis factor-alpha as a proliferative signal for an IL-2-dependent T cell line: strict species specificity of action.
• J Immunol. 1989; 142: 1203-8
• Display abstract

• The ability of TNF-alpha to stimulate T cell proliferation was examined. We demonstrate that murine rTNF-alpha induces the proliferation of CT6, a murine T cell line previously thought to be responsive only to IL-2. This activity appears to be the result of the direct action of murine rTNF-alpha on the CT6 cells because neither 1) murine IL-2 or murine IL-4, lymphokines also capable of inducing CT6 proliferation, were detected in culture supernatants from murine rTNF-alpha-treated CT6 cells nor 2) did antibodies specific for IL-2 or IL-4 inhibit murine rTNF-alpha-induced CT6 proliferation. Unlike many of the activities displayed by TNF-alpha, its ability to induce CT6 cell proliferation shows strict species specificity as indicated by the failure of human rTNF-alpha to stimulate these cells. Flow cytometric analysis and binding of radiolabeled TNF-alpha have indicated that receptors for TNF-alpha on these cells are specific for murine TNF-alpha. The ability of murine rTNF-alpha to induce the proliferation of certain T cell lines further indicates that this molecule plays an important role in regulation of T cell-mediated immune responses.

• Yamamoto R, Wang A, Vitt CR, Lin LS
• Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor.
• Protein Eng. 1989; 2: 553-8
• Display abstract

• The amino acids that are required for the cytotoxic activity of recombinant human tumor necrosis factor-alpha (TNF) were investigated by chemical modification and oligonucleotide-directed site-specific mutagenesis. TNF contains three histidine residues, located at positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate (DEP) was used to chemically modify TNF. The chemical inactivation of the in vitro cytotoxic activity of this lymphokine (using murine L929 target cells) was found to be time- and dose-dependent. Inactivated TNF failed to compete with fully bioactive [125I]TNF for human MCF-7 target cell receptors. Mutant polypeptides of TNF were genetically engineered by oligonucoleotide-directed site-specific mutagenesis. The cytotoxicity of a double histidine mutant, in which histidine-73 and histidine-78 were replaced with glutamine, was not altered and was chemically inactivated by DEP. Substituting glutamine for histidine-15 resulted in 10-15% of the wild-type bioactivity. Replacing histidine-15 with either asparagine, lysine or glycine resulted in a biologically inactive molecule. The data show that the histidine residue at position 15 is an amino acid that is required for the cytotoxic activity of TNF.

• Luettig B, Decker T, Lohmann-Matthes ML
• Evidence for the existence of two forms of membrane tumor necrosis factor: an integral protein and a molecule attached to its receptor.
• J Immunol. 1989; 143: 4034-8
• Display abstract

• Plasma membranes were isolated from thioglycolate-induced peritoneal mouse macrophages and tested directly in a 51Cr-release assay against WEHI 164 tumor cells. These membranes showed anti-TNF antibody inhibitable killing of the TNF-sensitive tumor cell line, indicating that membrane-associated TNF is present on mouse macrophages. In order to elucidate whether membrane TNF is an integral protein or a molecule attached to a receptor, cells and plasma membranes were treated with low pH buffer. A partial reduction in TNF activity was observed which could be restored by incubation with exogenous TNF. In a Western blot analysis the integral membrane TNF could be identified as the 26-kDa molecule on activated mouse macrophages. These results indicate that both forms of membrane-associated TNF exist on macrophages and are responsible for cell-mediated cytotoxicity against TNF-alpha-sensitive targets.

• Balkwill FR
• Tumour necrosis factor.
• Br Med Bull. 1989; 45: 389-400
• Display abstract

• Tumour necrosis factor, TNF, possesses the attributes of a typical cytokine, being a pleiotropic cell regulatory protein whose activity is largely determined by the cell type to which it binds and the presence of other protein regulators. Thus TNF can be a growth factor, cytotoxin, cytostatic agent or inducer of differentiation. This cytokine is also an important inflammatory mediator regulating the activity of neutrophils, eosinophils, T and B lymphocytes and modulating the properties of the vascular endothelium. All of these actions may play a role in the ability of TNF to induce necrosis of experimental animal tumours, but the potential of this cytokine in therapy of human cancer is uncertain. The increasing evidence for a role for TNF in the pathophysiology of acute and chronic disease makes the use of specific TNF antagonists an equally interesting therapeutic possibility.

• Eck MJ, Sprang SR
• The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding.
• J Biol Chem. 1989; 264: 17595-605
• Display abstract

• The three-dimensional structure of tumor necrosis factor (TNF-alpha), a protein hormone secreted by macrophages, has been determined at 2.6 A resolution by x-ray crystallography. Phases were determined by multiple isomorphous replacement using data collected from five heavy atom derivatives. The multiple isomorphous replacement phases were further improved by real space symmetry averaging, exploiting the noncrystallographic 3-fold symmetry of the TNF-alpha trimer. An atomic model corresponding to the known amino acid sequence of TNF-alpha was readily built into the electron density map calculated with these improved phases. The 17,350-dalton monomer forms an elongated, antiparallel beta-pleated sheet sandwich with a "jelly-roll" topology. Three monomers associate intimately about a 3-fold axis of symmetry to form a compact bell-shaped trimer. Examination of the model and comparison to known protein structures reveals striking structural homology to several viral coat proteins, particularly satellite tobacco necrosis virus. Locations of residues conserved between TNF-alpha and lymphotoxin (TNF-beta, a related cytokine known to bind to the same receptors as TNF-alpha) suggest that lymphotoxin, like TNF-alpha, binds to the receptor as a trimer and that the general site of interaction with the receptor is at the "base" of the trimer.

• Owen-Schaub LB, Crump WL 3rd, Morin GI, Grimm EA
• Regulation of lymphocyte tumor necrosis factor receptors by IL-2.
• J Immunol. 1989; 143: 2236-41
• Display abstract

• Activated lymphocytes are known to express TNF receptors. The precise stimuli involved in induction and regulation of these receptors have not been elucidated. Our findings demonstrate that IL-2, alone and in serum-free conditions, can trigger and regulate TNF receptor expression on normal lymphocytes. Flow cytometric analyses demonstrated that the receptor was rapidly induced on CD4, CD8, and CD16+ cells after IL-2 stimulation. Receptors increased with culture duration, became maximal between days 5 and 9, and were maintained for 18 to 20 days in the presence of IL-2. By using 125I-TNF and FITC-TNF binding, we present evidence that IL-2 concentration determines the magnitude of lymphoid TNF receptor expression--influencing both the percentage of TNF-positive cells within the population and the number of receptors/cell. Collectively, our results are persuasive for consideration of IL-2 as a central mediator in the regulation of lymphocyte TNF receptors.

• Kamijo R et al.
• Induction of differentiation of human monoblastic and myeloblastic leukemia cell lines by TNF muteins.
• Biochem Biophys Res Commun. 1989; 160: 820-7
• Display abstract

• The differentiation inducing activity of five recombinant human tumor necrosis factor (rHuTNF) muteins, prepared by protein engineering techniques, was compared by measuring the ability to reduce nitroblue tetrazolium dye of human myelogenous leukemia cells. TNF(C-Phe), in which the C-terminal leucine of TNF molecule was replaced by phenylalanine, was 20-times as potent in induction of differentiation of U-937 cells as the parent TNF(N-Met), in which methionine was added to N-terminal sequence of TNF. The differentiation inducing activity of TNF muteins was not always proportional to their binding activity to the receptors nor to their cytotoxicity on U-937 cells.

• Rosenblum MG, Donato NJ
• Tumor necrosis factor alpha: a multifaceted peptide hormone.
• Crit Rev Immunol. 1989; 9: 21-44
• Display abstract

• Homeostasis at the cellular level appears to be a complex state of equilibrium derived, in part, from multiple interactions among lymphokines, cytokines, growth factors, and hormones. Tumor necrosis factor (TNF-alpha) secreted by mitogen-stimulated macrophages and lymphotoxin (LT, TNF-beta) produced by mitogen-stimulated lymphocytes are two peptide hormones that share considerable homology in amino acid sequence and biological action. It has become increasingly evident that these factors, primarily involved in host defense, also mediate some responses to inflammatory, infectious, and neoplastic disease states. TNF-alpha is a low molecular weight (17,000-kdalton) peptide which seems to have a remarkably broad range of biological and immunologic effects, including antiviral action, growth regulation, and immunomodulation. These effects are shared by other lymphokines and cytokines such as interleukin 1 (IL-1), interferon alpha (IFN-alpha), interferon gamma (IFN-gamma), and TNF-beta. TNF-alpha also demonstrates both positive and negative cooperative growth-regulatory effects alone and in combination with antitumor drugs as well as with other bioactive peptides. Clinical trials are currently under way to determine the effectiveness of TNF-alpha as a single agent and in combination with biologic and chemotherapeutic agents against a variety of neoplastic diseases in man.

• Ishikura H, Hori K, Bloch A
• Differential biologic effects resulting from bimodal binding of recombinant human tumor necrosis factor to myeloid leukemia cells.
• Blood. 1989; 73: 419-24
• Display abstract

• Human recombinant tumor necrosis factor (rTNF) bound to ML-1 and HL-60 human myeloid leukemia cells in a bimodal manner. Saturable high-affinity binding was maximal at approximately 3 nmol/L rTNF, whereas saturable low-affinity binding reached its maximum at approximately 30 nmol/L. As analyzed by computer program, the observed data fit a two-receptor site model, with p less than .05. High-affinity binding concentrations of rTNF caused the differentiation of both cell lines, whereas low-affinity binding concentrations abolished this effect in a concentration-dependent manner. Thus, the type of biologic response elicited with rTNF in these cells is a function of the concentration at which the factor is applied. If generally applicable, this bimodal effect may require consideration when rTNF is to be used therapeutically.

• Engelmann H, Aderka D, Rubinstein M, Rotman D, Wallach D
• A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity.
• J Biol Chem. 1989; 264: 11974-80
• Display abstract

• Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.

• Kull FC Jr
• The TNF receptor in TNF-mediated cytotoxicity.
• Nat Immun Cell Growth Regul. 1988; 7: 254-65
• Display abstract

• The in vitro cytotoxic capacity (if not every pleiotropic property) of tumor necrosis factor (TNF) begins by interaction with specific high affinity cell surface receptors. The characterization of receptors and ligand kinetics is reviewed in relationship to cytotoxicity. Decreased receptor number and affinity correlate with sensitivity within a given cell line. In L-M cells (a sensitive tumorigenic fibroblast), TNF induces a biphasic downregulation of receptors. Internalized ligand and receptors are largely cleared before the onset of cell death. Drugs affecting cytotoxicity may act primarily on an early 'association' stage (ligand receptor interaction, internalization or perhaps signal transduction) or on a later 'lytic' stage. Phorbol myristate acetate is an example of the former, while chloroquine, cholera toxin and dibutyryl cyclic AMP are examples of the latter.

• Eades DK, Cornelius P, Pekala PH
• Characterization of the tumour necrosis factor receptor in human placenta.
• Placenta. 1988; 9: 247-51
• Display abstract

• A specific receptor for tumour necrosis factor present in a purified plasma membrane preparation from the villous tissue of human placenta has been characterized. The data fit a one-class-of-sites model exhibiting a KD = 28.4 (+/- 0.002) nM, with 8.1 (+/- 0.05) X 10(11) receptors mg-1 membrane protein. These data provide evidence for the existence of a specific receptor for a major endotoxin-induced cytokine in the placenta.

• Kull FC Jr
• Reduction in tumor necrosis factor receptor affinity and cytotoxicity by glucocorticoids.
• Biochem Biophys Res Commun. 1988; 153: 402-9
• Display abstract

• Micromolar concentrations of glucocorticoids rendered L-M cells (a murine tumorigenic fibroblast line) less sensitive to the cytotoxic activity of murine TNF. The potency of different steroids paralleled their known anti-inflammatory potency, and pretreatment was more effective than post treatment. Sex steroids and mineralocorticoids were ineffective. Dexamethasone also decreased the sensitivity of MCF-7 (a human mammary carcinoma line) to the cytotoxic activity of human recombinant TNF. Pretreatment of both cell lines reduced the affinity of specific cell surface receptors for the binding of their species 125I-TNF about 3-fold while retaining the same number of binding sites. The decrease in sensitivity was not due solely to the inhibition of early TNF-induced events (such as binding, internalization or signal transduction). Dexamethasone modestly enhanced inhibition beyond that of neutralizing antiserum alone when both were added midway in the L-M killing reaction (after receptor down regulation but before the onset of complete cell death).

• Kuriyama H et al.
• [Continuous internalization of TNF receptors in a human myosarcoma cell line]
• Gan To Kagaku Ryoho. 1988; 15: 325-33
• Display abstract

• The cell dynamics of the receptor on tumor necrosis factor (TNF) were studied with the use of TNF-sensitive KYM cells derived from human myosarcoma. With receptor synthesis inhibited by cycloheximide, the half-life of the surface TNF receptor was 2h in the absence of TNF and 30min in its presence, suggesting that the TNF receptor was non-recycling and that its internalization was accelerated by TNF. During cell incubation with suppression of TNF receptor degradation by chloroquine, the number of surface TNF receptors remained approximately constants, but the total number of surface and internal TNF receptors increased gradually, at 3h reaching 1.5 times of the initial number, thus suggesting continuous synthesis, externalization, internalization, and degradation of the TNF receptor in the absence of cycloheximide. When the cells were incubated with 125I-TNF, the intracellular quantity of the pulse-labeled TNF-receptor complex promptly increased, reaching a maximum at 20 min, and then declining gradually. Thus, it was confirmed that the TNF receptor is internalized as a TNF-receptor complex in the presence of TNF. In incubation with suppression of protein synthesis by cycloheximide following surface TNF receptor digestion by trypsin, TNF receptors reappeared on the cell surface, increasing in the number to a peak level at 60 min and gradually decreasing. The cells previously exposed to cycloheximide with or without TNF showed no recurrence of surface TNF receptors, suggesting that the TNF receptor is non-recycling. The results thus suggest that the TNF receptor is continuously internalized and degraded intracellularly by lysosomes without being recycled regardless of the presence or absence of TNF, and further that its internalization is accelerated when it is part of the TNF-receptor complex.

• Kramer SM et al.
• Characterization of the in vitro and in vivo species preference of human and murine tumor necrosis factor-alpha.
• Cancer Res. 1988; 48: 920-5
• Display abstract

• The species preference of human and murine tumor necrosis factor-alpha (TNF-alpha) was evaluated in human and murine systems for cytotoxic/cytostatic effects and receptor binding in vitro and murine systems for toxicity and antitumor activity in vivo. The in vitro cytotoxic/cytostatic effects of both species TNF-alpha on human and murine cell lines as well as the receptor binding studies using 125I-labeled recombinant human TNF-alpha demonstrated homologous species preferences. Species preference of TNF-alpha was also apparent in toxicity studies with BALB/c nu/nu and CB6F1 mice, and antitumor responses of CB6F1 mice to s.c. Meth A sarcoma implants. Moreover the growth of Meth A sarcoma implanted i.p. was not inhibited by either human or murine TNF-alpha. These results are discussed in view of the potential for underestimation of the biological potency of TNF-alpha from heterologous sources.

• Watanabe N et al.
• Continuous internalization of tumor necrosis factor receptors in a human myosarcoma cell line.
• J Biol Chem. 1988; 263: 10262-6
• Display abstract

• The cell dynamics of the receptor for tumor necrosis factor (TNF) were examined in TNF-sensitive KYM cells derived from human myosarcoma. With receptor synthesis inhibited by cycloheximide, the half-life of the surface TNF receptor was 2 h in the absence of TNF and 30 min in its presence, suggesting that the TNF receptor is non-recycling and that its internalization is accelerated by TNF. During cell incubation with TNF receptor degradation suppressed by chloroquine, the number of surface TNF receptors remained approximately constant, but the total number of surface and internal TNF receptors increased gradually, at 3 h reaching 1.5 times the initial number, thus suggesting continuous synthesis, externalization, internalization, and degradation of the TNF receptor in the absence of cycloheximide. On cell incubation with 125I-TNF, the intracellular quantity of the pulse-labeled TNF-receptor complex promptly increased, reaching a maximum at 20 min, and then gradually declined, thus confirming that the TNF receptor is internalized as a TNF-receptor complex in the presence of TNF. During incubations with protein synthesis suppressed by cycloheximide following surface TNF receptor digestion by trypsin, TNF receptors reappeared on the cell surface, increasing in number to a peak at 60 min and gradually decreasing, and cells previously exposed to cycloheximide with or without TNF showed no recurrence of surface TNF receptors, suggesting that the TNF receptor is non-recycling. The results of the study thus suggest that the TNF receptor is continuously internalized and degraded intracellularly by lysosomes without being recycled regardless of the presence or absence of TNF and, further, that its internalization is accelerated when it is part of the TNF-receptor complex.

• Beutler B, Cerami A
• The common mediator of shock, cachexia, and tumor necrosis.
• Adv Immunol. 1988; 42: 213-31

• Spriggs DR, Imamura K, Rodriguez C, Sariban E, Kufe DW
• Tumor necrosis factor expression in human epithelial tumor cell lines.
• J Clin Invest. 1988; 81: 455-60
• Display abstract

• Tumor necrosis factor (TNF) is a monokine with in vitro cytotoxicity for some but not all tumor cells. The basis for sensitivity and resistance to the antitumor effects of this agent remains unclear. The present studies have monitored the effects of TNF on 14 epithelial tumor cell lines. Eleven of these cell lines were resistant to the growth inhibitory effects of TNF (50% inhibitory concentration greater than 1,000 U/ml). 12 of the 14 tumor cell lines has detectable levels of high affinity cell surface TNF binding sites, thus suggesting that resistance was not often due to the absence of cell surface TNF receptors. Northern blot analysis demonstrated that three of the eleven resistant cell lines expressed detectable levels of TNF mRNA. Furthermore, both sensitive and resistant epithelial tumor cells had the capacity to express TNF transcripts in the presence of the protein synthesis inhibitor, cycloheximide. Finally, the presence of TNF expression at the RNA level is shown to be associated with the production of a TNF-like protein in the resistant Ov-D ovarian carcinoma cells. These findings suggest that certain human epithelial tumor cell lines inherently resistant to TNF also express this cytokine.

• Imamura K, Spriggs D, Kufe D
• Expression of tumor necrosis factor receptors on human monocytes and internalization of receptor bound ligand.
• J Immunol. 1987; 139: 2989-92
• Display abstract

• Tumor necrosis factor (TNF) is a polypeptide produced by monocytes and macrophages. Although TNF receptors have been identified on a variety of cell types, previous studies have not determined whether these receptors also exist on monocytes. In the present work, highly purified recombinant TNF was labeled with 125I. The 125I-labeled TNF bound specifically to receptors on human monocytes and monocyte membrane preparations. A curvilinear Scatchard plot indicated the presence of TNF-binding sites with two different affinities. The results also indicate that receptor-bound TNF is rapidly internalized by monocytes and then degraded intracellularly. These findings are in concert with recent studies demonstrating that TNF immunomodulates monocyte function by an autocrine mechanism.

• Creasey AA, Yamamoto R, Vitt CR
• A high molecular weight component of the human tumor necrosis factor receptor is associated with cytotoxicity.
• Proc Natl Acad Sci U S A. 1987; 84: 3293-7
• Display abstract

• We compared the molecular structure of the receptor to human recombinant tumor necrosis factor (HurTNF) on cells of different tissue origin that differ in their response to one of the known activities of TNF. We studied tumor cell lines that respond to the cytotoxic action of TNF and resistant variants that bind TNF, normal cell lines that are stimulated to proliferate by TNF and those that are not affected by TNF, and peripheral blood granulocytes whose activation is also augmented by TNF. Using 125I-labeled HurTNF, we found that it bound mainly to four cellular polypeptides (138, 90, 75, and 54 kDa), three of which were found in every cell type examined and one (138 kDa) that was observed only in a human breast carcinoma cell line (MCF-7) that is highly responsive to the cytotoxic action of TNF. The 138-kDa polypeptide was not found in resistant variants of MCF-7 that bind TNF. In contrast to the other polypeptides, the 138-kDa protein was detected 30 min after incubation at 4 degrees C, as compared to 5 min. Scatchard analysis and cross-linking data suggest a model for the TNF receptor structure whereby the receptor is composed of noncovalently linked membrane-bound polypeptides that bind TNF with high affinity (Kd, 0.05-0.8 X 10(-9) M) with the 138-kDa protein being the least abundant and/or even absent in most cells.

• Pfizenmaier K, Kronke M, Scheurich P, Nagel GA
• Tumor necrosis factor (TNF) alpha: control of TNF-sensitivity and molecular mechanisms of TNF-mediated growth inhibition.
• Blut. 1987; 55: 1-10

• Shalaby MR et al.
• Receptor binding and activation of polymorphonuclear neutrophils by tumor necrosis factor-alpha.
• J Leukoc Biol. 1987; 41: 196-204
• Display abstract

• The interaction of highly purified recombinant human tumor necrosis factor-alpha (rTNF-alpha) with human polymorphonuclear neutrophils (PMNs) was investigated. Binding of 125I-rTNF-alpha to PMN reached maximum levels in 30 min at 37 degrees C and in 2 h at 4 degrees C. Scatchard analysis of competitive binding data indicated approximately 6000 receptor sites per cell and a Kd of 1.37 nM. Binding data at 37 degrees C indicated a rapid internalization of rTNF-alpha. Following this receptor-mediated interaction, recombinant TNF-alpha was found to inhibit the migration of PMNs under agarose and to enhance PMN production of superoxide anion (O-2) in a dose-dependent manner. Furthermore, rTNF-alpha-activated PMNs caused a marked disruption of human umbilical-vein-derived endothelial cell monolayers and caused inhibition of their proliferative activities. These data substantiate the role of TNF-alpha as an activator of PMN functions and indicate that PMN/TNF-alpha/endothelial cell interactions may play a major role in inflammatory reactions.

• Tsujimoto M, Vilcek J
• Tumor necrosis factor-induced downregulation of its receptors in HeLa cells.
• J Biochem (Tokyo). 1987; 102: 1571-7
• Display abstract

• Tumor necrosis factor (TNF) induced loss of TNF receptors in HeLa cells was studied using acid elution technique, which could distinguish surface occupancy and real loss of receptors. Exposure of HeLa cells to TNF resulted in a rapid reduction in the number of TNF receptors without affecting the apparent binding affinity. The binding of transferrin after treatment with unlabeled TNF was unaffected. The TNF-mediated decrease in receptor number on the cells was reversible. Following removal of TNF from growth medium, binding activity was restored within 3 h. Cycloheximide prevented the restoration of TNF receptors, suggesting that de novo synthesis of receptors was required to restore the binding activity.

• Tsujimoto M et al.
• Comparative studies of the biological activities of human tumor necrosis factor and its derivatives.
• J Biochem (Tokyo). 1987; 101: 919-25
• Display abstract

• Biological activities of human tumor necrosis factor (TNF) and its derivatives were compared. In cytotoxicity assay with L929 cells, one derivative, designated as TNF(Asn), showed significantly lower activity than any other TNF examined. In binding assay, this derivative was also shown to have lower affinity for TNF receptors on L929 cells, suggesting that the cytotoxic activity of TNFs on L929 cells correlates with their affinity for receptors. We also found that the cytotoxic activity of TNF on A673 cells and its inhibitory effect on lipoprotein lipase were parallel with the cytotoxic activity on L929 cells, but the growth-enhancing activity on FS-4 cells and the cytotoxic activity on endothelial cells were not. It was also shown that TNF(Asn) had lower affinity than any other TNF for receptors on these target cells tested. These results suggested that there might be at least two types of cellular responses to TNF; one might correlate with the receptor-binding affinity of TNFs and the other not.

• Watanabe N et al.
• Cell cycle specificity of tumor necrosis factor and its receptor.
• Cell Biol Int Rep. 1987; 11: 813-7
• Display abstract

• Phase specificity in the TNF cytotoxic effect and the number of TNF binding receptors was investigated using L-M cells incubated synchronously from the S phase. TNF cytotoxicity was observed to occur at various levels during the cell cycle, with peak effect in the G2-M phase. Analysis with 125I-labeled TNF to determine the number of receptors binding TNF in the various cell phases shewed a phase specificity with the maximum number occurring in the G2-M phase, similar to the peak in cytotoxicity. The results suggest the existence of a cell cycle specificity in the cytotoxicity of TNF which is apparently related to changes in the number of receptors capable of binding TNF.

• Munker R, DiPersio J, Koeffler HP
• Tumor necrosis factor: receptors on hematopoietic cells.
• Blood. 1987; 70: 1730-4
• Display abstract

• Tumor necrosis factor (TNF) is a monokine that is cytotoxic/cytostatic for a variety of tumor cells and has multiple effects on normal cells. We demonstrate that normal and malignant human myeloid cells express a single class of high-affinity receptors (400 to 1,900 per cell, KD 20 to 90 pmol/L) for TNF. Mitogen-stimulated lymphocytes have a similar number of TNF receptors, whereas resting lymphoid cells have fewer receptors. RBCs and platelets have no detectable TNF receptors. No correlation is observed between the receptor number, receptor affinity, and the cytotoxic effect of TNF on myeloid cell lines. Significant cytotoxic effects of TNF on the most sensitive myeloid cell line (HL-60 promyelocytes) could be seen at concentrations tenfold lower than those concentrations at which one half of the TNF receptors were occupied. Our data show that significant biologic effects of TNF can occur at low levels of receptor occupancy and resistance to TNF is not related to the absence of TNF receptors on certain myeloid leukemic cell lines.

• Aggarwal BB, Aiyer RA, Pennica D, Gray PW, Goeddel DV
• Human tumour necrosis factors: structure and receptor interactions.
• Ciba Found Symp. 1987; 131: 39-51
• Display abstract

• Activation of lymphoid and myeloid cells causes the production of factors cytotoxic to various tumour cell types in vitro and in vivo. We have investigated the biochemistry, molecular biology and mechanism of action of two such factors. The factor derived from a myeloid cell line was named TNF-alpha (previously referred to as TNF) and that derived from lymphoid cells named TNF-beta (previously called lymphotoxin). Both proteins were purified from the conditioned media of the human cell lines and sequenced. Structural information revealed that TNF-alpha is 157 amino acid residues long and contains one disulphide bond. TNF-beta is a glycoprotein of 171 amino acids that contains no cysteine residues. Protein sequence information was used to isolate and characterize cDNAs for TNF-alpha and TNF-beta by recombinant DNA methods. The expression of the cDNAs in Escherichia coli made available large quantities of these proteins for biological studies. The two proteins are 31% identical and 52% homologous to each other. The genes for both cytokines are approximately three kilobases in size and are closely linked on human chromosome six. TNF-alpha and TNF-beta both bind to various cell types via a single class of high affinity receptors. On most cells the same receptor is recognized by both cytokines. The receptors for TNF-alpha can be up-regulated by both interferons and lectins. Up-regulation of receptors by interferons is accompanied by synergistic enhancement of the biological response whereas up-regulation by lectins results in an antagonistic response. Besides antiproliferative effects, both cytokines exhibit direct antiviral effects on infection by both DNA and RNA viruses.

• Pichyangkul S, Schick D, Jia FL, Berent S, Bollon A, Kahn A
• Binding of tumor necrosis factor alpha (TNF-alpha) to high-affinity receptors on polymorphonuclear cells.
• Exp Hematol. 1987; 15: 1055-9
• Display abstract

• The effects of tumor necrosis factor alpha (TNF-alpha) on human polymorphonuclear (PMN) cells were investigated. We found that 125I-TNF-alpha bound specifically to high-affinity receptors on PMN cells. At 4 degrees C, the binding occurred rapidly and reached steady state after 20 min. The Scatchard plot showed a single class of high-affinity receptors with approximately 2200 receptors/cell and a dissociation constant of 2 x 10(-10) M. There was a linear relationship between TNF-alpha binding and TNF-alpha-induced PMN cell adherence. The concentration of TNF-alpha required to achieve approximately 50% of maximum binding was also approximately the concentration required to reach 50% cell adherence. Auranofin was shown to inhibit TNF-alpha-induced PMN cell adherence at a dose of 5-10 micrograms/ml. This inhibitory effect was not due to the inhibition of TNF-alpha binding to PMN cells by the drug. These observations may have clinical implications.

• Beutler B, Cerami A
• Cachectin/tumor necrosis factor: an endogenous mediator of shock and inflammation.
• Immunol Res. 1986; 5: 281-93

• Tsujimoto M, Vilcek J
• Tumor necrosis factor receptors in HeLa cells and their regulation by interferon-gamma.
• J Biol Chem. 1986; 261: 5384-8
• Display abstract

• Incubation of HeLa cell cultures with human interferon-gamma (IFN-gamma) increased the binding of radioiodinated human tumor necrosis factor (TNF) to specific cell surface receptors (TNF-R). IFN-gamma also produced a proportionate increase in receptor-mediated endocytosis of TNF. TNF-R expression was significantly increased after 6 h of exposure to IFN-gamma (100 units/ml), and it remained elevated in the continuous presence of IFN-gamma for at least 20 h. Incubation of cells with IFN-gamma in the presence of cycloheximide, followed by treatment with actinomycin D and reversal of the inhibition of protein synthesis, also resulted in increased TNF-R expression as compared to cultures subjected to the same treatments in the absence of IFN-gamma. These results suggest that IFN-gamma can directly stimulate accumulation of the mRNA for TNF-R and that TNF-R is among the cellular proteins whose synthesis is increased by IFN-gamma.

• Tsujimoto M, Yip YK, Vilcek J
• Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor.
• J Immunol. 1986; 136: 2441-4
• Display abstract

• Incubation of several human tumor cell lines with human interferon-gamma (IFN-gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF.

• Lehmann V, Droge W
• Demonstration of membrane receptors for human natural and recombinant 125I-labeled tumor necrosis factor on HeLa cell clones and their role in tumor cell sensitivity.
• Eur J Biochem. 1986; 158: 1-5
• Display abstract

• Samples of natural and recombinant tumor necrosis factor (TNF) were labelled with 125I by use of Iodobeads and were used to study TNF receptor expression on different HeLa clones. Both labeled preparations retained cytotoxic activity after 125I-labelling and were indistinguishable from their unlabeled counterparts on sodium dodecylsulfate/polyacrylamide gel electrophoresis. They bound specifically to TNF-sensitive cells but not to HeLa clones that were resistant to TNF. The binding of 125I-TNF was competed up to 90% by the addition of non-radiolabeled TNF. The binding reached a half-maximal level at 10 pM and saturation at 100 pM. These concentrations approximated those required for cell destruction. Scatchard analysis of the binding data yielded linear plots suggesting that TNF binds to homogeneous TNF receptor sites. The number of receptor sites ranged between 770 and 2200 sites on TNF-sensitive cells. The surface expression of these TNF receptors appeared to be necessary but not sufficient for the cells to become sensitive against the cytotoxic action of TNF. One of the HeLa clones was found to express the same number of TNF receptors as the most sensitive clone, but was nevertheless resistant to the toxic effect of TNF.

• Tsujimoto M, Feinman R, Kohase M, Vilcek J
• Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells.
• Arch Biochem Biophys. 1986; 249: 563-8
• Display abstract

• Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.

• Aggarwal BB, Eessalu TE, Hass PE
• Characterization of receptors for human tumour necrosis factor and their regulation by gamma-interferon.
• Nature. 1985; 318: 665-7
• Display abstract

• Tumour necrosis factors, TNF-alpha and TNF-beta (previously called lymphotoxin), are the products of activated monocytes and lymphocytes, respectively, and both have recently been purified, sequenced and cloned by recombinant DNA methods, revealing 35% identity and 50% homology in the amino-acid sequence. Both proteins have been found to be specifically toxic to many tumour cells. Furthermore, it has been reported that various interferons are synergistic with TNF for anti-tumour effects in vitro, while activities attributed to the two proteins have also been shown to necrotize various tumours in vivo. We have now prepared 125I-labelled highly purified recombinant human TNF-alpha to study in detail its binding to the human cervical carcinoma cell line ME-180. Our results indicate that there is a single class of specific high-affinity receptors for TNF on this cell line which has a Kd of about 0.2 nM and an average of 2,000 receptor sites per cell. The binding of labelled TNF-alpha to these cells can be inhibited by both TNF-alpha and TNF-beta but not by gamma-interferon (IFN-gamma). However, preincubation of cells with IFN-gamma increases the total number of TNF receptors two to threefold without any significant change in the affinity constant. This is the first report that TNF-alpha and -beta share a common receptor and that the receptors can be up-regulated by interferon. Our results may explain previous observations regarding similar biological activities observed for these two cytotoxic proteins and also their synergistic action with interferons.

• Kull FC Jr, Jacobs S, Cuatrecasas P
• Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity.
• Proc Natl Acad Sci U S A. 1985; 82: 5756-60
• Display abstract

• Tumor necrosis factor (TNF) is a proteinaceous toxin shed by stimulated myeloid cells. Murine TNF was radioiodinated to a specific activity of 1 mCi/nmol (1 Ci = 37 GBq) of monomer. 125I-labeled TNF (125I-TNF) retained complete cytotoxic activity and it was immunochemically identical to the native toxin in a quantitative immunoprecipitation assay. It could be shown by competition binding that 125I-TNF bound to intact L929 cells with a specificity equal to that of native toxin. The conditions of time, temperature, and concentration involved in equilibrium specific binding to intact cells were studied in detail. When binding was carried out at 4 degrees C for 18 hr, four cell lines sensitive to 125I-TNF cytotoxicity demonstrated high-affinity binding. The binding reached half-maximal level at 3 pM and saturated at 30 pM. These concentrations approximated those required for cell death. Scatchard analysis gave approximately 1000 sites per cell. J774.1 cells, the source of the toxin, demonstrated similar binding but were not sensitive to 125I-TNF cytotoxicity. Other sensitive cell lines and freshly extracted tumor cells showed specific binding at 3 pM. Normal lymphoid organ cell suspensions and two human tumorigenic cell lines were not sensitive and failed to demonstrate specific binding. 125I-TNF, covalently cross-linked to its receptor on sensitive L-M cells with disuccinimidyl suberate, was isolated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. Two specific bands were identified. The most prominent band had a mobility corresponding to a molecular mass of 95 kDa and the second band had a molecular mass of 75 kDa. The presence of the binding site appears to be necessary but not sufficient to explain the sensitivity of cells to the cytotoxic action of TNF.

• Rubin BY, Anderson SL, Sullivan SA, Williamson BD, Carswell EA, Old LJ
• High affinity binding of 125I-labeled human tumor necrosis factor (LuKII) to specific cell surface receptors.
• J Exp Med. 1985; 162: 1099-104
• Display abstract

• 125I-labeled TNF(LuKII) (tumor necrosis factor) binds specifically to human and mouse cell lines sensitive to the cytotoxic effect of TNF, but not to cells made resistant to TNF. TNF-sensitive cells have cell surface receptors with a high affinity for TNF(LuKII). Mouse TNF competes with TNF(LuKII) for receptor binding. Scatchard analysis of the binding data yielded linear plots and suggests that TNF(LuKII) binds to homogeneous receptor sites. The number of TNF(LuKII) receptors on two TNF-sensitive cell lines is 200-300 per cell and the affinity constant of the receptor for TNF(LuKII) is approximately 1 X 10(-10) M.

• Marmenout A et al.
• Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor.
• Eur J Biochem. 1985; 152: 515-22
• Display abstract

• U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.

• Watanabe N, Sone H, Neda H, Niitsu Y, Urushizaki I
• [Induction of tumor necrosis factor (TNF) by OK-432]
• Gan No Rinsho. 1984; 30: 1290-2
• Display abstract

• The induction of tumor necrosis factor (TNF) by OK-432 was investigated. The results were as follows: Serum from OK-432 injected into NZW rabbits treated with LPS was cytotoxic to tumor cells in vitro. With respect to the target cell spectrum this factor was cytotoxic to L929, Lewis, M7609 and K562, but not to Chang liver cells, HMV cells or HeLa cells. The cytotoxic activity of this factor was found to be M.W. 48,000 on H.P.L.C., and the pI was revealed to be 5.4 by isoelectric focusing. These results were consistent with previously reported findings on TNF.

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