SMART MODE:

NORMAL GENOMIC

• Simon AK et al.
• Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes.
• Proc Natl Acad Sci U S A. 2001; 98: 5158-63
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• TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

• Inagaki-Ohara K et al.
• p53-dependent radiation-induced crypt intestinal epithelial cells apoptosis is mediated in part through TNF-TNFR1 system.
• Oncogene. 2001; 20: 812-8
• Display abstract

• Radiation induces apoptosis of crypt intestinal epithelial cells (IEC) through a pathway that is largely dependent on p53. However, exactly how p53 mediates IEC apoptosis is unclear. Studies in vitro suggest that one mechanism by which p53 mediates apoptosis is through its ability to transactivate members of the TNF receptor family of 'Death Receptors'. Here, we examined the role of one of its member, TNF receptor type 1 (TNFR1), in an in vivo model of p53-dependent radiation-induced IEC apoptosis. We demonstrate that mice genetically engineered to be deficient in TNF receptor type 1 (TNFR1(-/-)) and mice injected with TNFR1-fusion chimeric protein (TNFR1-Fc; a competitive inhibitor of TNFR1) were partially protected (30-40%) from p53-dependent radiation-induced IEC apoptosis. However, we found no evidence to support the possibility p53 transcriptionally regulates the expression of TNFR1 nor increases the susceptibility of IEC to TNF-mediated apoptosis. Interestingly, we found that injection of TNF readily induced IEC apoptosis and that radiation induced a p53-dependent increase in the intestinal level of TNF. Furthermore, injection of a neutralizing anti-TNF mAb reduced p53-dependent radiation-induced IEC apoptosis by approximately 60%. Overall, these results suggest that p53-dependent radiation-induced IEC apoptosis is mediated in part through ability of p53 to regulate TNF, which subsequently induces IEC apoptosis through TNFR1.

• Kassiotis G, Kollias G
• Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: implications for pathogenesis and therapy of autoimmune demyelination.
• J Exp Med. 2001; 193: 427-34
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• Multiple sclerosis (MS) is a disabling inflammatory demyelinating disease of the central nervous system, considered to result from self-reactivity to myelin antigens. Tumor necrosis factor (TNF) and the p55 TNF receptor (TNFR) have been strongly implicated in MS pathogenesis. We reveal in this study a dual role for TNF in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. In addition to its well-established proinflammatory effects, TNF exhibits potent immunosuppressive properties, providing one possible explanation for the immune and disease activating effect of anti-TNF treatment of MS. We show that in TNF-deficient mice, myelin-specific T cell reactivity fails to regress and expansion of activated/memory T cells is abnormally prolonged, leading to exacerbated EAE. Strikingly, immunosuppression by TNF and protection against EAE does not require the p55 TNFR, whereas the same receptor is necessary for the detrimental effects of TNF during the acute phase of the disease. Thus, blocking the function of the p55 TNFR in autoimmune demyelination may inhibit the noxious proinflammatory activities of TNF without compromising its immunosuppressive properties.

• Koniaris LG, Wand G, Wright TM
• TNF mediates a murine model of Addison's crisis.
• Shock. 2001; 15: 29-34
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• Addison's crisis is the most serious complication of adrenal insufficiency. To elucidate the mechanism of this disorder following infection, the role of TNF in adrenalectomized murine models of Addison's crisis and Addison's disease (chronic hypoglucocorticoidism) were examined. Adrenalectomy conferred a 40-fold increased sensitivity to the lethal effects of lipopolysacharride (LPS) (P < .001). Enhanced sensitivity to LPS was found to increase with duration of adrenal insufficiency (P < .02). Enhanced lethality to heat-killed Streptococcus pneumonia was also demonstrated (P < 0.02). Necropsy of endotoxin-killed adrenalectomized mice demonstrated similar pathologic findings to those found by others when the control mice were administered a lethal dose of either LPS or TNF. Adrenalectomized TNF receptor Ia and Ib double null mice were demonstrated to be resistant to the lethal effects of LPS (P < 0.02). Pretreatment with anti-TNF, but not control antisera, was found to prevent death in LPS-treated wild-type adrenalectomized mice as well (P < 0.02). Studies into the mechanism by which TNF was precipitating Addison's crisis demonstrated enhanced sensitivity to TNF (3-fold; P < 0.02), and a marked increase in serum TNF concentration (approximately 5-fold; P < 0.001) following LPS challenge. The effect of TNF upon long-term survival in adrenalectomized mice was examined in TNF-receptor Ia- and Ib-deficient mice. Deficiencies in either the TNF-receptor Ia or Ib was noted to confer a survival advantage relative to colony controls following adrenalectomy (P < 0.02). In summary, both LPS-induced Addison's crisis and chronic adrenal insufficiency are disorders of TNF disregulation. Based upon these data, therapeutic strategies targeted at controlling TNF in adrenal insufficiency are suggested.

• Luster MI, Simeonova PP, Gallucci RM, Bruccoleri A, Blazka ME, Yucesoy B
• Role of inflammation in chemical-induced hepatotoxicity.
• Toxicol Lett. 2001; 120: 317-21
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• The liver, which is the major organ responsible for the metabolism of drugs and toxic chemicals, is also the primary target organ for many toxic chemicals. Increasing evidence has indicated that inflammatory processes are intimately involved in chemical-induced hepatotoxic processes, and like other inflammatory diseases, such as autoimmunity, are responsible for producing mediators that can effect liver damage or repair. This review will summarize our current understanding of how inflammatory processes influence hepatic pathology and repair following exposure to established hepatotoxic chemicals including carbon tetrachloride, an industrial chemical, and acetaminophen, a widely used analgesic.

• Tatsuta K, Misawa H, Chikauchi K
• Biomimetric total synthesis of quinolactacin B, TNF production inhibitor, and its analogs.
• J Antibiot (Tokyo). 2001; 54: 109-12

• Schwartz DA, Christ WJ, Kleeberger SR, Wohlford-Lenane CL
• Inhibition of LPS-induced airway hyperresponsiveness and airway inflammation by LPS antagonists.
• Am J Physiol Lung Cell Mol Physiol. 2001; 280: 7718-7718
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• To determine whether the inflammatory effects of inhaled endotoxin could be prevented, we pretreated mice with synthetic competitive antagonists (975, 1044, and 1287) for lipopolysaccharide (LPS) before a LPS inhalation challenge. In preliminary studies, we found that these LPS antagonists did not act as agonists in vitro (THP-1 cells) or in vivo (after intratracheal instillation of 10 microg) and that these compounds (at least 1 microg/ml) effectively antagonized the release of tumor necrosis factor-alpha by LPS-stimulated THP-1 cells. Pretreatment of mice with 10 microg of either 1044 or 1287 resulted in a decrease in the LPS-induced airway hyperreactivity. Moreover, pretreatment of mice with 10 microg of 975, 1044, or 1287 resulted in significant reductions in LPS-induced lung lavage fluid concentrations of total cells, neutrophils, and specific proinflammatory cytokines compared with mice pretreated with sterile saline. Using residual oil fly ash to induce airway inflammation, we found that the action of the LPS antagonists was specific to LPS-induced airway disease. These results suggest that LPS antagonists may be an effective and potentially safe treatment for endotoxin-induced airway disease.

• Vallabhapurapu S, Ryseck RP, Malewicz M, Weih DS, Weih F
• Inhibition of NF-kappaB in T cells blocks lymphoproliferation and partially rescues autoimmune disease in gld/gld mice.
• Eur J Immunol. 2001; 31: 2612-22
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• The Fas ligand (FasL)/Fas pathway is crucial for the maintenance of homeostasis of the peripheral immune system. Its importance is illustrated by the spontaneous mouse mutants gld andlpr which lack functional FasL and Fas receptor, respectively. These animals develop lymphadenopathy, splenomegaly, increased serum Ig and autoantibodies, leading to an autoimmune syndromeand premature death. The Rel/NF-kappaB family of transcription factors plays an important role in peripheral lymphocyte proliferation and survival. In this report, we studied the consequences of T cell-specific inhibition of NF-kappaB on the development of the gld phenotype. Transgenic gld/gld mice expressing a non-degradable form of IkappaBalpha under the control of T cell-specific regulatory elements show dramatically reduced lymphadenopathy, splenomegaly, and an almost complete elimination of Thy-1(+)B220(+)CD4(-)CD8(-) abnormal T cells, correlating with reduced proliferative responses and increased apoptosis of peripheral T cells upon TCR triggering. Interestingly, the B cell abnormalities that are characteristic of gld/gld mice, such as the production of autoantibodies, high levels of serum Ig, and the development of glomerulonephritis, are partially corrected. These results suggest that the T cell-specific inhibition of NF-kappaB opens apoptotic pathways distinct from FasL/Fas which, along with a diminished proliferative response, blocks splenomegaly and lymphadenopathy and partially rescues autoimmune disease in gld/gld mice.

• Drutskaya MS, Kuprash DV, Nedospasov SA, Chertkov IL, Drize NI
• Abnormal hemopoiesis in long-term bone marrow culture from tumor necrosis factor-deficient mice.
• Bull Exp Biol Med. 2001; 131: 150-2
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• We studied hemopoiesis in mice deficient by the tumor necrosis factor gene. The total number of cells in long-term bone marrow cultures from these mice 2-fold surpassed that in wild-type and tumor necrosis factor p55 receptor-deficient animals. Increased cell production was related to the absence of tumor necrosis factor expression by hemopoietic precursors. The total cell production by explanted hemopoietic cells from tumor necrosis factor-deficient mice did not depend on the genotype of irradiated stromal sublayer in long-term cell cultures from wild-type mice and animals deficient by tumor necrosis factor or p55 receptor. These results suggest that tumor necrosis factor, but not its p55 receptor, is involved in transduction of signals regulating production of cultured cells. Tumor necrosis factor probably regulates hemopoiesis in long-term bone marrow cultures by initiating apoptosis of hemopoietic cells or inhibiting cell proliferation. Increased cell production probably attests to the absence of one or both effects.

• Birks EJ et al.
• Serum cytokines in left ventricular assist device candidates.
• Transplant Proc. 2001; 33: 1974-5

• Bozkurt B et al.
• Results of targeted anti-tumor necrosis factor therapy with etanercept (ENBREL) in patients with advanced heart failure.
• Circulation. 2001; 103: 1044-7
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• BACKGROUND: Previously, we showed that tumor necrosis factor (TNF) antagonism with etanercept, a soluble TNF receptor, was well tolerated and that it suppressed circulating levels of biologically active TNF for 14 days in patients with moderate heart failure. However, the effects of sustained TNF antagonism in heart failure are not known. METHODS AND RESULTS: We conducted a randomized, double-blind, placebo-controlled, multidose trial of etanercept in 47 patients with NYHA class III to IV heart failure. Patients were treated with biweekly subcutaneous injections of etanercept 5 mg/m(2) (n=16) or 12 mg/m(2) (n=15) or with placebo (n=16) for 3 months. Doses of 5 and 12 mg/m(2) etanercept were safe and well tolerated for 3 months. Treatment with etanercept led to a significant dose-dependent improvement in left ventricular (LV) ejection fraction and LV remodeling, and there was a trend toward an improvement in patient functional status, as determined by clinical composite score. CONCLUSION: Treatment with etanercept for 3 months was safe and well-tolerated in patients with advanced heart failure, and it resulted in a significant dose-dependent improvement in LV structure and function and a trend toward improvement in patient functional status.

• Donnelly EF, Geng L, Wojcicki WE, Fleischer AC, Hallahan DE
• Quantified power Doppler US of tumor blood flow correlates with microscopic quantification of tumor blood vessels.
• Radiology. 2001; 219: 166-70
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• P:URPOSE: To evaluate the ability of a quantified power Doppler ultrasonography (US) system to help quantitate differences in tumor vascularity after radiation therapy and administration of tumor necrosis factor (TNF). MATERIALS AND METHODS: Murine glioblastoma tumors were grown in the thighs of two sets of 25 mice each. Each mouse was assigned to one of four treatment groups: control (no treatment), radiation therapy, TNF therapy, or combination therapy (both radiation and TNF therapies). Mice were then evaluated with quantified power Doppler US, and a vascularity index (color area) was calculated for different tumor regions in each group. The tumors were then excised, and histologic evaluation was performed by using an immunofluorescence-tagged monoclonal antibody against blood vessel endothelium. The number of stained blood vessels per high-power field was correlated with the sonographically determined vascularity index. RESULTS: The color area of the total tumor decreased to 37% of that in the control group in mice treated with radiation therapy alone (P: =.02), 26% of that in the control group in mice treated with TNF alone (P: =.05), and 8% of that in the control group in those treated with both TNF and radiation (P: =.006). These results correlated well with the quantified results from immunofluorescent staining (r = 0.98). CONCLUSION: Quantified power Doppler US is a noninvasive method for the evaluation of tumor vascularity and blood flow.

• Dbaibo GS et al.
• Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death.
• FEBS Lett. 2001; 503: 7-12
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• Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor alpha. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.

• Mann DL
• Tumor necrosis factor and viral myocarditis: the fine line between innate and inappropriate immune responses in the heart.
• Circulation. 2001; 103: 626-9

• Xu S, Lam KP
• B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses.
• Mol Cell Biol. 2001; 21: 4067-74
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• B-cell maturation protein (BCMA) is a member of the tumor necrosis factor (TNF) receptor family and is expressed in B lymphocytes. BCMA binds two TNF family members, BAFF and APRIL, that stimulate cellular proliferation. BAFF in particular has been shown to influence B-cell survival and activation, and transgenic mice overexpressing BAFF have a lupus-like autoimmune disorder. We have inactivated BCMA in the mouse germ line. BCMA(-/-) mice have normal B-cell development, and the life span of mutant B lymphocytes is comparable to that of wild-type B cells. The humoral immune responses of BCMA(-/-) mice to T-cell-independent antigens as well as high and low doses of T-cell-dependent antigens are also intact. In addition, mutant mice have normal splenic architecture, and germinal centers are formed during an ongoing immune response. These data suggest a functional redundancy of BCMA in B-cell physiology that is probably due to the presence of TACI, another TNF receptor family member that is expressed on B cells and that can also bind BAFF and APRIL.

• Eigler A, Loher F, Endres S
• [Suppression of synthesis of tumor necrosis factor]
• Internist (Berl). 2001; 42: 28-34

• ten Hagen TL, Eggermont AM, Lejeune FJ
• TNF is here to stay--revisited.
• Trends Immunol. 2001; 22: 127-9

• Jin Y, Atkinson SJ, Marrs JA, Gallagher PJ
• Myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1.
• J Biol Chem. 2001; 276: 30342-9
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• Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.

• Goldberg GS et al.
• Global effects of anchorage on gene expression during mammary carcinoma cell growth reveal role of tumor necrosis factor-related apoptosis-inducing ligand in anoikis.
• Cancer Res. 2001; 61: 1334-7
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• Anchorage-independent growth is a hallmark of tumor cells. We compared gene expression profiles of anchored and nonanchored human mammary carcinoma cells to study this phenomenon. In this study, we show that anchorage had striking effects on cell growth and morphology but altered transcript levels from a limited number of genes. Only about 1% of mRNA transcripts detected in these cells was altered by anchorage. These include genes related to amino acid and polyamine metabolism, apoptosis, ion channels, cytoskeletal and stress proteins, transcription factors, and growth factors. Some of these may be crucial for the survival of transformed cells. For example, clusterin and the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) were suppressed by anchorage, which could help prevent programmed cell death of these tumor cells. In addition to suppressing TRAIL expression, anchorage also decreased the susceptibility of these tumor cells to TRAIL-induced apoptosis as determined by poly(ADP-ribose) phosphorylase cleavage, annexin-V binding (P < 0.01), and cell cycle analysis (P < 0.0001). These data may help explain mechanisms by which anchorage prevents apoptosis of cells that would otherwise experience anoikis. Thus, genes found to be altered by this analysis could serve as potential targets for anticancer therapy. These findings suggest that TRAIL may be used as a means to target circulating epithelial tumor cells before their attachment and colonization at new sites.

• Zakhartchouk AN, Godson DL, Babiuk LA, Tikoo SK
• 121R protein from the E3 region of bovine adenovirus-3 inhibits cytolysis of mouse cells by human tumor necrosis factor.
• Intervirology. 2001; 44: 29-35
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• Based on the DNA sequence and known mRNA structures, the early region 3 (E3) of bovine adenovirus (BAV)-3 has the potential to encode four proteins. One of them (121R) is produced as a 14.5-kD protein throughout infection. Analysis of the 121R protein showed limited homology to a 14.7K protein of human adenovirus (HAV)-5. Interestingly, both anti-14.7K and anti-121R sera immunoprecipitated a 14.5-kD protein from cells infected with BAV-3. To determine if 121R is functionally related to 14.7K, we constructed a recombinant E3-deleted HAV-5 (AdKV121) expressing the BAV-3 E3 121R protein. Mouse C3HA cells infected with HAV-5 mutant dl 758 (deletion of 14.7K) were sensitive to TNF lysis. However, wild-type HAV-5- or recombinant AdKV121-infected cells were resistant to TNF-induced cytolysis. Our result show that the BAV-3 E3 121R protein is serologically and functionally related to the 14.7K protein encoded by the E3 region of HAV-5.

• Simon A, Drenth JP, van der Meer JW
• [From gene to disease; tumor necrosis factor receptor and a syndrome of familial periodic fever]
• Ned Tijdschr Geneeskd. 2001; 145: 77-8
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• Familial Hibernian fever (FHF) is a rare hereditary syndrome that causes periodic attacks of fever and inflammation. It is an autosomal dominantly inherited disorder. The gene involved in FHF encodes for a receptor for tumour necrosis factor (TNFR1). These mutations are thought to result in impaired shedding of the receptor from the cell membrane, leading to deficient curtailing of the inflammatory reaction. The acronym TRAPS (TNF-receptor associated periodic syndrome) has been proposed as a more accurate name.

• Connolly K et al.
• In vivo inhibition of Fas ligand-mediated killing by TR6, a Fas ligand decoy receptor.
• J Pharmacol Exp Ther. 2001; 298: 25-33
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• TR6, a member of the tumor necrosis factor (TNF) receptor superfamily, has recently been shown to bind to Fas ligand (FasL) and inhibit FasL-mediated cell killing in vitro. In the current study, we demonstrate that TR6 can block the lethal activity of FasL in multiple in vitro systems, and extend this finding to an in vivo model of hepatitis. The binding of human TR6 to human FasL was verified with BIAcore chip technology. Human primary hepatocytes, HT-29 cells and Jurkat cells were assayed for viability to demonstrate TR6 inhibition of FasL-mediated cytotoxicity in vitro. Human TR6 was also shown to cross-react with membrane-bound mouse FasL, since the in vitro cytotoxic activity of L929 cells transfected with murine FasL was inhibited in the presence of human TR6. In vivo, FasL-induced acute, lethal, fulminant hepatic apoptosis resulting in death within 2 h of intravenous injection into Fas+ mice, but not Fas- MRL/lpr mice. Pretreatment of mice with TR6 blocked FasL-induced mortality, presumably by attenuating FasL-induced hepatic apoptosis. Thus, in both in vitro and in vivo systems, TR6 acts as a functional FasL decoy receptor and may be clinically useful in the treatment of hepatitis and other diseases associated with FasL-mediated tissue injury.

• Siegel RM, Lenardo MJ
• To B or not to B: TNF family signaling in lymphocytes.
• Nat Immunol. 2001; 2: 577-8
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• BLyS and family are known to affect B cells in a positive fashion. Knock-outs of BLyS receptors indicate some new functions, including negative regulation by one BLyS receptor, TACI.

• Eichhorst ST, Krammer PH
• Derangement of apoptosis in cancer.
• Lancet. 2001; 358: 345-6

• Henn AD, Berleth ES, Mihich E, Ehrke MJ
• Changes in cytosolic and membrane TNF inhibitory protein-B1 (TIP-B1) levels associated with protection from TNF-induced cytotoxicity.
• FASEB J. 2001; 15: 1315-7

• Locksley RM, Killeen N, Lenardo MJ
• The TNF and TNF receptor superfamilies: integrating mammalian biology.
• Cell. 2001; 104: 487-501

• Melnick M, Chen H, Zhou Y, Jaskoll T
• Embryonic mouse submandibular salivary gland morphogenesis and the TNF/TNF-R1 signal transduction pathway.
• Anat Rec. 2001; 262: 318-30
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• TNF is a pleiotropic cytokine that modulates cell proliferation and apoptosis. The objective of the present study was to investigate the possible function(s) of the TNF/TNF-R1 signaling pathway in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro experiments to investigate the phenotypic outcomes of enhanced and deficient ligand. The results of these experiments indicate that the TNF/TNF-R1 signal transduction pathway plays an important role in balancing cell proliferation and apoptosis during SMG duct and presumptive acini formation. Copyright 2001 Wiley-Liss, Inc.

• Josephs MD et al.
• Lipopolysaccharide and D-galactosamine-induced hepatic injury is mediated by TNF-alpha and not by Fas ligand.
• Am J Physiol Regul Integr Comp Physiol. 2000; 278: 1196201-1196201
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• Tumor necrosis factor (TNF)-alpha and Fas ligand (FasL) are trimeric proteins that induce apoptosis through similar caspase-dependent pathways. Hepatocytes are particularly sensitive to inflammation-induced programmed cell death, although the contribution of TNF-alpha and/or FasL to this injury response is still unclear. Here, we report that D-galactosamine and lipopolysaccharide-induced liver injury in C57BL/6 mice is associated with increased hepatic expression of both TNF-alpha and FasL mRNA. Pretreatment of mice with a TNF-binding protein improved survival, reduced plasma aspartate aminotransferase concentrations, and attenuated the apoptotic liver injury, as determined histologically and by in situ 3' OH end labeling of fragmented nuclear DNA. In contrast, pretreatment of mice with a murine-soluble Fas fusion protein (Fasfp) had only minimal effect on survival, and apoptotic liver injury was either unaffected or exacerbated depending on the dose of Fasfp employed. Similarly, mice with a spontaneous mutation in FasL (B6Smn.C3H-Fasl(gld) derived from C57BL/6) were equally sensitive to D-galactosamine/lipopolysaccharide-induced shock. We conclude that the shock and apoptotic liver injury after D-galactosamine/lipopolysaccharide treatment are due primarily to TNF-alpha release, whereas increased FasL expression appears to contribute little to the mortality and hepatic injury.

• Baumgarth N
• Secreted IgM versus BLyS in germinal center formation.
• Nat Immunol. 2000; 1: 179-179

• Ware CF
• APRIL and BAFF connect autoimmunity and cancer.
• J Exp Med. 2000; 192: 358-358

• Nagata S
• Steering anti-cancer drugs away from the TRAIL.
• Nat Med. 2000; 6: 502-3

• Thompson JS et al.
• BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.
• J Exp Med. 2000; 192: 129-35
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• The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

• Yan M, Marsters SA, Grewal IS, Wang H, Ashkenazi A, Dixit VM
• Identification of a receptor for BLyS demonstrates a crucial role in humoral immunity.
• Nat Immunol. 2000; 1: 37-41
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• B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) superfamily. BLyS stimulates proliferation of, and immunoglobulin production by, B cells. However, the relative importance of BLyS in physiological B cell activation is unclear. We identified a B cell receptor for BLyS through expression cloning as TACI, an orphan TNF receptor homologue of unknown function. Binding of BLyS to TACI activated signaling by nuclear factor-kappa B (NF-kappa B). In vitro soluble TACI-Fc fusion protein blocked BLyS-induced NF-kappa B activation in B lymphoma cells and IgM production in peripheral blood B cells. In vivo treatment of immunized mice with TACI-Fc inhibited production of antigen-specific IgM and IgGI antibodies and abolished splenic germinal center (GC) formation. Thus, BLyS activity must play a critical role in the humoral immune response.

• Korner H et al.
• Tumor necrosis factor sustains the generalized lymphoproliferative disorder (gld) phenotype.
• J Exp Med. 2000; 191: 89-96
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• Tumor necrosis factor (TNF) and Fas ligand (FasL) play major roles in the homeostasis of the peripheral immune system. This becomes dramatically obvious in the absence of a functional FasL. Mice with such a deficiency develop a profound lymphadenopathy, splenomegaly, hypergammaglobulinemia, and strain-dependent systemic autoimmune disease, and succumb to premature death. It is consequently termed generalized lymphoproliferative disorder (gld). By contrast, TNF deficiency alone does not result in a striking phenotype. Thus, we sought to determine what role TNF might play in contributing to the gld phenotype by creating C57BL/6.gld.TNF(-/-) mice. Contrary to the expected outcome, mice deficient for both FasL and TNF had a substantially milder gld phenotype with regard to mortality, lymphoaccumulation, germinal center formation, and hypergammaglobulinemia. To confirm these data in a strain highly permissive for the phenotype, C3H/HeJ.gld and C3H.HeJ.lpr mice were treated with a TNF-specific monoclonal antibody. This transient neutralization of TNF also resulted in a significantly attenuated lymphoproliferative phenotype. We conclude that TNF is necessary for the full manifestation of the lymphoproliferative disorder, in particular playing a critical role in lymphoaccumulation. Most importantly, absence of TNF protects gld mice against premature death.

• Kuwano K, Hara N
• Signal transduction pathways of apoptosis and inflammation induced by the tumor necrosis factor receptor family.
• Am J Respir Cell Mol Biol. 2000; 22: 147-9

• Schwenzer R, Zimmermann G, Fotin M, Wajant H, Grell M
• No RISC, no fun: assembly of receptor-induced signalling complexes in the tumor necrosis factor system.
• Eur Cytokine Netw. 2000; 11: 519-20

• Keith M, Norwich KH, Wong W, Jeejeebhoy KN
• The tissue distribution of tumor necrosis factor-alpha in rats: a compartmental model.
• Metabolism. 2000; 49: 1309-17
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• Tumor necrosis factor (TNF) is widely accepted to be the mediator of the cascade of metabolic abnormalities associated with both critical and chronic illness. TNF binding to cell surface receptors mediates its physiologic actions, although the exact mechanism of TNF action is unknown. Therefore, this study was designed to investigate the in vivo metabolism of TNF using a mathematical model to examine tissue uptake and loss of TNF over time. Two distinct patterns of TNF uptake were observed. Muscle tissues were found to accumulate TNF over the entire experimental period, whereas the visceral organs were found to have a rapid initial accumulation of TNF followed by a rapid loss of TNF back to the plasma or out into the bile or the urine. These patterns of TNF binding and retention may reflect the number of TNF receptors or their affinity for TNF, as well as the balance between cell surface and soluble TNF receptors. Furthermore, TNF binding patterns provide insight into the biologic action of TNF at these sites.

• Clark A
• Post-transcriptional regulation of pro-inflammatory gene expression.
• Arthritis Res. 2000; 2: 172-4
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• The cytokine tumour necrosis factor (TNF)alpha is a vital mediator of the innate immune response, and a pleiotropic regulator of cellular function. Its involvement in rheumatoid arthritis is illustrated by the clinical benefits of TNF alpha blockade. Post-transcriptional regulation (the control of mRNA stability and translation) appears to play a critical role in the regulation of TNF alpha expression by mitogen activated protein kinase signal transduction pathways and by anti-inflammatory agents. The aim of this article is to review some recent advances in our understanding of these processes, and to speculate on mechanisms of regulation of TNF alpha and other pro-inflammatory genes.

• Pfeffer K
• Morphogenic and host protective functions executed by tumor necrosis factor receptors, lymphotoxin receptors and their ligands.
• Eur Cytokine Netw. 2000; 11: 514-514

• Menart V, Gaberc-Porekar V, Jevsevar S, Pernus M, Meager A, Stalc A
• Early events in TNFa-p55 receptor interations--experiments with TNF dimers.
• Pflugers Arch. 2000; 439: 1135-1135
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• The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule. To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer. For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E. coli and prepared in pure form. By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared. The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity. To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces. These dimers showed substantially reduced cytotoxicity on the L929 cell line. In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line.

• Kokot T, Muc-Wierzgon M, Zubelewicz B, Wierzgon J, Nowakowska E, Brodziak A
• [Soluble tumor necrosis factor receptors--their properties and role in diagnosis and therapy of acute and chronic diseases]
• Postepy Hig Med Dosw. 2000; 54: 585-96
• Display abstract

• The review presents the properties and significance of soluble forms of tumor necrosis factor-alpha receptors in physiology and pathology of human organism. Special attention was turned to the possibility of practical application of those forms of the receptors in the therapy of diseases caused by excessive activity of TNF-alpha. The significance of the soluble receptors as prognostic factors of disease progression and outcome was stressed.

• Yu G et al.
• APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.
• Nat Immunol. 2000; 1: 252-6
• Display abstract

• We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.

• Fischer A, Rieux-Laucat F, Le Deist F
• Autoimmune lymphoproliferative syndromes (ALPS): models for the study of peripheral tolerance.
• Rev Immunogenet. 2000; 2: 52-60
• Display abstract

• Lymphocyte cell death is a key event in the homeostasis of the immune system. Lymphocytes can be induced to die because of exposure to toxic agents, because of cytokine withdrawal or because specific cell surface receptors are engaged by their ligands. A number of such receptors belonging to the TNF receptor have been described in the recent past. Among these, the role of the Fas ligand/receptor interaction in the induction of lymphocyte cell death has been enlightened by the study of natural mutants, first described in mouse strains, then in humans. This review discusses the main findings provided by murine studies and clinical observations.

• Grammer AC, Lipsky PE
• CD40-mediated regulation of immune responses by TRAF-dependent and TRAF-independent signaling mechanisms.
• Adv Immunol. 2000; 76: 61-178

• Mohler KM, Murray KM, Mann DL, Francis G
• Use of targeted anticytokine treatments in heart failure.
• Circulation. 2000; 102: 65-65

• Gerspach J, Gotz A, Zimmermann G, Kolle C, Bottinger H, Grell M
• Detection of membrane-bound tumor necrosis factor (TNF): an analysis of TNF-specific reagents.
• Microsc Res Tech. 2000; 50: 243-50
• Display abstract

• Tumor necrosis factor (TNF) exists in two bioactive forms, the membrane integrated form and the proteolytically derived soluble cytokine. Both forms of TNF are involved in a variety of different physiological and pathophysiological situations. Here we analyzed different human and mouse TNF-specific reagents for their ability to determine the expression of membrane-expressed TNF. The data prove some antibodies to be very useful for the analysis of transmembrane TNF expression because these antibodies distinguish between the transmembrane form of TNF and soluble TNF bound to cellular TNF receptors. In addition, we found that recombinant human TNF receptor fusion proteins are advantageous tools to analyze both human and mouse transmembrane TNF expression.

• Wargula JC, Lovell DJ
• Use of etanercept in children.
• Bull Rheum Dis. 2000; 49: 1-4

• Kaplan MJ, Ray D, Mo RR, Yung RL, Richardson BC
• TRAIL (Apo2 ligand) and TWEAK (Apo3 ligand) mediate CD4+ T cell killing of antigen-presenting macrophages.
• J Immunol. 2000; 164: 2897-904
• Display abstract

• The human marrow produces approximately 1010 monocytes daily, and this production must be balanced by a similar rate of destruction. Monocytes/macrophages can undergo apoptosis after activating CD4+ T cells, suggesting one mechanism that may contribute to macrophage homeostasis. Previous reports indicate that Fas-Fas ligand interactions are the principle molecules mediating this response. However, D10, an Iak-restricted cloned Th2 line, will similarly induce apoptosis in Ag-presenting macrophages, and D10 cells lack Fas ligand. To confirm that D10 cells kill macrophages through Fas-independent pathways, D10 cells were shown to kill MRL lpr/lpr (Iak) macrophages in an Ag-dependent fashion, indicating additional mechanisms. Recent reports demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), interacting with Apo2, and TNF-like weak inducer of apoptosis (TWEAK), interacting with Apo3, will induce apoptosis in some cells. Using Abs to TRAIL and an Apo3-IgG Fc fusion protein, we demonstrated that D10 cells express both TRAIL and TWEAK. The Apo3 fusion protein, but not human IgG, inhibited D10-induced macrophage apoptosis, as did anti-TRAIL. Further studies demonstrated that AE7, a cloned Th1 line, and splenic T cells express TWEAK, TRAIL, and Fas ligand, and inhibiting these molecules also inhibited macrophage killing. These results indicate that D10 cells induce macrophage apoptosis through TRAIL- and TWEAK-dependent pathways. Because normal T cells also express these molecules, these results support the concept that T cells have multiple pathways by which to induce macrophage apoptosis. These pathways may be important in immune processes such as macrophage homeostasis as well as in down-regulation of immune responses and elimination of macrophages infected with intracellular organisms.

• Papadakis KA, Targan SR
• Tumor necrosis factor: biology and therapeutic inhibitors.
• Gastroenterology. 2000; 119: 1148-57

• Kaufman DR, Choi Y
• Signaling by tumor necrosis factor receptors: pathways, paradigms and targets for therapeutic modulation.
• Int Rev Immunol. 1999; 18: 405-27

• McWhirter SM et al.
• Structural and biochemical analysis of signal transduction by the TRAF family of adapter proteins.
• Cold Spring Harb Symp Quant Biol. 1999; 64: 551-62

• Francis GS
• TNF-alpha and heart failure. The difference between proof of principle and hypothesis testing.
• Circulation. 1999; 99: 3213-4

• Robins HI et al.
• Whole body hyperthermia induction of soluble tumor necrosis factor receptors: implications for rheumatoid diseases.
• J Rheumatol. 1999; 26: 2513-6
• Display abstract

• OBJECTIVE: To test the hypothesis that 41.8 degrees C x 60 min whole body hyperthermia (WBH) induces increased serum levels of soluble necrosis factor receptors (sTNF-R). METHODS: We tested the serum of cancer patients for changes in sTNF-RI and RII levels, as a function of time, pre and post: (1) WBH alone, (2) WBH and chemotherapy, i.e., melphalan (L-PAM), and (3) L-PAM alone. RESULTS: For sTNF-RI there was a marked increase (over pre-treatment values, i.e., 86%) in serum levels after WBH alone (n = 3), which peaked 2.5 h post-WBH; L-PAM (iv) only resulted in a dip in sTNF-RI seen 40 min postadministration; the combination (WBH + L-PAM), resulted in both the dip at 40 min and the increase at 2.5 h post-treatment. For sTNF-RII both WBH alone (n = 3) and WBH + L-PAM (n = 2), there was an increase in receptor serum levels of 25% and 30%, respectively, which peaked 5.5 h post-treatment, and remained elevated at 24 h. L-PAM alone resulted in a dip in levels only at 40 min post-treatment. sTNF-RI and RII levels returned to baseline values within 7 days post-treatment. CONCLUSION: 41.8 degrees C WBH results in transient increases in TNF-RI and RII. These results may have therapeutic implications for the application of WBH to TNF mediated disease processes.

• Visioli F
• Tumour necrosis factor as a potential target for the treatment of congestive heart failure.
• Pharmacol Res. 1999; 40: 91-91

• Kwon B, Youn BS, Kwon BS
• Functions of newly identified members of the tumor necrosis factor receptor/ligand superfamilies in lymphocytes.
• Curr Opin Immunol. 1999; 11: 340-5
• Display abstract

• Rapid progress in the discovery of members of the tumor necrosis factor receptor/ligand superfamilies has been made, mainly with the help of massive DNA sequencing and bioinformatic studies. Biological studies of the new members have not only indicated overlapping roles with other members but also provided insights into novel functions. In particular, multiple pairings of receptors and ligands highlight a complex control mechanism of immune responses by these superfamilies.

• Chen AI et al.
• Ox40-ligand has a critical costimulatory role in dendritic cell:T cell interactions.
• Immunity. 1999; 11: 689-98
• Display abstract

• The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.

• French LE, Tschopp J
• The TRAIL to selective tumor death.
• Nat Med. 1999; 5: 146-7

• Walczak H et al.
• Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.
• Nat Med. 1999; 5: 157-63
• Display abstract

• To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.

• Goeddel DV
• Signal transduction by tumor necrosis factor: the Parker B. Francis Lectureship.
• Chest. 1999; 116: 6973-6973

• Newton RC, Decicco CP
• Therapeutic potential and strategies for inhibiting tumor necrosis factor-alpha.
• J Med Chem. 1999; 42: 2295-314

• Ottaway CA, Fong IW, da Silva B, Singer W, Karrass L
• Integrative aspects of a human model of endotoxemia.
• Can J Physiol Pharmacol. 1998; 76: 473-8
• Display abstract

• The production of tumor necrosis factor (TNF)-alpha is a key step in the response to sepsis and has powerful local and systemic effects on the host. These systemic responses include a complex cascade of centrally mediated endocrine and neural responses. An integrative model of these regulatory cytokine-neuroendocrine interactions in humans is presented. The rapid kinetics of these responses are illustrated by data showing the response of normal human subjects to experimental endotoxemia. Appreciation of the integrative biology of the in vivo response to experimental endotoxemia can provide a framework for the design of experiments aimed at examining the effects of physical training paradigms on particular cytokine and neuroendocrine pathways.

• Chung TD et al.
• Tumor necrosis factor-alpha-based gene therapy enhances radiation cytotoxicity in human prostate cancer.
• Cancer Gene Ther. 1998; 5: 344-9
• Display abstract

• The purpose of the present study was to determine the therapeutic potential of combining radiotherapy with tumor necrosis factor (TNF)-alpha-based gene therapy in the human prostate cancer PC-3 xenograft. PC-3 cells are highly resistant to TNF-alpha-induced cytotoxicity in vitro. A modest enhancement of radiation killing was observed with the addition of TNF-alpha in clonogenic survival assays. Combined treatment with Ad.Egr-TNF, a replication-deficient adenovirus modified to express TNF-alpha following the exposure of infected cells to ionizing radiation (40 Gy administered at 5 Gy per fraction) in vivo, resulted in increased tumor control, as defined by a reduction of tumor volume, when compared with treatment with Ad.Egr-TNF alone or with radiation alone (P < .03). The improvement in tumor control was achieved without increasing acute normal tissue damage when compared with tissue injury from radiation alone. The results of these studies support further development and clinical application of genetic radiotherapy for human prostate cancer.

• Matsumoto N, Oida H, Aze Y, Akimoto A, Fujita T
• Intratumoral tumor necrosis factor induction and tumor growth suppression by ONO-4007, a low-toxicity lipid A analog.
• Anticancer Res. 1998; 18: 4283-9
• Display abstract

• BACKGROUND: ONO-4007 is a lipid A analog with low toxicity. MATERIALS AND METHODS: The antitumor activity and tumor necrosis factor (TNF)-inducing activity of ONO-4007 were compared with those of lipopolysaccharide (LPS) in WKAH rats bearing KDH-8 hepatoma cells. RESULTS: Weekly injections of ONO-4007 (3 and 10 mg/kg i.v.) suppressed tumor growth, but LPS (0.01 and 0.1 mg/kg i.v.) did not. A single injection of ONO-4007 (3 and 10 mg/kg i.v.) into tumor-bearing rats induced higher levels of endogenous TNF production in tumor tissues than LPS (0.001, 0.01 and 0.1 mg/kg i.v.). Repeated injections of LPS caused a reduction of TNF production in tumor tissues, whereas the reduction by ONO-4007 was less remarkable than that by LPS. Intratumoral injections of anti-rat TNF-alpha antibody attenuated the antitumor effect of ONO-4007. CONCLUSION: The antitumor effect of ONO-4007 is more pronounced than that of LPS and the effect is mediated by TNF produced in tumor tissues.

• Griffith TS, Lynch DH
• TRAIL: a molecule with multiple receptors and control mechanisms.
• Curr Opin Immunol. 1998; 10: 559-63
• Display abstract

• Apoptosis research is benefiting from bioinformatic approaches to identify new components of the cell death machinery and novel cell death inducers/receptors. Over the past year, knowledge of the system involving TNF-related apoptosis-inducing ligand (TRAIL) and its receptors has increased via genomic database analysis to include four distinct receptors that interact with a single ligand. Currently, these molecules are of major interest due to their potential roles and application in cancer therapy.

• Piguet PF, Vesin C, Guo J, Donati Y, Barazzone C
• TNF-induced enterocyte apoptosis in mice is mediated by the TNF receptor 1 and does not require p53.
• Eur J Immunol. 1998; 28: 3499-505
• Display abstract

• Injection of recombinant mouse TNF into mice is known to induce a shrinkage of the duodenal villi, which becomes evident 30-90 min later and is associated with a detachment of enterocytes in the lumen. These cells can be collected by lavage and are all apoptotic, i.e. hypodiploid as seen by flow cytometric analysis. Thus the count of detached cells was used as an evaluation of the TNF-induced cell loss and apoptosis in the mucosa. TNF injection induced a cell loss of similar magnitude in wild-type (+/+) or in mice lacking the TNF receptor (TNFR)2 (p75, TNFR2-/-), while mice lacking the TNFR1 (p55, TNFR1-/-) were completely resistant to this effect. TNF increased the expression of p53 tumor suppressor gene in the enterocytes from the crypts but not from the villi, as seen by Western blots and histochemistry. TNF increased the expression of p53 in both TNFR2-/- and TNFR1-/- mice. Furthermore, enterocyte cell loss was not attenuated in p53-/- mice. The results indicate that TNF, acting on its receptor 1, induces an apoptotic detachment of the enterocytes from the tip of the villi (i.e. the old enterocytes), while in the enterocytes from the crypts (the young enterocytes) TNF increases, via either TNFR1 or TNFR2, the expression of p53, without inducing apoptosis.

• Ferrari R
• Tumor necrosis factor in CHF: a double facet cytokine.
• Cardiovasc Res. 1998; 37: 554-9

• Wu R, Ji X, Ding S, Wei Z, Zhang M
• Expression of tumor necrosis factor mRNA, protein, and receptor protein products in cervical neoplasms.
• Chin Med J (Engl). 1998; 111: 1046-7

• Mueller H
• Tumor necrosis factor as an antineoplastic agent: pitfalls and promises.
• Cell Mol Life Sci. 1998; 54: 1291-8
• Display abstract

• The discovery and cloning of the cytokine tumor necrosis factor alpha (TNF) gave rise to new hopes for a significant victory in the war against cancer. Preclinical in vitro studies in cell cultures and in vivo studies in animal models demonstrated the antitumor capacities of TNF. Although clinical studies were largely made possible by the availability of recombinant TNF, phase I and II clinical trials showed very quickly that the systemic administration of TNF induced severe side effects mainly due to its pleiotropic action on immunocompetent cells. The clinical manifestations of the side effects were similar to those observed during a severe infection and inflammation. Very recently, lessons from these clinical studies yielded refined approaches whereby the toxicity of TNF is limited through local administration, a combination with other therapeutic regimens and targeted gene therapy. These new approaches are slated for larger clinical trials and in the near future might demonstrate the limited but powerful usefulness of TNF as an antineoplastic agent for different types of cancer.

• Gravestein LA, Borst J
• Tumor necrosis factor receptor family members in the immune system.
• Semin Immunol. 1998; 10: 423-34
• Display abstract

• The tumor necrosis factor (TNF) receptor family contains death receptors, which have a cytoplasmic death domain and can induce apoptosis, as well as receptors with no apparent homology in the cytoplasmic tail. This second group of receptors binds TNF receptor-associated factors (TRAFs), which are implicated in gene regulation and anti-apoptotic signaling. A bewildering variety of TNF receptor family members and their ligands are expressed on cells of the immune system. Based on the pattern of expression of receptors and ligands and based on the phenotype of available knock out mice, we summarize at which stages in lymphoid development and/or the peripheral immune response the various TNF receptor family members may play a role.

• Powell CB, Scott JH, Collins JL
• Comparison of TNFalpha and TNFbeta cytolytic mechanisms in human ovarian and cervical carcinoma cell lines.
• Gynecol Oncol. 1998; 71: 258-65
• Display abstract

• OBJECTIVES: The aim of this study was to determine the potential and mechanism of tumor necrosis factor beta (TNFbeta) mediated cytolysis in human ovarian and cervical carcinoma cells. METHODS: The cytolytic potential of tumor necrosis factor alpha (TNFalpha) and TNFbeta was determined using the TNF reference cell line L929 and human ovarian (SK-OV-3, CaOV-3) and cervical (SiHa, HT-3) carcinoma cell lines. We have previously reported the effects of the lipoxygenase enzyme inhibitor, nordihydroguaiaretic acid, the oxygen radical scavenger glutathione, and fragmented DNA-specific staining with diamidino-2-phenylindole and ApopTag on TNFalpha-mediated cytolysis in these cells. The effects of these agents on TNFbeta-mediated cytolysis were determined. RESULTS: All of the cell lines express a protein-synthesis-dependent TNFalpha and TNFbeta resistance mechanisms. When protein synthesis is inhibited the cytolytic activity of TNFbeta was fivefold greater than that of TNFalpha in L929 cells. In contrast, the cytolytic activity of TNFalpha was fivefold greater than that of TNFbeta in the human cells. Like the TNFalpha cytolytic mechanism, the TNFbeta cytolytic mechanism is dependent on lipoxygenase enzymes, but not oxygen radicals, and results in apoptosis. CONCLUSIONS: To date there is little information about the cytolytic potential of TNFbeta in human cells. The fact that the cytolytic mechanism of TNFbeta appears very similar to that of TNFalpha could be important to our understanding of the potential of these closely related cytokines in anticancer therapies. Although the cytolytic potential of TNFbeta is greater than that of TNFalpha in mouse cells, this is not true in human cells and could limit the efficacy of TNFbeta in anticancer therapies.

• Warzocha K, Salles G
• The tumor necrosis factor signaling complex: choosing a path toward cell death or cell proliferation.
• Leuk Lymphoma. 1998; 29: 81-92
• Display abstract

• Signal transduction pathways which are initiated by the tumor necrosis factor (TNF) utilize receptors which are devoid of intrinsic catalytic activity. Recently identified two families of proteins that directly associate with the cytoplasmic domains of the TNF receptor family members, have partially bridged a molecular gap within the TNF-induced signaling pathways. Clearly, there are numerous alternate routes that originate from the TNF ligand-receptor assembly and terminate on the diverse cellular responses, including proliferation, differentiation, or death. This review focuses on recent advances characterizing the TNF ligand-receptor signaling network, which allow to better understand its participation in a life-death balance within the target cell.

• De Valck D, Vercammen D, Fiers W, Beyaert R
• Differential activation of phospholipases during necrosis or apoptosis: a comparative study using tumor necrosis factor and anti-Fas antibodies.
• J Cell Biochem. 1998; 71: 392-9
• Display abstract

• Phospholipases generate important secondary messengers in several cellular processes, including cell death. Tumor necrosis factor (TNF) can induce two distinct modes of cell death, viz. necrosis and apoptosis. Here we demonstrate that phospholipase D (PLD) and cytosolic phospholipase A2 (cPLA2) are differentially activated during TNF-induced necrosis or apoptosis. Moreover, a comparative study using TNF and anti-Fas antibodies as cell death stimuli showed that PLD and cPLA2 are specifically activated by TNF. These results indicate that both the mode of cell death and the type of death stimulus determine the potential role of phospholipases as generators of secondary messengers.

• Vandevoorde V, Haegeman G, Fiers W
• Induced expression of trimerized intracellular domains of the human tumor necrosis factor (TNF) p55 receptor elicits TNF effects.
• J Cell Biol. 1997; 137: 1627-38
• Display abstract

• The various biological activities of tumor necrosis factor (TNF) are mediated by two receptors, one of 55 kD (TNF-R55) and one of 75 kD (TNF-R75). Although the phenotypic and molecular responses elicited by TNF in different cell types are fairly well characterized, the signaling pathways leading to them are so far only partly understood. To further unravel these processes, we focused on TNF-R55, which is responsible for mediating most of the known TNF effects. Since several studies have demonstrated the importance of receptor clustering and consequently of close association of the intracellular domains for signaling, we addressed the question of whether clustering of the intracellular domains of TNF-R55 (TNF-R55i) needs to occur in structural association with the inner side of the cell membrane, where many signaling mediators are known to reside. Therefore, we investigated whether induced intracellular clustering of only TNF-R55i would be sufficient to initiate and generate a full TNF response, without the need for a full-length receptor molecule or a transmembrane region. Our results provide clear evidence that inducible forced trimerization of either TNF-R55i or only the death domain elicits an efficient TNF response, comprising activation of the nuclear factor kappaB, induction of interleukin-6, and cell killing.

• Yamauchi N, Niitsu Y
• [TNF]
• Gan To Kagaku Ryoho. 1997; 24: 1486-94
• Display abstract

• TNF exerts its biological activities through two distinct receptors of TNF-R55 and TNF-R75. Recent studies have identified TNF receptor-associated molecules, and intracellular signal transduction for apoptosis induced by TNF has been elucidated. Various TNF mutants with higher affinity to TNF-R55 and lower affinity to TNF-R75 are being clarified biogenetically. These mutants show lower adverse effect and higher antitumor activity. Furthermore, TNF gene transduction into lymphocytes and tumor cells shows a decrease in tumorigenicity and enhancement of host immunogenicity. This approach appears to be promising for cancer treatment.

• Brandts CH, Mordmuller BG, Lehman LG, Kremsner PG
• [The dual role of tumor necrosis factor (TNF) during a malarial attack]
• Sante. 1997; 7: 271-4
• Display abstract

• The roles of the various factors implicated in the pathogenesis of severe malaria are not well understood. Tumor necrosis factor (TNF), a cytokine produced mainly by macrophages, seems to play a crucial role in both the host's defence against the parasite and the development of severe complications. This review investigates the dual role of TNF in acute malaria, summarizing current knowledge of the beneficial and detrimental effects of this molecule. Recent work has suggested a possible explanation for this dualism, involving a complex interaction between TNF and its soluble receptors.

• Roth J, Goldbach JM, Storr B, Zeisberger E
• Studies on the role of tumor necrosis factor alpha in the responses to bacterial pyrogenic stimuli.
• Ann N Y Acad Sci. 1997; 813: 255-63

• Liu Z, Zou W
• [The relationship between tumor necrosis factor and glomerular mesangial cells]
• Zhonghua Bing Li Xue Za Zhi. 1997; 26: 203-6
• Display abstract

• OBJECTIVE: To study the autocrine function of tumor necrosis factor (TNF) in glomerular mesangial cells (MC) and the action of TNF on MC. METHODS: Cultured rat and human MC were studied by RT-PCR, in situ hybridization and immunohistochemical methods. RESULTS: The expression of both TNF and TNF receptor mRNA and protein synthesis in MC were manifested. CONCLUSION: (1) MC is the target cell for TNF, MC have specific cell surface receptor to TNF. (2) TNF acts on MC through both autocrine and paracrine mechanism.

• Kastan M
• On the TRAIL from p53 to apoptosis?
• Nat Genet. 1997; 17: 130-1

• Utsumi T
• [Role of cellular processing of TNF-alpha in the expression of its biological function]
• Seikagaku. 1997; 69: 1004-10

• Duckworth WC
• Tumor necrosis factor and insulin resistance: specificity of sequence accounts of inhibition of insulin action.
• J Lab Clin Med. 1997; 130: 114-5

• Stephens FO
• Tumor necrosis factor: is it a breakthrough?
• J Clin Oncol. 1996; 14: 688-9

• Brouckaert P, Fiers W
• Tumor necrosis factor and the systemic inflammatory response syndrome.
• Curr Top Microbiol Immunol. 1996; 216: 167-87

• Aggarwal BB, Natarajan K
• Tumor necrosis factors: developments during the last decade.
• Eur Cytokine Netw. 1996; 7: 93-124

• Lee RK, Spielman J, Zhao DY, Olsen KJ, Podack ER
• Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.
• J Immunol. 1996; 157: 1919-25
• Display abstract

• Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

• Hunt JS, Chen HL, Miller L
• Tumor necrosis factors: pivotal components of pregnancy?
• Biol Reprod. 1996; 54: 554-62
• Display abstract

• Tumor necrosis factor alpha is a major component of the infection-stimulated cytokine milieux associated with preterm labor and pregnancy termination. As a consequence, this potent factor is thought by some to have exclusively negative effects on the course of pregnancy. Yet in humans and other mammals, messenger RNA hybridizing with tumor necrosis factor alpha cRNA probes and protein detected by anti-tumor necrosis factor alpha have been identified in normal cycling and pregnant uteri, placentas, and embryos, consistent with an important role for tumor necrosis factor in female reproduction and embryonic development. Here, evidence for this intriguing dichotomy is presented, unresolved aspects are discussed, and potential roles for uteroplacental and embryonic tumor necrosis factor are proposed.

• Kristiansson M, Soop M, Shanwell A, Sundqvist KG
• Presence of tumor necrosis factor alpha and tumor necrosis factor soluble receptors in erythrocyte concentrates.
• Anesthesiology. 1996; 84: 243-4

• Rothwell NJ, Luheshi GN
• Brain TNF: damage limitation or damaged reputation?
• Nat Med. 1996; 2: 746-7

• Kretzschmar C
• [The evaluation of tumor necrosis factor-alpha concentration in body fluids in local and systemic endotoxin exposure]
• Zentralbl Veterinarmed [B]. 1996; 43: 525-38
• Display abstract

• Tumor-necrosis factor alpha (TNF) has been shown to have a pivotal role in mediating biological effects of endotoxins. Therefore, TNF measurement in the circulation is thought to be a useful diagnostic and prognostic instrument in patients exposed to endotoxin. However, the character of the TNF response depends on the mode of endotoxin exposure and is influenced by a variety of factors. Furthermore, to interpret the TNF state, the regulative effects of the two soluble TNF receptors have to be considered. Finally, the current techniques of TNF determination have to be improved and standardized to obtain reliable and comparable results and to establish TNF measurement as a valuable laboratory parameter.

• Wallach D
• A decade of accumulated knowledge and emerging answers. The 6th international congress on TNF Rhodes, Greece. May 1996.
• Eur Cytokine Netw. 1996; 7: 713-24

• Lynch DH
• The role of FasL and TNF in the homeostatic regulation of immune responses.
• Adv Exp Med Biol. 1996; 406: 135-8

• Sytwu HK, Liblau RS, McDevitt HO
• The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice.
• Immunity. 1996; 5: 17-30
• Display abstract

• The possible involvement of Fas/APO-1 (CD95) and TNF in antigen-specific AICD of thymocytes and mature T cells has been investigated. Antigenic stimulation in vivo of influenza hemagglutinin (HA)-specific TCRtg mice was used to demonstrate that the kinetics of thymocyte and peripheral CD4+ T cell deletion are similar in mice with normal (+/+) or defective Fas (lpr/lpr) background, indicating that a Fas-independent pathway(s) is responsible for the deletion of activated T cells. TCRtg-+/+ or TCRtg-lpr/lpr mice injected with murine TNF-blocking MAb (TN3) showed rapid apoptosis of thymocytes after HA stimulation, indicating that death signaling through Fas and TNF receptors is not essential for HA-induced thymocyte deletion. CDC peripheral T cells in TCRtg-lpr/lpr mice did not undergo apoptosis following injection with HA and TN3, indicating that TNF-mediated apoptosis is involved in the deletion of mature T cells after antigenic stimulation. However, apoptosis still occurred in TCRtg-+/+ mice injected with TN3, indicating that both Fas- and TNF-mediated cell death can contribute to the deletion of activated peripheral T cells.

• Bazzoni F, Beutler B
• The tumor necrosis factor ligand and receptor families.
• N Engl J Med. 1996; 334: 1717-25

• Bemelmans MH, van Tits LJ, Buurman WA
• Tumor necrosis factor: function, release and clearance.
• Crit Rev Immunol. 1996; 16: 1-11
• Display abstract

• Tumor Necrosis Factor (TNF) is a multifunctional cytokine. It plays an important role in the pathophysiology of several diseases. Recently, it has been discovered that TNF is circulating in two different forms, a bioactive form and an immunologically detectable form. These two forms of TNF show different clearance kinetics. The immunological form is supposed to be an inactivated TNF protein. For this inactivation, proteolytic degradation or TNF binding by inactivating proteins is necessary. In this review we have focused on TNF inactivation by TNF binding proteins. Recent data show that there are soluble TNF receptors circulating which can bind and inactivate TNF. These receptors are membrane-bound TNF receptors which have been proteolytically cleaved from the cell membrane. Two TNF receptors are circulating, the soluble TNF receptor of 55 kDa (P55) and the receptor of 75 kDa (P75). The receptors are held responsible not only for inactivation of the TNF, but also for the clearance of TNF. Recent data show that the kidney is the most important organ for TNF clearance, followed by the liver. All other organs are of less importance. In this review, function, release, and clearance of TNF are discussed.

• Russell DA, Tucker KK, Chinookoswong N, Thompson RC, Kohno T
• Combined inhibition of interleukin-1 and tumor necrosis factor in rodent endotoxemia: improved survival and organ function.
• J Infect Dis. 1995; 171: 1528-38
• Display abstract

• The interleukin (IL)-1 receptor antagonist (Ra) and a polyethylene glycol-linked dimer of the type I soluble receptor of tumor necrosis factor (TNF), PEG-(rsTNF-RI)2, were used to determine whether maximal protection against lethality and organ dysfunction is achieved by single or dual cytokine inhibition under rigorous conditions of rodent endotoxemia. Inhibition of IL-1 or TNF alone protected maximally against lethality when inhibitors were given simultaneously with lipopolysaccharide (LPS) under minimal lethal conditions. Combined inhibition of IL-1 and TNF was necessary to maximally protect against lethality when treatment was delayed until 7 h after LPS injection under minimal lethal conditions or when treatment was begun immediately after LPS injection under supralethal conditions. Improved survival in IL-1Ra- plus PEG-(rsTNF-RI)2-treated rats was associated with enhanced protection against renal and metabolic dysfunctions. Thus, under very severe conditions of endotoxemia, TNF and IL-1 may act independently to mediate lethality and some organ dysfunctions.

• Zhang Y, Kolesnick R
• Signaling through the sphingomyelin pathway.
• Endocrinology. 1995; 136: 4157-60

• Richter J, Gullberg U, Lantz M
• TNF-induced superoxide anion production in adherent human neutrophils involves both the p55 and p75 TNF receptor.
• J Immunol. 1995; 154: 4142-9
• Display abstract

• TNF, a potent activator of neutrophil granulocytes, acts via two cell-surface receptors: the p55-TNF receptor (TNF-R55) and the p75-TNF receptor (TNF-R75), which can be cleaved from the cell surface and thus form soluble TNF-binding proteins (TNF-BP). The role of the two receptors in activation of the neutrophil respiratory burst was investigated. Two mAbs reacting with TNF-R55 (H398 and TBP2) induced O2 release in a similar manner but to a lesser extent than TNF. TBP2, however, required preincubation at 4 degrees C to exert its effect. Preincubation of neutrophils (both at 4 and 37 degrees C) with mAb to TNF-R75 decreased TNF-induced superoxide anion production by 67 and 64%, respectively, indicating the essential role also for TNF-R75 in neutrophil activation. This inhibitory effect could not be explained by cross-down-regulation of TNF-R55 because the TNF-R75 mAb had no effect on TNF binding to TNF-R55 as determined by binding of 125I-labeled TNF or release of TNF-R55-BP as measured by ELISA. Furthermore, the TNF-R75 mAb did not decrease superoxide anion generation induced by the TNF-R55 mAb H398, thus ruling out that the inhibitory effect of the TNF-R75 mAb is due to inhibition of the signaling pathway downstream of TNF-R55. In contrast to the TNF-R75 mAb, TNF-R55 mAbs induced down-regulation of TNF-R75 and shedding of both TNF-R55-BP and TNF-R75-BP. We conclude that both TNF-R55 and TNF-R75 are involved in TNF-induced activation of the neutrophil respiratory burst.

• Fabian TC et al.
• Reduced tumor necrosis factor production in endotoxin-spiked whole blood after trauma: experimental results and clinical correlation.
• Surgery. 1995; 118: 63-72
• Display abstract

• BACKGROUND. The overproduction of tumor necrosis factor-alpha (TNF) plays a key role in virtually every experimental model of septic shock, which has led to the development of several therapies that target TNF and other cytokines in clinical sepsis. However, our previous work showed that plasma TNF was reduced, rather than increased, when a septic challenge was administered 3 days after hemorrhagic shock. In this study we compared whole-blood TNF production ex vivo in human beings and animals after trauma. METHODS. TNF was measured before and after a 4-hour incubation of whole blood with 0 or 5 micrograms/ml Escherichia coli endotoxin (LPS) at 37 degrees C ex vivo. Samples were obtained from trauma patients with (n = 8) and without (n = 14) sepsis and compared with those obtained in healthy volunteers (n = 11). In parallel experiments in a pig model TNF was measured before and after fluid resuscitation from trauma after an ex vivo (0 or 5 micrograms/ml LPS) or an in vivo (5 micrograms/kg LPS, 30 minutes intravenously) challenge. RESULTS. With either an immunoassay or a bioassay in either human beings or pigs before or after trauma, TNF was at or below the threshold of detection, unless the blood sample was spiked with LPS. After spiking, TNF was markedly elevated, but the increment was reduced after trauma. In pigs an LPS challenge in vivo delayed 3 days after trauma evoked an increment in plasma TNF that was blunted compared with that in an uninjured control. This trauma-induced reduction in blood TNF could not be attributed to a simple reduction in the number of monocytes nor to changes in cortisol, nor to increased numbers of neutrophils, whose proteolytic enzymes can impair production or increase the degradation of TNF. Although the plasma concentration of soluble TNF-binding proteins (60 kd) was elevated in nonsepsis (p = 0.0358) and sepsis trauma patients (p = 0.0148), the correlation with TNF production was relatively weak (R2 = 0.260). CONCLUSIONS. There was no evidence of TNF overproduction in whole blood after trauma. If these results could be generalized to other tissues, it would be difficult to justify therapeutic targeting of TNF in exaggerated inflammatory response (or septic complications) after trauma.

• Packer M
• Is tumor necrosis factor an important neurohormonal mechanism in chronic heart failure?
• Circulation. 1995; 92: 1379-82

• Kern WV, Engel A, Kern P
• Soluble tumor necrosis factor receptors in febrile neutropenic cancer patients.
• Infection. 1995; 23: 64-5

• Eason JD et al.
• Inhibition of the effects of TNF in renal allograft recipients using recombinant human dimeric tumor necrosis factor receptors.
• Transplantation. 1995; 59: 300-5
• Display abstract

• Tumor necrosis factor alpha (TNFa) and lymphotoxin (LT) or TNF beta are closely linked cytokines produced by macrophages and activated T lymphocytes, which play important regulatory roles in the immune response to allografts. They have also been implicated as mediators of the adverse reactions observed during OKT3 therapy. Therefore, anti-TNF agents could be useful both for immunosuppression and for limiting the systemic response observed in patients receiving OKT3. Recombinant TNFR:Fc is a fusion protein that binds TNFa and LT, thereby neutralizing their effects in vitro. The present study investigates the potential clinical application of TNFR:Fc in a nonhuman primate renal allograft model. Cynomolgus renal allograft recipients were treated with TNFR:Fc induction therapy alone or in combination with subtherapeutic doses of cyclosporine. Control animals received no immunosuppression or subtherapeutic cyclosporine. TNFR:Fc, administered as the only immunosuppressive agent, successfully prolonged renal allograft survival in the majority of treated animals. The prolongation of allograft survival was even more impressive when TNFR:Fc was combined with subtherapeutic doses of cyclosporine. Onset of rejection was significantly delayed as well in the TNFR:Fc treated groups. No adverse side effects were observed in any of the TNFR:Fc treated animals. Precursor cytotoxic T cells were detected in peripheral blood samples of treated recipients but the level of effector CTLs in vivo was below the threshold of detection. These results demonstrate that TNFR:Fc can be safely administered and is effective in prolonging renal allograft survival and in delaying the onset of rejection when administered alone or in combination with cyclosporine.

• Diez-Ruiz A et al.
• Neopterin and soluble tumor necrosis factor receptor type I in alcohol-induced cirrhosis.
• Hepatology. 1995; 21: 976-8
• Display abstract

• Alcohol-induced cirrhosis (AC) is accompanied by disturbances of immune function and cytokine production. To better define the pattern of cytokine synthesis in this disease and to relate it to the immune activation state, we measured circulating levels of soluble tumor necrosis factor receptor p55 (sTNFR-55) and neopterin in a group of 85 patients with AC (classified according to the Child-Pugh score of severity of liver disease) and 43 healthy volunteers. Serum concentrations of sTNFR-55 and neopterin were significantly raised in patients with AC. Moreover, concentrations of sTNFR-55 were significantly higher in patients with more severe disease compared with the group with lower severity. There were significant correlations between sTNFR-55 and neopterin levels in patients and controls. The results contribute to affirm the existence of an immune activation state in AC that could be responsible for the development of the disease and clinical complications.

• Carty TJ, Gilman SC
• The biochemistry and pharmacology of TNF alpha and beta, 21 April 1995, New York.
• Inflamm Res. 1995; 44: 455-7

• Tracey KJ
• TNF and Mae West or: death from too much of a good thing.
• Lancet. 1995; 345: 75-6

• Ware CF, VanArsdale TL, Crowe PD, Browning JL
• The ligands and receptors of the lymphotoxin system.
• Curr Top Microbiol Immunol. 1995; 198: 175-218

• Bazzoni F, Beutler B
• How do tumor necrosis factor receptors work?
• J Inflamm. 1995; 45: 221-38
• Display abstract

• Quite suddenly, a new level of understanding has been attached to the TNF ligand and receptor families. Many of the proximal transducers that signal the presence of TNF or its homologs have been identified, and certain components of the distal signaling pathway have emerged as well. We lack, however, a "movie" of the events that transpire when TNF binds its receptors on the surface of a cell. The facts in hand permit an educated approximation.

• Selmaj K, Papierz W, Glabinski A, Kohno T
• Prevention of chronic relapsing experimental autoimmune encephalomyelitis by soluble tumor necrosis factor receptor I.
• J Neuroimmunol. 1995; 56: 135-41
• Display abstract

• We have evaluated the effect of the type I (p-55, type beta) soluble tumor necrosis factor receptor (sTNFrI) in an animal model of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) was induced in SJL/J mice by adoptive transfer of T lymphocytes sensitized to myelin basic protein (MBP). sTNFrI completely blocked both clinical signs of disease and pathological changes that included CNS demyelination and inflammatory cell infiltration. Effective inhibition of disease expression was obtained using several different regimens of subcutaneous (s.c.) injection. These included daily doses starting at day 0, every other day injections starting at day 0, daily doses starting on day 4, and two doses separated by 12 h on day 1 and 2. Furthermore, treatment with sTNFrI for 15 days completely protected these animals from the recurrent episodes of disease normally associated with adoptively transferred EAE. These findings suggest that TNF plays a major causative role in EAE and that the sTNFrI may prove to be a useful therapeutic approach in multiple sclerosis.

• Peitsch MC, Tschopp J
• Comparative molecular modelling of the Fas-ligand and other members of the TNF family.
• Mol Immunol. 1995; 32: 761-72
• Display abstract

• A number of proteins with significant similarity to the tumour necrosis factor (TNF) have been identified over the last years. Upon interaction with their cognate receptor (members of the TNF-receptor family), all members of this protein family induce either cell death or proliferation/differentiation of the receptor-bearing cells. One of the last identified members of the TNF family is the apoptosis-inducing ligand of the Fas-receptor, termed Fas-ligand (FasL). Here we report the cloning and sequencing of the mouse cDNA for the FasL. Using knowledge-based protein modelling, we demonstrate that all members of the TNF family form trimeric complexes, and define the residues located at the subunit interfaces. The resulting structurally corrected multiple sequence alignment allows the identification of residues potentially involved in receptor recognition, and should help design mutagenesis experiments for structure-function relationship studies.

• Sarin A, Conan-Cibotti M, Henkart PA
• Cytotoxic effect of TNF and lymphotoxin on T lymphoblasts.
• J Immunol. 1995; 155: 3716-8
• Display abstract

• Recombinant TNF and lymphotoxin trigger the apoptotic death of normal mouse and human T lymphocyte blasts in vitro. This cytotoxic effect does not involve the Fas death pathway and differs from the TNF-triggered death of tumor cells in several respects: 1) It is a slower process, requiring 2 to 3 days; 2) it is blocked, rather than enhanced, by cycloheximide; and 3) based on the agonistic effect of anti-TNF receptor Abs, it involves a synergistic effect of both the 55-kDa TNFR1 and the 75-kDa TNFR2, as opposed to the dominance of TNFR1 for tumor cytotoxicity. The TNF-induced death of blasts is potently inhibited by IL-2, as well as by IL-1, IL-4, IFN-gamma, and IL-12. Because activated T cells secrete both TNF and LT, these findings reveal a new pathway for Ag-induced down-modulation of T cell responses.

• Leist M, Gantner F, Jilg S, Wendel A
• Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release.
• J Immunol. 1995; 154: 1307-16
• Display abstract

• The systemic inflammatory response is characterized by release of circulating TNF which may cause multiorgan failure including septic liver failure. We studied TNF signaling in an appropriate in vitro system with primary murine hepatocyte cultures from normal and genetically altered animals. Either one of the three different TNF species, huTNF-alpha, huTNF-beta, or muTNF-alpha (at concentrations > 1 ng/ml) induced direct hepatocytotoxicity preceded by DNA fragmentation in cells prepared from wild-type C57BL mice. TNF-induced cytotoxicity was preceded by oligonucleosomal DNA fragmentation. Further cellular responses to TNF exposure were induction of nitric oxide synthase and secretion of serum amyloid A. None of the above events occurred in hepatocytes lacking the gene for the 55-kDa TNF receptor (TNF-R1), even after stimulation with > 1 micrograms/ml TNF. However, selective stimulation of the TNF-R1 in wild-type hepatocytes with huTNF-alpha elicited a pattern of responses essentially similar to that seen with muTNF-alpha. We obtained analogous results when we examined the hepatotoxicity of TNF in D-galactosamine-sensitized mice, i.e., DNA fragmentation and liver failure was noted in wild-type mice, whereas TNF-R1-deficient mice were completely resistant. We conclude that the TNF-R1 is not only necessary, but also sufficient for TNF signaling in murine hepatocytes.

• Beutler B
• Summary of the 5th International Congress on TNF and related cytokines: scientific advances and their medical applications.
• J Leukoc Biol. 1995; 57: 11-2

• Crowe PD, Walter BN, Mohler KM, Otten-Evans C, Black RA, Ware CF
• A metalloprotease inhibitor blocks shedding of the 80-kD TNF receptor and TNF processing in T lymphocytes.
• J Exp Med. 1995; 181: 1205-10
• Display abstract

• TNF is synthesized as a 26-kD membrane-anchored precursor and is proteolytically processed at the cell surface to yield the mature secreted 17-kD polypeptide. The 80-kD tumor necrosis factor (TNF) receptor (TNFR80) is also proteolytically cleaved at the cell surface (shed), releasing a soluble ligand-binding receptor fragment. Since processing of TNF and TNFR80 occurs concurrently in activated T cells, we asked whether a common protease may be involved. Here, we present evidence that a recently described inhibitor of TNF processing N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)L- 3-(2'naphthyl)- alanyl-L-alanine, 2-aminoethyl amide (TAPI) also blocks shedding of TNFR80, suggesting that these processes may be coordinately regulated during T cell activation. In addition, studies of murine fibroblasts transfected with human TNFR80, or a cytoplasmic deletion form of TNFR80, reveal that inhibition of TNFR80 shedding by TAPI is independent of receptor phosphorylation and does not require the receptor cytoplasmic domain.

• Kayagaki N et al.
• Metalloproteinase-mediated release of human Fas ligand.
• J Exp Med. 1995; 182: 1777-83
• Display abstract

• Fas ligand (FasL) is a type II integral membrane protein homologous with tumor necrosis factor (TNF). Recent studies indicate that TNF is processed to yield the soluble cytokine by metalloproteinases at the cell surface of activated macrophages and T cells. In the present study, we investigated whether FasL is also released by metalloproteinases. Treatment with hydroxamic acid inhibitors of matrix metalloproteinases specifically led to accumulation of membrane-type FasL (p40) on the surface of human FasL cDNA transfectants and activated human T cells, as estimated by surface immunofluorescence and immunoprecipitation with newly established anti-human FasL monoclonal antibodies. This surface accumulation of mFasL was associated with the decrease of soluble FasL (p27) in the supernatant as estimated by quantitative ELISA and immunoprecipitation with anti-human FasL monoclonal antibodies. These results indicate that human FasL is efficiently released from the cell surface by metalloproteinases like TNF.

• Moosmayer D et al.
• A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity.
• Ther Immunol. 1995; 2: 31-40
• Display abstract

• Tumour necrosis factor (TNF) is an important mediator of immune and inflammatory responses and has been recognized as a major pathogenic factor in several autoimmune and inflammatory diseases. TNF receptor TR60 plays a critical role in signalling the pathogenic activities of TNF. We here describe molecular cloning and bacterial production of a single-chain antibody (scFv H398) directed against TR60 which possesses antagonistic activity. VH and VL encoding sequences were isolated by PCR from the murine hybridoma cell line H398, cloned into a scFv expression vector and expressed in Escherichia coli. The recombinant antibody (Ab) fragment was found as an active soluble protein in the periplasm but also formed inclusion bodies. Re-folded scFv H398 purified from inclusion bodies was shown to be functional and stable at 37 degrees C with a half-life of 50 h. Comparison of the antigen binding characteristics of scFv with the parental enzymatically produced Fab H398 revealed that both Ab fragments have the same epitope specificity and an identical antigen binding affinity of 1.5 nM. In an in vitro assay it was demonstrated that scFv H398 is an efficient inhibitor of TNF mediated cytotoxicity with an IC50 of 22 nM, which is comparable to the antagonistic activity of natural Fab H398 with an IC50 of 12 nM. As scFv H398 possesses the high affinity TR60 binding and receptor antagonistic activity of the parental Ab H398 but is expected to be less antigenic in man, it provides a valuable tool for the development of novel therapeutic reagents against TNF mediated diseases.

• Tanaka M, Suda T, Takahashi T, Nagata S
• Expression of the functional soluble form of human fas ligand in activated lymphocytes.
• EMBO J. 1995; 14: 1129-35
• Display abstract

• Fas is a type I membrane protein which mediates apoptosis. Fas ligand (FasL) is a 40 kDa type II membrane protein expressed in cytotoxic T cells upon activation that belongs to the tumor necrosis factor (TNF) family. Here, we found abundant cytotoxic activity against Fas-expressing cells in the supernatant of COS cells transfected with human FasL cDNA but not with murine FasL cDNA. Using a specific polyclonal antibody against a peptide in the extracellular region of human FasL, a protein of 26 kDa was detected in the supernatant of the COS cells. The signal sequence of granulocyte colony-stimulating factor was attached to the extracellular region of human FasL. COS cells transfected with the cDNA coding for the chimeric protein efficiently secreted the active soluble form of human FasL (sFasL). Chemical crosslinking and gel filtration analysis suggested that human sFasL exists as a trimer. Human peripheral T cells activated with phorbol myristic acetate and ionomycin also produced functional sFasL, suggesting that human sFasL works as a pathological agent in systemic tissue injury.

• Beyaert R et al.
• Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis.
• J Biol Chem. 1995; 270: 23293-9
• Display abstract

• Cellular responses initiated by tumor necrosis factor (TNF) are mediated by two different cell surface receptors with respective molecular masses of 55 kDa (p55) and 75 kDa (p75). p55 is functional in almost every cell type and can independently transmit most biological activities of TNF. In contrast, TNF signaling via p75 seems so far largely restricted to cells of lymphoid origin, where it can induce proliferation, cytokine production, and/or apoptosis. The mechanisms that regulate TNF receptor activity are largely unknown. Here we report that the p75 of unstimulated p75-responsive PC60 T cells is phosphorylated on serine by a kinase activity present in p75 immune complexes. Several lines of evidence indicate that the latter kinase is casein kinase-1 (CK-1). Previous results have shown that the p75 TNF receptor is constitutively phosphorylated in vivo. Our data show that the latter in vivo phosphorylation is also at least partially due to CK-1. Pretreatment of cells with TNF had no detectable effect on p75 phosphorylation in vitro or in vivo. However, a specific CK-1 inhibitor potentiated TNF-induced apoptosis mediated by p75, suggesting an inhibitory role for phosphorylation by CK-1. Although in vivo p75 phosphorylation could be seen in both p75-unresponsive and p75-responsive cell lines, in vitro p75 phosphorylation in p75 coimmunoprecipitates could not be observed in cell lines that were biologically unresponsive to p75 stimulation. The latter observation further indicates a regulatory role for p75 phosphorylation in p75-mediated signaling. Taken together, our data demonstrate that the p75 TNF receptor is phosphorylated and associated with CK-1, which negatively regulates p75-mediated TNF signaling.

• Gruss HJ, Dower SK
• Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas.
• Blood. 1995; 85: 3378-404
• Display abstract

• The TNF receptor superfamily members are all type I membrane glycoproteins with typical homology in the extracellular domain of variable numbers of cysteine-rich repeats (overall homologies, 25% to 30%). In contrast, the TNF ligand superfamily members (with the exception of LT alpha) are type II membrane glycoproteins with homology to TNF in the extracellular domain (overall homologies, 20%). TNF and LT alpha are trimeric proteins and are composed of beta-strands forming a beta-jellyroll. The homology of the beta-strand regions for the TNF ligand superfamily members suggest a similar beta-sandwich structure and possible trimeric or multimeric complex formation for most or all members. A genetic linkage, as evidence for evolutionary relatedness, is found by chromosomal cluster of TNFR p80, CD30, 4-1BB, and OX40 for 1p36; TNFR p60, TNFR-RP, and CD27 for 12p13; TNF, LT alpha, and LT beta for 6 (MHC locus); CD27L and 4-1BBL for 19p13; and FASL and OX40L for 1q25. Of the TNF ligand superfamily, TNF, LT alpha, and LT beta and their receptors (TNFR p60, TNFR p80, and TNFR-RP) interact in a complex fashion of cross-binding. However, the other family members presently have a one ligand/one receptor binding principle (CD27/CD27L, CD30/CD30L, CD40/CD40L, 4-1BB/4-1BBL, OX40/gp34, and FAS/FASL). In general, the members of the TNF ligand superfamily mediate interaction between different hematopoietic cells, such as T cell/B cell, T cell/monocyte, and T cell/T cell. Signals can be transduced not only through the receptors but also through at least some of the ligands. The transduced signals can be stimulatory or inhibitory depending on the target cell or the activation state. Taken together, TNF superfamily ligands show for the immune response an involvement in the induction of cytokine secretion and the upregulation of adhesion molecules, activation antigens, and costimulatory proteins, all known to amplify stimulatory and regulatory signals. On the other hand, differences in the distribution, kinetics of induction, and requirements for induction support a defined role for each of the ligands for T-cell-mediated immune responses. The shedding of members of the TNF receptor superfamily could limit the signals mediated by the corresponding ligands as a functional regulatory mechanism. Induction of cytotoxic cell death, observed for TNF, LT alpha, CD30L, CD95L, and 4-1BBL, is another common functional feature of this cytokine family. Further studies have to identify unique versus redundant biologic and physiologic functions for each of the TNF superfamily ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

• Takahashi N, Brouckaert P, Fiers W
• Mechanism of tolerance to tumor necrosis factor: receptor-specific pathway and selectivity.
• Am J Physiol. 1995; 269: 398405-398405
• Display abstract

• Repetitive administration of low doses of tumor necrosis factor (TNF) induces a selective tolerance to some, but not all, of its effects. The aim of the present study was to define the pathways involved in tolerance. We observed that the induction of tolerance is mediated by TNF-R55 triggering. TNF-R75 triggering or the addition of sensitizers can interfere with this induction but does not break an acquired tolerance, inasmuch as tolerant animals were also tolerant to otherwise lethal challenges with the combination of human TNF and the sensitizers interleukin-1 and RU-38486. We further defined the selectivity of the tolerance by examining changes in quantitative parameters such as interleukin-6 induction, hypothermia, and hemoconcentration. The differences between tolerant and nontolerant animals mimicked those observed after administration of human TNF vs. murine TNF and were to be found in the duration rather than in the amplitude of the induced changes. We conclude that tolerance selectively blocks the TNF-R75-mediated pathway, especially the part mimicked by interleukin-1 and RU-38486; this pathway normally leads to a state of unresponsiveness to glucocorticoids.

• Kato T, Sawamura Y, Tada M, Sakuma S, Sudo M, Abe H
• p55 and p75 tumor necrosis factor receptor expression on human glioblastoma cells.
• Neurol Med Chir (Tokyo). 1995; 35: 567-74
• Display abstract

• Expression of the two types of tumor necrosis factor (TNF) receptor, p55 and p75, in 12 human glioblastoma cell lines was studied. Reverse-transcription polymerase chain reaction detected messenger ribonucleic acid (mRNA) transcripts of p55 TNF receptor in all 12 cell lines tested, but p75 TNF receptor mRNA in only four cell lines. Flow cytometric analysis with anti-p55 and anti-p75 TNF receptor monoclonal antibodies demonstrated both p55 and p75 proteins in these four cell lines, but the level of expression of p75 molecule was very low. Correlation of p55 and p75 TNF receptor expression with TNF-induced growth suppression and production of bioactive molecules (interleukin-6, interleukin-8, manganase-superoxide dismutase, prostaglandin E2) showed that p55 TNF receptor mediates these TNF actions, but none of the responses were influenced by the presence of the p75 TNF receptor, which apparently has no specific role.

• Redl H, Schlag G, Adolf GR, Natmessnig B, Davies J
• Tumor necrosis factor (TNF)-dependent shedding of the p55 TNF receptor in a baboon model of bacteremia.
• Infect Immun. 1995; 63: 297-300
• Display abstract

• We have tested the hypothesis that the tumor necrosis factor (TNF) plays a significant role in vivo in TNF receptor shedding and studied the release of TNF-binding protein 1 (TNF-BP1), the soluble fragment of the 55- to 60-kDa TNF alpha (TNF) receptor, in a baboon model of Escherichia coli bacteremia, using three different doses of bacteria in acute infection (8 h) experiments (n [animals] = 11) and a single dose in a subchronic infection (72 h) experiment. In the subchronic infection study, one group of animals (n = 6) was pretreated with a neutralizing murine monoclonal antibody to TNF (CB0006). Concentrations of TNF and TNF-BP1 in plasma were determined in specific, monoclonal antibody-based immunoassays. Untreated animals (n = 6) showed undetectable TNF concentrations (< 10 pg/ml at baseline), whereas TNF-BP1 levels in plasma were in the range of 2 ng/ml, similar to concentrations observed in humans. Infusion of bacteria resulted in a rapid, dose-dependent increase in plasma TNF concentrations that reached a maximal level after 2 h and returned to baseline within 6 h. TNF-BP1 concentrations also showed a dose-dependent increase to peak concentrations three- to fivefold above baseline within 2 h but, in contrast to TNF levels, remained significantly elevated for up to 48 h. In animals pretreated with antibody CB0006, circulating TNF was completely neutralized, and TNF-BP1 was significantly reduced. We conclude that TNF-BP1 is released in bacteremia and that release in vivo is partially dependent on the presence of TNF.

• Chambaut-Guerin AM, Martinez MC, Hamimi C, Gauthereau X, Nunez J
• Tumor necrosis factor receptors in neuroblastoma SKNBE cells and their regulation by retinoic acid.
• J Neurochem. 1995; 65: 537-44
• Display abstract

• The human neuroblastoma cell line SKNBE can be differentiated either by serum removal or by adding to the culture medium different morphogens, for instance, retinoic acid (RA), cyclic AMP derivatives, and phorbol esters. Both the differentiated and undifferentiated cells express the two types of membrane tumor necrosis factor (TNF) receptors (TNFRs) of 55 and 75 kDa (p55 and p75 TNFR, respectively) and also their soluble forms. After RA addition the number of the surface TNFRs per cell is increased approximately twofold, but the kinetics of expression are different, depending on the receptor type. The level of the mRNAs of 2.4 and 4.2 kb, which, respectively, encode the p55 and p75 TNFRs, is also increased during the time course of differentiation, and the kinetics of their expression are biphasic. In contrast, the number of TNFRs and the level of their encoding mRNAs remain unchanged after exposure of the cells to both a phorbol and a cyclic AMP derivative.

• Morgavi P et al.
• Sensitization of human glioblastoma T98G cells to VP16 and VM26 by human tumor necrosis factor.
• Anticancer Res. 1995; 15: 1423-8
• Display abstract

• The effect of Tumor Necrosis Factor (TNF) on VP16 or VM26 cytotoxicity was studied in a human glioblastoma cell line T98G, which expresses TNF-receptors. Although T98G cells did not produce TNF endogenously they were resistant to the cytolytic effect of TNF. T98G cells were also moderately sensitive to the action of VP16 or VM26. TNF given at 1000 U/ml was able to increase the cell drug-sensitivity significantly. This effect was due to an increase in the VP 16-induced cleavable-complexes by TNF. These findings suggest that TNF specifically sensitizes human glioma T98G cells to the effects of VP 16 of VM26.

• Su X, Yu W, Wang S
• [Tumor necrosis factor and clinical diseases]
• Zhonghua Nei Ke Za Zhi. 1995; 34: 842-5

• Alzani R et al.
• Mechanism of suramin-induced deoligomerization of tumor necrosis factor alpha.
• Biochemistry. 1995; 34: 6344-50
• Display abstract

• Deoligomerization of human tumor necrosis factor alpha (TNF), spiked with 125I-labeled form, was studied quantitatively using size-exclusion chromatography and off-line monitoring with a gamma-counter. A detailed investigation of the oligomeric state of TNF was carried out as a function of its own concentration (0.3-7500 nM referred to the subunit, M(r) 17,000) in the absence or in the presence of various amounts (10, 100, 1000 microM) of suramin, an inhibitor of TNF biological activity in vitro, which promotes TNF deoligomerization. The dependence of trimeric form content on total TNF concentration was modeled with a sequential dissociation process (trimer-->dimer-->monomer) assuming an identical dissociation constant for each step, Kd1 = 0.2 nM. This model was used as the simplest for data fitting although, generally, no chromatographic resolution of dimeric species could be obtained. Best fitting of all data could be achieved with a model including a conformational change of TNF trimer into a state more prone to deoligomerization (Kd2 = 400 nM), which was favored by suramin binding. A kinetic study of TNF dissociation by the same method produced values for the deoligomerization rate of trimer: on the average, koff approximately 4 x 10(-5) S-1 (t1/2 approximately 5 h) between 4 and 20 degrees C with little dependence on suramin concentration; at 37 degrees C, a sizable increase is observed in the presence of 1 mM suramin (koff = 2.3 x 10(-4) S-1, t1/2 = 0.8 h). Data of suramin inhibition on TNF receptor binding, as obtained after incubation times much shorter than the above half-life of trimer, indicate that suramin binding to TNF trimer is the early mechanism of receptor binding inhibition.

• Terranova PF, Hunter VJ, Roby KF, Hunt JS
• Tumor necrosis factor-alpha in the female reproductive tract.
• Proc Soc Exp Biol Med. 1995; 209: 325-42
• Display abstract

• Tumor necrosis factor-alpha (TNF), originally identified as an inflammation-associated cytokine, is synthesized throughout the female reproductive tract as well as in placentas and embryos. Development, female sex steroid hormones, and lipopolysaccharide influence expression of this gene. The functions of TNF may be determined in part by differential expression of the two species of TNF receptors, both of which seem to be regulated by female sex steroid hormones. Evidence has accumulated that supports a role for this potent, pleiotropic cytokine in autocrine and paracrine processes central to reproduction, including gamete and follicle development, steroidogenesis, uterine cyclicity, placental differentiation, development of the embryo, and parturition.

• Kupferminc MJ et al.
• Soluble tumor necrosis factor receptors in maternal plasma and second-trimester amniotic fluid.
• Am J Obstet Gynecol. 1995; 173: 900-5
• Display abstract

• OBJECTIVES: We assessed maternal plasma and second-trimester amniotic fluid for levels of the p55 and p75 soluble tumor necrosis factor receptors. STUDY DESIGN: Blood was drawn from 61 healthy pregnant women (group A) before second-trimester genetic amniocentesis, and an aliquot of amniotic fluid was also obtained for this study. An additional blood sample was obtained from 13 of these patients at 36 to 40 weeks' gestation. Twenty-three healthy, nonpregnant women of reproductive age donated blood as a control group (group B). All plasma and amniotic fluid specimens were collectively assayed for the p55 and p75 soluble tumor necrosis factor receptors by specific enzyme-linked immunoassays. Additionally, tumor necrosis factor-alpha concentrations were measured in second-trimester plasma and amniotic fluid of 22 patients in group A and in all 23 of the nonpregnant women. RESULTS: The p55 and p75 soluble tumor necrosis factor receptors were detectable in all plasma samples from both groups of patients. The concentrations of both soluble receptors were significantly higher in second-trimester plasma compared with nonpregnant measurements (p < 0.01), and the plasma concentrations of both soluble receptors increased significantly from the second to third trimester (p < 0.01). The p55 and p75 soluble tumor necrosis factor receptors were also detectable in all amniotic fluid samples. Tumor necrosis factor-alpha was detected in the plasma of 15 of 22 patients in the second trimester but in none of the amniotic fluid samples and in none of the plasma samples from the nonpregnant cohort. CONCLUSIONS: Both the p55 and p75 soluble tumor necrosis factor receptors are physiologic constituents of second-trimester maternal plasma and amniotic fluid. Concentrations are elevated in pregnancy and further increase from the second to third trimester.

• Grell M
• Tumor necrosis factor (TNF) receptors in cellular signaling of soluble and membrane-expressed TNF.
• J Inflamm. 1995; 47: 8-17

• Vella AT, McCormack JE, Linsley PS, Kappler JW, Marrack P
• Lipopolysaccharide interferes with the induction of peripheral T cell death.
• Immunity. 1995; 2: 261-70
• Display abstract

• In mice injected with superantigens, T cells specific for that antigen proliferate and then die. It has been suggested that the target cells die because they encounter superantigen on the surfaces of nonprofessional presenting cells, such as B cells, which cannot deliver costimulatory signals to T cells. A number of reagents that induce costimulatory molecules on B cells were tested. Lipopolysaccharide very effectively prevented T cell death driven by superantigen. Perhaps surprisingly, the action of lipopolysaccharide was not mediated through the expected costimulatory molecule, B7. Rather, the effects of lipopolysaccharide involved the production of inflammatory cytokines, in particular TNF alpha. The rescued cells survived in vitro culture and were resistant to Fas-induced killing. These data demonstrate that LPS can block antigen-induced T cell death perhaps by interfering with Fas signaling.

• Vercammen D, Vandenabeele P, Declercq W, Van de Craen M, Grooten J, Fiers W
• Cytotoxicity in L929 murine fibrosarcoma cells after triggering of transfected human p75 tumour necrosis factor (TNF) receptor is mediated by endogenous murine TNF.
• Cytokine. 1995; 7: 463-70
• Display abstract

• We compared the biological function of the human tumor necrosis factor receptors p55 (hTNF-R55) and p75 (hTNF-R75) expressed in the murine (m) fibrosarcoma cell line L929. Receptor-specific triggering of hTNF-R55 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-R32WS86T, a hTNF-R55-specific mutant of hTNF, resulted in cytotoxicity. Specific clustering of hTNF-R75 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-D143F, a hTNF-R75-specific mutant of hTNF also induced cytotoxicity, albeit at low level. In both cases, the cytotoxic activity of receptor clustering could be synergized by addition of 20 mM LiCl. Remarkably, cytotoxicity induced after R75 triggering in transfected L929 cells could be completely abolished by addition of neutralizing anti-mTNF antibodies, in contrast to cell killing seen after specific R55 clustering. No soluble mTNF could be demonstrated using a sensitive biological assay, although L929 cells were expressing low levels of mTNF-specific mRNA as shown by PCR. These data clearly demonstrate that minute amounts of endogenously produced TNF can be a key mediator in R75-mediated cytotoxicity. Presumably, the latter efficiently traps the ligand and transfers it to TNF-R55, and/or by binding it, protects the endogenously made TNF from inactivation.

• Naismith JH, Devine TQ, Brandhuber BJ, Sprang SR
• Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor.
• J Biol Chem. 1995; 270: 13303-7
• Display abstract

• Activation of the cell surface receptors for tumor necrosis factor (TNF) is effected by the aggregation of cytoplasmic domains that occurs when the extracellular domains of two or three receptors bind to trimeric TNF alpha or TNF beta. The structure of the type I TNF receptor extracellular domain (sTNF-R1), crystallized in the absence of TNF, has now been determined at 2.25-A resolution. The receptor itself is an elongated molecule comprising four disulfide-rich domains in a nearly linear array. Contrary to expectations, the unliganded domains are found to associate into dimers of two distinct types, in which monomers are related by local two-fold axes of symmetry. In one case, the receptors are antiparallel to each other and associate through an interface that overlaps the TNF binding site. If intact receptors were capable of such an association, their cytoplasmic domains would be separated by over 100 A. This interaction could inhibit signaling in the absence of TNF. Parallel dimers are also observed in which the dimer interface is well separated from the TNF binding site. Associations among TNF-bound parallel dimers could cause receptor clustering. Both dimers bury substantial areas of protein surface and are formed by polar and non-polar interactions.

• Selmaj KW, Raine CS
• Experimental autoimmune encephalomyelitis: immunotherapy with anti-tumor necrosis factor antibodies and soluble tumor necrosis factor receptors.
• Neurology. 1995; 45: 449-449
• Display abstract

• Multiple sclerosis (MS) is a demyelinating disease in which an inflammatory cell infiltrate represents a characteristic pathologic feature of active lesions within the CNS. The possibility has been raised that cell-mediated immune mechanisms orchestrate the pathogenesis of MS. Cytokines play a particularly important role in cellular immune mechanisms. These soluble glycoproteins, nonimmunoglobulin in nature, act nonenzymatically to regulate immune cell function. A unique family of cytokines, the tumor necrosis factors (TNFs), demonstrate immunoregulatory activity but are also involved in the effector arm of cellular immune responses. Recently, studies both in vitro and in vivo have suggested a role for TNFs in the pathology of MS. This report summarizes data implicating TNFs in the mechanisms of MS and attempts to apply the anti-TNF approach in the future therapeutic strategy for this disease.

• Conroy DM, Francischi JN, Sirois P
• Effect of tumor necrosis factor receptor binding protein on cell infiltration induced by lipopolysaccharide and Sephadex beads in guinea pig lung.
• Inflammation. 1995; 19: 233-43
• Display abstract

• In the present study, the effect of a tumor necrosis factor receptor binding protein (TNFbp) on the cell infiltration induced by lipopolysaccharide (LPS) and Sephadex beads in guinea pig lung was examined. The intratracheal injection of LPS (2.5 micrograms) induced a six-fold increase in total cell number recovered in bronchoalveolar lavage (BAL) fluid at 24 hr. This increase in bronchopulmonary inflammation was mainly due to a neutrophil and macrophage infiltration, representing 60% and 35% of the total cells, respectively. The intravenous or intratracheal injection of Sephadex beads to guinea pigs induced a three-fold increase in total cell number recovered in BAL at 24 h and was characterized by a prominent eosinophil, macrophage, and neutrophil infiltration representing 36%, 42%, and 16% of the total cells, respectively. In addition, bronchial tissues isolated from Sephadex-treated guinea pigs showed an increased in vitro reactivity to both histamine and acetylcholine. TNFbp (1-50 micrograms) induced a dose-dependent inhibition of cell infiltration induced by LPS. In contrast TNFbp neither attenuated the bronchopulmonary cell infiltration observed 24 h following intravenous or intratracheal administration of Sephadex beads nor inhibited the increase in bronchial reactivity. These results show that TNF plays an important role in cell infiltration induced by LPS, but not that induced by Sephadex, in the guinea pig lung.

• Beutler B
• TNF, immunity and inflammatory disease: lessons of the past decade.
• J Investig Med. 1995; 43: 227-35

• Smart T
• Inhibiting TNF to preserve lean body mass.
• GMHC Treat Issues. 1995; 9: 3-3

• Clark IA, Rockett KA
• TNF, malaria, and sepsis.
• Lancet. 1995; 345: 929-929

• Meekins JW
• Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia.
• Br J Obstet Gynaecol. 1995; 102: 842-3

• St Pierre BA, Granger DA, Wong JL, Merrill JE
• A study on tumor necrosis factor, tumor necrosis factor receptors, and nitric oxide in human fetal glial cultures.
• Adv Pharmacol. 1995; 34: 415-38

• Trentin L et al.
• Tumour-infiltrating lymphocytes bear the 75 kDa tumour necrosis factor receptor.
• Br J Cancer. 1995; 71: 240-5
• Display abstract

• Tumour necrosis factor alpha (TNF-alpha) is a cytokine with a variety of immunological properties. The identification of two receptors for this molecule, i.e. the 75 kDa and the 55 kDa TNF receptors (TNF-R), recently clarified the mechanisms through which this cytokine provides its wide range of immunomodulatory activities. In this study we have investigated the expression and the functional properties of these receptors on tumour-infiltrating lymphocytes (TILs) recovered from 17 patients with solid cancers (melanoma, colorectal carcinoma and lung cancer). To this end, TIL lines and freshly isolated TILs were evaluated for (a) the expression and the functional role of TNF receptors following culture in the presence of interleukin 2 (IL-2) and (b) the production of TNF-alpha following culture with IL-2 and the role of this cytokine in IL-2-driven TIL proliferation. Flow cytometry analysis demonstrated that TILs bear the 75 kDa TNF-R. Moreover, TIL lines express detectable messages for TNF-alpha and release this cytokine. Functional in vitro studies have shown that anti-TNF-alpha, as well as anti-75 kDa TNF-R antibodies, are able to inhibit the IL-2-induced TIL proliferation. These data demonstrate that TILs are equipped with a fully functional TNF-R system and suggest a putative role for this receptor and its ligand in the activation and expression of TILs following immunotherapy with IL-2.

• Boiko VN, Shadrin OV, Derevianchenko IG, Buntsevich AM, Bykovskaia SN
• [Radioprotective effect of a complex preparation of lymphokinin cytokines]
• Biull Eksp Biol Med. 1994; 118: 38-40

• Koshita Y et al.
• Mechanisms of tumor regression of TNF gene-transduced Meth-A cells transplanted in mice.
• Ann N Y Acad Sci. 1994; 716: 334-5

• Brouckaert P et al.
• Receptor-selective mutants of tumour necrosis factor in the therapy of cancer: preclinical studies.
• Circ Shock. 1994; 43: 185-90
• Display abstract

• The use of TNF-mutants that are selective agonists of the TNF-R55 is one strategy that is being explored to broaden the therapeutic margin of TNF. Several problems still have to be overcome before they can be used in clinical trials. Regarding the sensitizing effect of some infections and some tumours, we identified IFN-gamma as a mediator in BCG- but not in tumour-induced sensitization. In both models, the vessel wall is most probably the key tissue as alpha-LFA-1 antibodies could protect against lethality. Studies in primates showed that an unexpected feature, namely, the longer half-life of such mutants, might interfere with this strategy. Recent observations also indicate that the mechanism of tolerance-induction, another way to separate antitumour and toxic effects of TNF, might reside in the functional ablation of the TNF-R75. Using IL-60/0 knockout mice, we could not find any causal role for IL-6 in TNF-mediated lethality, this in contrast to results obtained previously with neutralizing antibodies. Finally, we identified the acute phase protein alpha 1-acid glycoprotein as a protein with protective properties towards TNF-induced lethality and liver damage.

• Yamazaki M
• [TNF-alpha]
• Gan To Kagaku Ryoho. 1994; 21: 2679-87
• Display abstract

• TNF-alpha, a cytokine with tumor necrosis activity produced by many types of cells including macrophages, was recently recognized as an important host defense factor that affects not only malignant cells, but many kinds of normal cells. Clinical applications of TNF-alpha are now limited, but local injection or local production of TNF-alpha should be given closer consideration in antitumor trials. Here we described the TNF receptor family and TNF-alpha-driven inflammation, and also discussed the physiological roles of TNF-alpha.

• Wada HG, Fok KS, Fendly BM, Chiang NY, Sussman HH
• Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells.
• J Cell Physiol. 1994; 161: 597-605
• Display abstract

• The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-alpha has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling.

• Totpal K, LaPushin R, Kohno T, Darnay BG, Aggarwal BB
• TNF and its receptor antibody agonist differ in mediation of cellular responses.
• J Immunol. 1994; 153: 2248-57
• Display abstract

• TNF binds to two distinct receptors designated p60 and p80. Because Abs to the p60 receptor (anti-p60) can mimic TNF, we therefore compared the cellular signaling of TNF with that of anti-p60. We demonstrate both qualitative and quantitative differences between TNF and anti-p60. HepG2 cells, which express the p60 receptor, were found to be completely resistant to TNF but highly sensitive to the antiproliferative effects of anti-p60. In contrast, normal fibroblasts were found to be several fold more sensitive to TNF than to anti-p60. Several other epithelial cell lines that also express primarily the p60 receptor showed quantitative differences in mediation of cellular responses by TNF and anti-p60. The blocking of the p60 receptor by TNF had no effect on the response of HepG2 cells to anti-p60, suggesting a difference in their binding sites. Anti-p60, however, inhibited the effect of TNF on fibroblasts. Ab against the p80 receptor had no effect by itself or on the effect of TNF and anti-p60. The difference in the response to TNF and anti-p60 could not be correlated to the differences in the level of expression of p60 receptor on these cells. Furthermore, cycloheximide potentiated the TNF-mediated effect but not that mediated through anti-p60, thus also indicating a difference in the mechanism of action of these two agents. Overall, these results demonstrate that TNF and anti-p60, although both working through the p60 receptor, differ in their cellular signaling.

• Giroir BP, Horton JW, White DJ, McIntyre KL, Lin CQ
• Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock.
• Am J Physiol. 1994; 267: 11824-11824
• Display abstract

• Tumor necrosis factor-alpha (TNF) is a pluripotent cytokine that mediates many of the hemodynamic manifestations of endotoxic shock. To determine whether TNF is responsible for postburn myocardial dysfunction, we compared cardiac function (Langendorff preparation) in 49 guinea pigs 18 h after thermal injury. Group 1 (n = 15) was sham burned; all remaining animals received a 43% surface area burn under anesthesia. Group 2 (n = 15) received lactated Ringer solution (LR, 4 ml.kg-1.%burn-1). Group 3 (n = 9) received LR and drug vehicle. Group 4 (n = 10) received LR plus 1 mg of TNF inhibitor consisting of the human p80 TNF receptor linked to the Fc portion of human immunoglobulin G1, which was shown to specifically bind and neutralize TNF secreted by guinea pig peritoneal macrophages in vitro. Burn injury caused a significant fall in left ventricular pressure (LVP, from 86 +/- 2 to 62 +/- 3 mmHg, P < 0.05) and maximal rate of LVP rise (+) and fall (-) (+/- dP/dtmax) [from 1,365 +/- 42 to 1,109 +/- 44 mmHg/s (P < 0.05) and from 1,184 +/- 31 to 881 +/- 40 mmHg/s (P < 0.05), respectively], a decrease in time to peak systolic LVP (from 111 +/- 2 to 102 +/- 2 ms, P < 0.05), and a decrease in time to +dP/dtmax (from 57 +/- 1 to 48 +/- 1 ms, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

• Saito T, Kato H, Saito M, Karaki Y, Tazawa K, Fujimaki M
• TNF receptor number-dependent cytotoxicity to TNF-resistant human esophageal cancer cell lines by combination with recombinant human necrosis factor and hyperthermia.
• Hum Cell. 1994; 7: 55-61
• Display abstract

• The synergistic effect of recombinant human tumor necrosis factor (rh-TNF) and hyperthermia on five established cell lines of human esophageal cancer (SGF series) was analyzed by in vitro assays. The SGF cell lines were either resistant or slightly sensitive to rh-TNF. However, they became highly sensitive to rh-TNF even at as low a concentration as 10 U/ml and dose-dependently when combined with hyperthermia (43.5 degrees C, 60 min). This result was due to synergistic effects of rh-TNF and hyperthermia, and the degree of the effect increased with the hyperthermic temperature and TNF concentration. The number of TNF receptors per cell varied widely from cell line to cell line, from 4,400 to 23,100, which did not correlate to the extent of cytotoxicity by rh-TNF alone. The degree of synergistic effect of rh-TNF and hyperthermia was evaluated quantitatively in terms of a Synergistic Index (S. I. = Predicted cell viability/Experimental cell viability): A significant correlation was found between the logarithmic S. I. and the number of TNF receptors. These findings indicated that the combination of rh-TNF and hyperthermia produced a significant antitumor effect even on the cell lines poorly sensitive to TNF in a receptor-dependent manner, although the cellular sensitivity to TNF alone was not directly correlated to the number of TNF receptors.

• Grazioli L, Casero D, Restivo A, Cozzi E, Marcucci F
• Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates.
• J Biol Chem. 1994; 269: 22304-9
• Display abstract

• We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been partially purified by gel filtration chromatography yielded two prominent bands of M(r) 60,000 and 75,000 corresponding to the two known TNFR (types I and II, respectively). In addition to these, types I and II TNFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting that TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that had been partially purified by gel filtration chromatography and incubated with TNF before electrophoretic separation. Indeed, type II TNFR aggregates, but not type I TNFR aggregates, were generated at optimal TNF concentrations. Formation of type II TNFR aggregates in this last experimental setting and of both type I and type II TNFR aggregates during affinity purification could be prevented if an alkylating agent (N-ethylmaleimide) was added during the TNFR-TNF incubation step. Similar results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggregates were generated at TNF concentrations ranging from 10(-9) to 10(-10) M and their formation was prevented in the presence of N-ethylmaleimide.

• Blankenstein T
• Observations with tumour necrosis factor gene-transfected tumours.
• Folia Biol (Praha). 1994; 40: 19-28
• Display abstract

• Tumour necrosis factor (TNF) produced by genetically engineered tumour cells can lead to very effective tumour rejection. Tumour suppression does not result from a direct effect of TNF on tumour cells but rather is mediated by the induction of an anti-tumour immune response. It requires a local and continuous presence of TNF at the tumour site. Tumour rejection induced by TNF is dose-dependent and even tumour cell-derived TNF in amounts which cause complete tumour eradication must not be accompanied by toxic side effects. A complex pattern of tumour infiltrating cells has been observed in TNF-producing tumours consisting of macrophages, CD4+ and CD8+ T cells. For tumour suppression macrophages and CD8+ T cells are needed whereas CD4+ T cells seem to reflect innocent bystander cells. Consistently, TNF is active in T cell deficient mice but in most cases T cells are needed for complete tumour elimination. TNF was active in a number of different tumour models but recent experiments also showed that local TNF failed to induce an anti-tumour response in certain tumour models. Moreover, either depending on the tumour cell line used or on the level of TNF secreted by the tumour, systemic toxicity has been observed, leading to cachexia or wasting of the mice. In one case it has been shown that a TNF gene-transfected tumour showed no tumour growth inhibition in vivo but that TNF augmented metastasis of these cells. Experiments conducted to demonstrate systemic protective immunity by TNF-producing tumours used as a vaccine have not yet been successful.

• Sleijfer S, Mulder NH
• Tumour necrosis factor: the decline and fall of a biological agent and its resurrection.
• Clin Oncol (R Coll Radiol). 1994; 6: 127-32

• Hallahan DE, Virudachalam S, Kufe DW, Weichselbaum RR
• Ketoconazole attenuates radiation-induction of tumor necrosis factor.
• Int J Radiat Oncol Biol Phys. 1994; 29: 777-80
• Display abstract

• PURPOSE: Previous work has demonstrated that inhibitors of phospholipase A2 attenuate ionizing radiation induced arachidonic acid production, protein kinase C activation and prevent subsequent induction of the tumor necrosis factor gene. Because arachidonic acid contributes to radiation-induced tumor necrosis factor expression, we analyzed the effects of agents which alter arachidonate metabolism on the regulation of this gene. METHODS AND MATERIALS: Phospholipase A2 inhibitors quinicrine, bromphenyl bromide, and pentoxyfylline or the inhibitor of lipoxygenase (ketoconazole) or the inhibitor of cyclooxygenase (indomethacin) were added to cell culture 1 h prior to irradiation. RESULTS: Radiation-induced tumor necrosis factor gene expression was attenuated by each of the phospholipase A2 inhibitors (quinicrine, bromphenyl bromide, and pentoxyfylline). Furthermore, ketoconazole attenuated X ray induced tumor necrosis factor gene expression. Conversely, indomethacin enhanced tumor necrosis factor expression following irradiation. CONCLUSION: The finding that radiation-induced tumor necrosis factor gene expression was attenuated by ketoconazole suggests that the lipoxygenase pathway participates in signal transduction preceding tumor necrosis factor induction. Enhancement of tumor necrosis factor expression by indomethacin following irradiation suggests that prostaglandins produced by cyclooxygenase act as negative regulators of tumor necrosis factor expression. Inhibitors of tumor necrosis factor induction ameliorate acute and subacute sequelae of radiotherapy. We propose therefore, that ketoconazole may reduce acute radiation sequelae such as mucositis and esophagitis through a reduction in tumor necrosis factor induction or inhibition of phospholipase A2 in addition to its antifungal activity.

• Erickson SL et al.
• Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice.
• Nature. 1994; 372: 560-3
• Display abstract

• Tumour necrosis factor (TNF) elicits multiple biological effects through two distinct cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75). Most TNF-mediated biological responses, such as cell death, gene induction, antiviral activity and cytokine production, have been attributed to TNF-R1 (refs 1-5). Gene targeting of this receptor confirms its role in the lethality attributable to low doses of lipopolysaccharide after sensitization with D-galactosamine; surprisingly, the toxicity of high doses of lipopolysaccharide was unaffected. The function of TNF-R2 is less well understood, although there are data supporting a role in T-cell development and the proliferation of cytotoxic T lymphocytes. To clarify the physiological role of TNF-R2, we have generated mice deficient in this receptor by gene targeting. The TNF-R2-/- mice show normal T-cell development and activity, but we find that they have increased resistance to TNF-induced death. Additionally, such mice injected subcutaneously with TNF show a dramatic decrease in tissue necrosis, indicating that this receptor plays a role in the necrotic effects of TNF.

• Bebo BF Jr, Linthicum DS
• Nucleotide sequence of the TNF type I receptor from a mouse endothelioma cell line.
• Immunogenetics. 1994; 39: 450-1

• Isobe K, Fan ZH, Emi N, Nakashima I
• Gene transfer of TNF receptor for treatment of cancer by TNF.
• Biochem Biophys Res Commun. 1994; 202: 1538-42
• Display abstract

• Tumor necrosis factor-alpha receptors (TNFR-55 and TNFR-75) were inserted into retrovirus derived vector. They were transfected into a packaging cell line. The high titer of transfectants, which produced virus containing TNFR-55 or TNFR-75, was obtained. TNFR-55 and TNFR-75 negative human colon cancer cells were infected with this virus as a model experiment of gene therapy. Although the original colon cancer cell line did not express TNFR-55 or TNFR-75, the colon cancer cells, which were infected by recombinant virus, expressed a high level of TNFR-55 or TNFR-75. TNFR-55 or TNFR-75 transformed colon cancer cells were killed by recombinant TNF.

• Lidbury BA, Rathjen DA, Ramshaw IA, Cowden WB
• The effect of enhancing antibodies on TNF interactions with its specific receptor: consequences for in vitro TNF antiviral activity.
• Biotechnol Ther. 1994; 5: 27-45
• Display abstract

• This study examines the interaction of TNF with its receptor(s) in the presence of antibodies which have been previously found to enhance the in vivo antiviral and antitumor activities of this cytokine. The presence of Mab 32 has been previously shown to enhance the binding of 125I-TNF to the surface L929 cells (13), and this property was also found in the present study for HeLa cells. In addition to this, Mab 32 was found to enhance the internalization of the TNF ligand into both L929 and HeLa cells compared to control treated cultures. The consequences of such enhanced TNF-binding on TNF antiviral activity were examined in both L929 cells and HeLa cells. It was discovered that the similarities in antibody-enhanced TNF binding and internalization contrasted dramatically with the sensitivities of these two cell lines to the antiviral actions of TNF, both with and without Mab 32 (viz., L929 cells were sensitive; HeLa cells were resistant). It has been proposed that the modulation of TNF-R expression, particularly by IFNs, is an important factor in TNF's biological effects. It has been shown that the presence of IFN-gamma, with TNF plus specific enhancing antibodies, further augmented antiviral activity in vivo (13). This finding stimulated interest in examining IFN-gamma modulation of TNF-R as a factor in the antiviral activity of TNF. The expression of TNF receptor(s) in TNF- and/or IFN-gamma-exposed cells, both with and without HSV-1 infection, was therefore examined. TNF alone could induce a dose-dependent increase in receptor expression which was not significantly increased by Mab 32. Exposure of L929 cells to IFN-gamma alone also induced TNF receptor expression in mock-infected cells. HSV-1 infection of L929 cells resulted in a significant upregulation of TNF-R expression which was reversed if the cells had been preexposed to IFN-gamma. The inclusion of TNF with IFN-gamma before infection restored TNF-R expression but did not show any further synergistic or additive effects on TNF-R expression. Some minor increases in TNF-R expression were seen if Mab 32 was included with these two cytokines.

• Bass DA, Seeds MC, Jones DF, Chilton FH, Bauldry SA
• Priming of phospholipases A2 of human neutrophils by tumor necrosis factor.
• Chest. 1994; 105: 99100-99100

• Newton CR et al.
• Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.
• Protein Expr Purif. 1994; 5: 449-57
• Display abstract

• We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.

• Vandenabeele P et al.
• Functional requirement of the two TNF receptors for induction of apoptosis in PC60 cells and the role of mitochondria in TNF-induced cytotoxicity.
• Circ Shock. 1994; 44: 196-200
• Display abstract

• The rat/mouse T-cell hybridoma PC60 was transfected either with hTNF-R55 cDNA, hTNF-R75 cDNA, or both. Receptor-specific stimulation was achieved using agonistic monoclonal antibodies or receptor-specific muteins of hTNF. Either hTNF-R55 or hTNF-R75 could mediate the activation of NF-kappa B and the induction of GM-CSF, IL-6, and IFN-gamma. But only in cells carrying both hTNF-R55 and hTNF-R75, was TNF able to induce apoptosis. This apoptosis could be inhibited almost completely by cotransfection with human bcl-2 cDNA. Functional cooperation was observed between liganded and unliganded receptors for the induction of apoptosis. In vitro protein kinase activity was detected only in TNF-R75 immunoprecipitates from cells in which the receptor was signaling. Direct evidence was obtained for reactive oxygen intermediates of mitochondrial origin responsible for TNF-induced cytotoxicity in L929 cells.

• Darnay BG, Reddy SA, Aggarwal BB
• Identification of a protein kinase associated with the cytoplasmic domain of the p60 tumor necrosis factor receptor.
• J Biol Chem. 1994; 269: 20299-304
• Display abstract

• Tumor necrosis factor (TNF) has been shown to bind two distinct receptors, designated p60 and p80, with high affinity, resulting, within minutes, in phosphorylation of several proteins. The receptors themselves do not exhibit protein kinase activity nor have any associated proteins been identified. We employed the glutathione-S-transferase (GST) fusion protein system consisting of the cytoplasmic domain of p60 (GST-p60CD delta 1) as a probe to help us identify receptor-associated proteins from human histiocytic lymphoma U-937 cells. We found that a protein of approximately 52 kDa (pp52) bound to GST-p60CD delta 1 from [35S]methionine- and 32P-labeled cells. The associated protein was phosphorylated on serine and threonine residues. Furthermore, we identified serine/threonine kinase activity associated with p60CD delta 1 that required either Mn2+ or Mg2+ for optimal activity. The preferred substrates for this kinase, in addition to p60CD delta 1, included casein and histone H1, but not histone H2B, myelin basic protein, enolase, or the cytoplasmic domain of p80. As was the case in U-937 cells, p60CD delta 1-associated kinase activity was also identified in human breast adenocarcinoma MCF-7 cells and human foreskin fibroblasts. TNF stimulation of MCF-7 and foreskin fibroblasts for 5-15 min induced approximately 50 and 240% increases in phosphorylation of p60CD delta 1, respectively. Thus, our results provide the first evidence for protein kinase activity that is specifically associated with the cytoplasmic domain of the p60 form of the TNF receptor and causes its phosphorylation. This p60 TNF receptor-associated protein and the associated kinase described here are referred to as p60-TRAP and p60-TRAK, respectively.

• Wong GH, Goeddel DV
• One-day northern blotting for detection of mRNA: NDGA inhibits the induction of MnSOD mRNA by agonists of type 1 TNF receptor.
• Methods Enzymol. 1994; 234: 244-52

• Wells JA
• Structural and functional basis for hormone binding and receptor oligomerization.
• Curr Opin Cell Biol. 1994; 6: 163-73
• Display abstract

• Most single-pass transmembrane receptors undergo a change in oligomeric state upon hormone binding. Recent mutational, biophysical and structural studies of the human growth hormone and tumor necrosis factor receptor complexes have revealed much about the mechanisms and molecular bases for binding and oligomerization. Principles learned from these examples and others should apply to many other hormone-receptor complexes.

• Zhang L, Aggarwal BB
• Role of sulfhydryl groups in induction of cell surface down-modulation and shedding of extracellular domain of human TNF receptors in human histiocytic lymphoma U937 cells.
• J Immunol. 1994; 153: 3745-54
• Display abstract

• The interaction of different polypeptide growth factors with their cell surface receptors, which are typically rich in cysteine, is modulated by sulfhydryl (SH) reagents. Because the extracellular domain of both human TNFRs are likewise rich in cysteine residues, we examined the effect of SH reagents on these receptors in human histiocytic lymphoma U937 cells, which express both p60 and p80 forms. Iodoacetamide induced down-regulation of cell surface TNFRs in a dose- and time-dependent manner. Down-regulation was complete at 10 mM IAA for 1 h at 37 degrees C. The decrease was minimal at 4 degrees C. The down-modulation was also observed with other cell-permeable and -impermeable thiol reagents. The expression of other cell surface molecules such as CD3, CD11b, CD23, and CD25 were not affected. IAA induced the down-regulation of TNFR on cells of both epithelial and myeloid origin. With the use of receptor-specific Abs, we found that the kinetics of down-modulation of the p60 and p80 receptors by IAA were very similar. We also found that down-modulation was not a result of internalization but rather of the shedding of the receptors, as ascertained by receptor-ligand cross-linking analysis, immunoassay, Western blot analysis, and ligand-blot analysis. When TNFRs with a molecular mass of 80 kDa disappeared from the cell surface, a 42-kDa polypeptide appeared in the medium. Thus, we demonstrate that SH reagents down-regulate TNFRs by inducing shedding of both receptors from the cell surface, most likely by activating an endopeptidase that cleaves the receptor.

• Toyoshima M et al.
• [Arterial infusion chemotherapy with human tumor necrosis factor (TNF): an experimental study using rabbits bearing VX2 tumor]
• Nippon Igaku Hoshasen Gakkai Zasshi. 1994; 54: 1041-3
• Display abstract

• To evaluate the direct effects of TNF on tumor vessels, three groups of Japanese white rabbits bearing VX2 tumor received 5000 U, 25000, U, and 5000 U of TNF respectively. Angiograms were obtained before, and at 1, 15 and 30 minutes after arterial TNF injection. At 1 minute after TNF injection, dilated tumor vessels and dense tumor stains, depending on the dose of TNF, were demonstrated. At 30 minutes after injection these findings had disappeared However, there were no changes in normal vessels. It is suggested that arterial TNF injection influenced tumor vessels without damaging on normal vessels, indicating that it is effective as cancer therapy.

• Lynch DH et al.
• The mouse Fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster.
• Immunity. 1994; 1: 131-6
• Display abstract

• The gene for the mouse Fas ligand was cloned and its chromosomal position determined. Fasl was tightly linked to gld (no crossovers in 567 meiotic events) on mouse chromosome 1 and closely linked with a novel member of the same TNF family of ligands, the Ox40 ligand (Ox40l, 1 crossover in 567 meiotic events). Southern blot analysis did not reveal any difference between the Fasl gene from gld and wild-type mice and levels of Fasl mRNA transcripts were similar in PMA and ionomycin induced wild-type and coisogenic gld T cells. Sequence analysis of the gld gene indicated a single amino acid change (Phe Leu) in the COOH terminal portion of this type II transmembrane protein, and COS cells transfected with Fasl cDNA from gld mice failed to induce apoptosis of Fas-expressing target cells. Thus, the data demonstrate that the gld phenotype is the result of a point mutation in the Fasl gene and that Fasl is part of a complex of ligands structurally related to TNF mapping within a small region of mouse chromosome 1.

• Takeda K, Iwamoto S, Takeda M
• Lymphotoxin and TNF differ greatly in capacity to induce differentiation of human myeloblastic leukemia ML-1 cells.
• Biochem Mol Biol Int. 1994; 32: 1109-19
• Display abstract

• We found that recombinant and natural lymphotoxin differ from TNF in that they only marginally induce differentiation of human myeloblastic leukemia ML-1 cells. Even at 1,000-fold concentrations, lymphotoxin was significantly less potent than TNF. Lymphotoxin competitively, but not completely, inhibited TNF-induced differentiation. Based on studies of the two types of TNF receptors (55 kDa and 75 kDa) using monoclonal antibodies, the binding activity of lymphotoxin to the receptors was less than 1/3 that of TNF, but lymphotoxin could bind to both TNF receptors. Both receptors were involved in induction of differentiation by TNF. However, lymphotoxin, differed from TNF; it seemed to be weak in sending signals for differentiation through the 75 kDa receptor.

• Boka G, Anglade P, Wallach D, Javoy-Agid F, Agid Y, Hirsch EC
• Immunocytochemical analysis of tumor necrosis factor and its receptors in Parkinson's disease.
• Neurosci Lett. 1994; 172: 151-4
• Display abstract

• Activated glial cells observed in the substantia nigra in Parkinson's disease may participate in the mechanism of nerve cell death by providing toxic substances such as cytokines. Among these compounds, tumor necrosis factor-alpha (TNF) is of interest because it can provoke cell death. We detected TNF-immunoreactive glial cells in the substantia nigra of parkinsonian patients but not in those of control subjects. Immunoreactivity for TNF receptors was found in cell bodies and processes of most dopaminergic neurons of control and parkinsonian subjects, suggesting that nigral dopaminergic neurons might be sensitive to TNF produced in Parkinson's disease. These results suggest that TNF may participate in the degenerative processes occurring in Parkinson's disease, at least after a primary insult inducing a reactive gliosis.

• Brakebusch C, Varfolomeev EE, Batkin M, Wallach D
• Structural requirements for inducible shedding of the p55 tumor necrosis factor receptor.
• J Biol Chem. 1994; 269: 32488-96
• Display abstract

• Induced shedding of the p55 tumor necrosis factor receptor (p55-R) was previously shown to be independent of the amino acid sequence properties of the intracellular domain of this receptor. We now find it also independent of the sequence properties of the transmembrane domain and of the cysteine-rich region that constitutes most of the extracellular domain of the receptor. The shedding is shown to depend solely on the sequence properties of a small region within the spacer that links the cysteine-rich region in the extracellular domain to the transmembrane domain. Detailed tests of effects of mutations in the spacer on the shedding indicate that the process is independent of the amino acid side-chain identity in this region except for a limited dependence on the identity of 1 residue (Val-173), located downstream to the putative major cleavage site of the receptor. It is strongly affected, however, by some mutations that seem to change the conformation of the spacer region. These findings suggest that a short amino acid sequence in the p55-R is essential and sufficient for its shedding and that the shedding is mediated either by a protease with limited sequence specificity or by several different proteases that recognize different amino acid sequences, yet it strictly depends on some conformational features of the cleavage region in the receptor.

• Abe Y, Kimura K, Horiuchi A, Watanabe Y, Kimura S
• The elevation of plasma soluble tumor necrosis factor receptor levels by TNF induction therapy for patients with malignancy.
• Surg Today. 1994; 24: 506-11
• Display abstract

• Soluble tumor necrosis factor receptor (sTNF-R) is known to inhibit patient immunity via specific binding with the TNF molecule. To examine the possible involvement of sTNF-R in cancer immunotherapy, the plasma levels of sTNF-R of both 55 kDa and 75 kDa origins were estimated when TNF was induced in patients with malignancy using both a polysaccharide preparation (Lentinan) and a streptococcal preparation (OK-432). The pretreatment plasma levels of the 55 kDa and 75 kDa sTNF-R were 1.04 +/- 0.53 and 1.06 +/- 0.34 ng/ml (mean +/- SE), respectively. The plasma levels of TNF were undetectable before treatment. The plasma sTNF-R levels peaked 2 h after the administration of OK-432 and followed the same pattern as the TNF levels in plasma. Both TNF and sTNF-R nearly returned to pretreatment levels at 16 h after the induction of TNF. The peak plasma levels of the 55 kDa and 75 kDa sTNF-R were 2.46 +/- 0.95 and 3.03 +/- 0.88 ng/ml, respectively, but they did not correlate with the plasma TNF levels. When peripheral white blood cells were cultured with the addition of lipopolysaccharide in vitro, an elevation of the 72 kDa sTNF-R was detected. Thus, the plasma source of this soluble receptor can at least be partly attributed to the white blood cells. However, the 55 kDa sTNF-R showed little increase in the cultures, and its source remains unknown. We should therefore be aware of the elevation of plasma sTNF-R levels by the induction therapy of TNF for patients with malignancies because of the immunosuppressive effect of sTNF-R.

• Butler DM et al.
• TNF receptor fusion proteins are effective inhibitors of TNF-mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells.
• Cytokine. 1994; 6: 616-23
• Display abstract

• KYM-1D4 cells are a subline derived from a human rhabdomyosarcoma which are highly sensitive to TNF-mediated cytotoxicity. They were selected for this study because they express human TNF-R and are therefore a more relevant target for comparing the potential therapeutic value of human TNF-inhibitory agents than the usual murine cell lines. Two recombinant soluble TNF-R-IgG fusion proteins, one containing p55 TNR-R, the other containing p75 TNF-R, and a recombinant monomeric soluble p55 TNF-R were all found to block the cytotoxicity generated by human TNF-alpha and LT as well as also murine TNF. The p55 TNF-R-IgG fusion protein (p55-sf2) was the most effective of the antagonists tested, requiring an equimolar, (based on a monomeric configuration of TNF-alpha) or a 3-fold higher (based on a trimeric configuration of TNF-alpha) molar concentration to inhibit the cytotoxicity mediated TNF-alpha by 50%. p55-sf2 was also as effective at inhibiting the cytotoxicity mediated by LT or murine TNF in the KYM-1D4 assay. In contrast, the monomeric soluble p55 TNF-R was the least effective inhibitor, requiring a > 4000-fold higher molar concentration than p55-sf2 to achieve a similar degree of protection. The fusion proteins, particularly p55-sf2, may be useful as human therapeutic agents, as at low concentrations they can prevent both TNF-alpha-mediated and LT-mediated effects on human cells. As TNF-R-IgG fusion proteins also block the action of murine TNF in vitro, they may also be useful in the investigation of murine models of human inflammatory disease.

• Bemelmans MH, Gouma DJ, Buurman WA
• Tissue distribution and clearance of soluble murine TNF receptors in mice.
• Cytokine. 1994; 6: 608-15
• Display abstract

• In this study, clearance of sTNFR was investigated. The data show that bilateral nephrectomy results in an increase of the levels of both sTNFR after which a new steady state situation develops, suggesting that other organs, apart from the kidneys, are involved in clearing of sTNFR. Bilateral nephrectomy also leads to an increase in circulating TNF. This TNF was detected by ELISA and appeared to be not biologically active. To investigate whether the endotoxin induced increase in sTNFR is dependent of renal function, endotoxin was injected in nephrectomized mice. The data show that nephrectomy followed by endotoxin injection resulted in a further increase of the levels of both sTNFR. However, the endotoxin induced increase in nephrectomized mice was similar to the situation in normal mice after LPS indicating that the endotoxin induced increase is kidney independent in these mice. To investigate the relative participation of various organs in sTNFR clearance, 125I labelled sTNFR-P75 was injected. The data reveal that the majority of the sTNFR is removed from the circulation by the kidneys although indications for involvement of the liver and the lungs were also obtained. Calculation of the parametric clearance revealed that nephrectomy resulted in a 50% reduction of sTNFR-P75 clearance. Furthermore, the data presented strongly suggest that sTNFR release seems to be a continuous process, which is in balance with clearance of the sTNFR by the kidney, although other organs such as the liver and the lungs are involved.

• Takahashi N, Brouckaert P, Bemelmans MH, Buurman WA, Fiers W
• Mechanism of induction of tolerance to tumour necrosis factor (TNF): no involvement of modulators of TNF bioavailability or receptor binding.
• Cytokine. 1994; 6: 235-42
• Display abstract

• The repetitive administration of low doses of hTNF to mice induces tolerance to the lethal effects of mTNF. The underlying mechanism is unknown. In this study we have investigated whether changes in bioavailability and receptor binding could account for the observed differences. To that end we compared the pharmacokinetics of mTNF, the antibody response to TNF, the levels of soluble TNF receptors and the receptor binding of TNF in tolerant and control mice. No differences in pharmacokinetic parameters were observed. An antibody response towards hTNF occurred but the antibodies did not neutralize the mTNF used as a challenge. Furthermore, tolerance failed to protect mice against lethality induced by TNF in the presence of galactosamine, where 100- to 1000-fold lower dose of TNF is required. Also, tolerance could be induced in athymic nude mice where the antibody response is absent. These results show that the mechanism of induction of tolerance is not due to an antibody response. No differences in levels of soluble receptors or receptor binding could be observed in tolerant vs control mice. We conclude that the induction of tolerance involves mechanisms operating at the post-receptor pathways.

• Rothe J, Mackay F, Bluethmann H, Zinkernagel R, Lesslauer W
• Phenotypic analysis of TNFR1-deficient mice and characterization of TNFR1-deficient fibroblasts in vitro.
• Circ Shock. 1994; 44: 51-6
• Display abstract

• In order to analyse the physiological relevance of the 55 kDa tumor necrosis factor receptor 1 (TNFR1) and its role in various TNF related pathological conditions, such as septic shock, we have generated mice by gene targeting deficient for TNFR1 expression. The TNFR1-deficient mice are unable to cope with Listeria monocytogenes infections but mount an apparently normal immune response when challenged with Vaccinia or LCMV viruses. They are resistant to the lethal effects of lipopolysaccharide (LPS) after sensitization with D-galactosamine (D-GalN) but remain sensitive to very high doses of LPS given alone. We have analyzed functions relevant to inflammatory processes, such as adhesion, secondary factor release, and proliferation in fibroblasts derived from these mice. We show that the TNFR1 virtually monopolises TNF-mediated signaling in all these situations and that the 75 kDa TNFR2 seems to be largely restricted to an accessory role, which is compatible with the previously established "ligand passing" hypothesis.

• Schreiber M, McFadden G
• The myxoma virus TNF-receptor homologue (T2) inhibits tumor necrosis factor-alpha in a species-specific fashion.
• Virology. 1994; 204: 692-705
• Display abstract

• The myxoma virus T2 protein shares extensive homology with the ligand binding domains of tumor necrosis factor (TNF) receptors. To characterize the T2-TNF interaction, myxoma T2 protein and rabbit, mouse, and human TNF alpha were expressed independently from vaccinia virus vectors. Growth of the TNF alpha-expressing viruses was significantly attenuated in TNF-hypersensitive L929-8 cells, and these cells were rapidly lysed by all three TNF alpha s. Rabbit cells showed strict species specificity in that RK-13 and SIRC cells were only sensitive to lysis by the homologous rabbit TNF alpha. Although RK-13 cells were hypersensitive to rabbit TNF alpha even in the absence of protein synthesis inhibitors, growth of T2 nonexpressing myxoma and vaccinia viruses in RK-13 cells was only modestly reduced in the presence of rabbit TNF alpha, suggesting that poxviruses possess additional anti-TNF mechanisms. When the ability of the myxoma T2 protein to inhibit biological activities of TNF alpha was assayed, T2 protein effectively protected L929-8 cells from lysis by rabbit, but not human or mouse TNF alpha. These studies show that rabbit TNF receptors may be species-specific for rabbit TNF alpha and we conclude that the myxoma T2 protein evolved to specifically inhibit TNF activities from its natural host, the South American rabbit.

• Boockfor FR, Harris SE, Barto JM, Bonner JM
• Placental cell release of transferrin: analysis by reverse haemolytic plaque assay.
• Placenta. 1994; 15: 501-9
• Display abstract

• Recent demonstrations of transferrin (TF) mRNA in placental tissue raised the possibility that the placenta may serve as an extra-hepatic source of this iron-binding protein during development. In this study, we first confirmed these findings using cRNA probes for TF, and then adapted the TF reverse haemolytic plaque assay for use with rat placental cells to identify the location and functional characteristics of cells secreting this product. We found TF releasing cells in placentae (day 19-21) with greater proportions present in cultures from basal than labyrinth zone regions. These cells appeared quite stable as evidenced by observations that fresh populations and 24, 48 and 72 h cultures from the same placental regions all contained similar percentages of secretors. The rate of TF plaque formation was greatly enhanced in the presence of tumour necrosis factor (0.1 ng/ml) for basal zone and yolk sac cells, but not for labyrinth zone cells, suggesting a potent but regionally specific modulation of TF release. When taken together, these findings demonstrate clearly that TF is released from cells of the placenta. Moreover, the regional differences in frequency and modulation of these cells suggests that the release of placental TF is a dynamic and carefully controlled process.

• Heller RA, Kronke M
• Tumor necrosis factor receptor-mediated signaling pathways.
• J Cell Biol. 1994; 126: 5-9

• Tracey KJ, Cerami A
• Tumor necrosis factor: a pleiotropic cytokine and therapeutic target.
• Annu Rev Med. 1994; 45: 491-503
• Display abstract

• Advances in the molecular biology of human diseases indicate that the most striking manifestations of illness may be caused not by exogenous pathogenic or tumor products, but rather by toxic peptides produced by the host itself. Tumor necrosis factor (TNF), a polypeptide cytokine produced during infection, injury, or invasion, has proved pivotal in triggering the lethal effects of septic shock syndrome, cachexia, and other systemic manifestations of disease. Because removing TNF from the diseased host may prevent development of the illness, this factor has recently been the focus of intensive research. This review discusses the biology of this cytokine, with particular emphasis on its potential therapeutic role in septic shock and cachexia.

• Lai TL, Zhang L
• Statistical analysis of ligand-binding experiments.
• Biometrics. 1994; 50: 782-97
• Display abstract

• After a brief review of commonly used methods for parameter estimation from ligand-binding data in the biochemistry literature, we propose some diagnostic checks and statistical tests of the underlying assumptions and develop methods for evaluating the biases and variances of the estimates and for constructing confidence intervals. Examples on the analysis of data from two radioligand-binding experiments are presented to illustrate these methods.

• Reid T, Louie P, Heller RA
• Mechanisms of tumor necrosis factor cytotoxicity and the cytotoxic signals transduced by the p75-tumor necrosis factor receptor.
• Circ Shock. 1994; 44: 84-90
• Display abstract

• We have here provided evidence that TNF mediated cytotoxicity is genetically, pharmacologically, and temporally distinct from the cytotoxicity mediated by TNF/CHX. Most studies on TNF cytotoxicity have been done by the combined use of TNF/CHX. The relevance of this approach to the physiological mechanisms underlying cytotoxicity by TNF alone is at present unclear. We have described a system in which overexpression of the p75-TNFR causes TNF-resistant cells to become TNF-sensitive. These cells are killed by TNF alone in a very short period of time and they are a useful system to study the mechanism of TNF-cytotoxicity.

• Suzuki J, Tomizawa S, Arai H, Seki Y, Maruyama K, Kuroume T
• Purification of two types of TNF inhibitors in the urine of the patient with chronic glomerulonephritis.
• Nephron. 1994; 66: 386-90
• Display abstract

• In order to clarify whether TNF inhibitory activity in the urine of glomerulonephritis (GN) patients depends on the existence of TNF inhibitors/soluble TNF receptors, we purified two types of TNF inhibitors from the urine of chronic renal failure (CRF) with GN patient. Using four steps of chromatography (anion exchange chromatography, preparative reverse phase chromatography, TNF affinity chromatography, analytical reverse phase chromatography), two types of 99% pure TNF inhibitors were purified. Amino terminal amino acid sequence revealed that one inhibitor was identical to soluble TNF receptor, p55, however, the other was homologous but not identical to soluble TNF receptor, p75. Our experiments demonstrated that TNF inhibitory activity in the urine of CRF patients depended partly on the existence of soluble TNF receptors in the urine.

• Moosmayer D et al.
• Coexpression of the human TNF receptors TR60 and TR80 in insect cells: analysis of receptor complex formation.
• Lymphokine Cytokine Res. 1994; 13: 295-301
• Display abstract

• For investigation of a possible physical interaction between the two human tumor necrosis factor receptors, TR60 (type I) and TR80 (type II), the baculovirus expression system was used. Each of the receptors was expressed as a membrane-integrated protein in insect cells, able to specifically bind the two ligands, tumor necrosis factor (TNF) and lymphotoxin (LT alpha). Typically, about 150,000 membrane receptors per cell could be detected 40 h after infection, exerting high affinity ligand binding capacity with Kd values virtually identical to that of human cell lines. The baculovirus system allowed coexpression of both TNF membrane receptors at very high and about equal numbers to investigate the existence of heteromultimeric receptor complexes, either formed spontaneously or ligand induced. Neither saturation binding studies nor immunoprecipitation experiments gave an indication for the existence of TNF receptor heteromers. These data are in accordance with the current view of TNF signaling, in which homonultimerization, rather than heteromer formation of TNF receptors is the initial activating event.

• Zentner A, Rendl J, Grelle I, Dummer R, Brocker EB, Zillikens D
• Elevated levels of soluble tumour necrosis factor receptor I in blister fluids of bullous pemphigoid and suction blisters.
• Arch Dermatol Res. 1994; 286: 355-7

• Abe Y, Watanabe Y, Kimura S
• The role of tumor necrosis factor receptors in cell signaling and the significance of soluble form levels in the serum.
• Surg Today. 1994; 24: 197-202
• Display abstract

• Two types of tumor necrosis factor membrane receptors (TNF-R) have been identified, namely 55 and 75 kDa TNF-R. Soluble forms of these receptors are present in the human serum. Recent findings on the role of these two TNF-R in biological cell signaling and the clinical significance of the serum levels of soluble TNF-R (sTNF-R) were reviewed. It is not the uptake of TNF molecules into cells but rather the molecular capping of TNF-R on the cell membrane that initiates the biological activity of TNF. The 55 kDa TNF-R mediates major bioactivities of TNF, while the significance of 75 kDa TNF-R remains unclear. We herein suggest a new concept of the role of these two TNF-R: The 75 kDa TNF-R signal appeared to enhance that of 55 kDa TNF-R in the induction of ICAM-1 expression on HL-60 human promyelocytic leukemic cells. High serum levels of sTNF-R are reported in patients with malignancy, endotoxin shock, pneumonia, and autoimmune diseases. However, the source of elevated serum sTNF-R remains unclear. Studies on the clinical usefulness of serum sTNF-R levels as cancer and inflammation markers are now being carried out.

• Menegazzi R, Cramer R, Patriarca P, Scheurich P, Dri P
• Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor.
• Blood. 1994; 84: 287-93
• Display abstract

• Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF-induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF-induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr-9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin-coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.

• Bigda J et al.
• Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity.
• J Exp Med. 1994; 180: 445-60
• Display abstract

• Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75-R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain--a region at which a conformational change seems to take place upon TNF binding--decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.

• Zhu XJ, Cao XT
• [New advances in the research of tumor necrosis factor receptor]
• Sheng Li Ke Xue Jin Zhan. 1994; 25: 117-20

• Jansen J, van der Poll T, van Deventer SJ
• Endotoxin and the release of tumor necrosis factor receptors.
• Prog Clin Biol Res. 1994; 388: 383-97

• Cinat ME, Waxman K, Granger GA, Pearce W, Annas C, Daughters K
• Trauma causes sustained elevation of soluble tumor necrosis factor receptors.
• J Am Coll Surg. 1994; 179: 529-37
• Display abstract

• BACKGROUND: Soluble tumor necrosis factor receptors (sTNF-R) are thought to modulate the systemic effects of tumor necrosis factor (TNF) by binding to serum TNF and preventing its interaction with target organs. Recently, it has been shown that traumatic injury causes the early release of the soluble forms of the 55 and 75 kDa membrane receptors for TNF. This study was done to determine the magnitude of TNF receptor elevation after trauma, to delineate the duration of this elevation, and to determine if sTNF-R levels correlate with severity of injury and outcome. STUDY DESIGN: One hundred injured patients treated at a Level I Trauma Center were included in the study (74 males, 26 females, mean age of 29.4 years [range of ten to 72 years], mean injury severity score of 16.8 [range of zero to 75]). Serum samples were drawn from these patients beginning within one hour of injury and continuing for as many as 15 days. Samples were analyzed using polyclonal ELISA assays for TNF and sTNF 55 and 75 kDa receptor levels; control levels of receptor were determined from healthy volunteers. RESULTS: Tumor necrosis factor was not measurable, but trauma caused immediate elevation of both receptor levels (within one hour of injury). Receptor levels remained elevated for as many as 15 days after injury. Late variations in levels were related to complications, that is, hypoxia, infection, and sepsis. Levels were significantly more elevated in critically ill patients and nonsurvivors. CONCLUSIONS: We conclude that sTNF-R levels are significantly elevated after trauma, in the absence of measurable TNF. Levels are elevated for variable periods of time, which seem to depend on the severity of injury and complications.

• Min WQ, Gu CH, Wang YM
• [Relation of plasma soluble tumor necrosis factor receptor to tumor necrosis factor and clinical features of hepatitis B]
• Zhonghua Yi Xue Za Zhi. 1994; 74: 602-4
• Display abstract

• Plasma soluble tumor necrosis factor receptor (sTNFR) was detected by radioimmunoprecipitation-polyethylene glycol assay in 64 patients with viral hepatitis B. The levels of two distinct receptors (sTN-FR1 and sTNFR2) were significantly higher in chronic severe hepatitis (CSH) followed by chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and acute hepatitis (AH) or controls (P < 0.01). A more markedly increased sTNFR was observed in patients with high SB (> 342 mumol/L), low Pa (< 20%), and secondary infection or fatal outcome. For patients with 20% below of sTNFR levels, the increase of TNF was proportional to that of sTNFR. But, for patients with exceeding 20% of sTNFR, the ratio of TNF/sTNFR became higher. The ratio of TNF to sTNFR may be greatly indicative to determine the clinical severity and outcome. Administration of sTNFR could prevent the adverse pathologic sequence caused by the exaggerated TNF and open a new therapeutical field.

• van Tits LJ, Bemelmans MH, Steinshamn S, Waage A, Leeuwenberg JF, Buurman WA
• Non-signaling functions of TNF-R75: findings in man and mouse.
• Circ Shock. 1994; 44: 40-4

• Edgington SM
• Molecular crosstalk. Will virology and growth-factor research aid cytokine drug discovery?
• Biotechnology (N Y). 1993; 11: 465-7

• Sidhu RS, Bollon AP
• Tumor necrosis factor activities and cancer therapy--a perspective.
• Pharmacol Ther. 1993; 57: 79-128
• Display abstract

• Tumor necrosis factor (TNF) is a multifunctional cytokine which has excited and fascinated numerous investigators and commercial entities due to its promise as a therapeutic agent against cancer and as a target for drugs treating septic shock. TNF is a protein having cytotoxic, cytostatic, immunomodulatory as well as several other activities and is also involved in septic shock. This review covers the structure of TNF and its receptors, various in vitro activities and in vivo activities based on studies in animal model systems. The role of TNF as an anticancer therapeutic agent, based on various phase I and phase II clinical studies, has also been considered. The review concludes with several considerations for increasing the therapeutic utility of TNF in terms of targeting, toxicity and half-life.

• Balkwill F
• Tumour necrosis factor. Improving on the formula.
• Nature. 1993; 361: 206-7

• Baumann H, Morella KK, Wong GH
• TNF-alpha, IL-1 beta, and hepatocyte growth factor cooperate in stimulating specific acute phase plasma protein genes in rat hepatoma cells.
• J Immunol. 1993; 151: 4248-57
• Display abstract

• In rat hepatoma H-35 cells, TNF-alpha, like IL-1 beta, stimulates the synthesis and secretion of type 1 acute phase plasma proteins, including alpha 1-acid glycoprotein (AGP), complement component 3 (C3), hemopexin, and haptoglobin. TNF and IL-1 in combination act additively to synergistically on the expression of AGP and C3 but not on hemopexin and haptoglobin. The cytokine stimulation of AGP and C3 genes is further enhanced by hepatocyte growth factor. The effect of TNF is mediated by the type I TNF receptor as judged from the TNF-like effect elicited by the agonist antibodies to type I but not type II TNF receptor. The data suggest that, although TNF and IL-1 share considerable overlap in their signal transduction mechanism, cytokine-specific signal pathways exist that affect a subset of acute phase plasma protein genes. A potential regulatory role is attributed to the transcription factors C/EBP beta and NF-kB based on studies on transiently transfected H-35 cells.

• Peppel K, Poltorak A, Melhado I, Jirik F, Beutler B
• Expression of a TNF inhibitor in transgenic mice.
• J Immunol. 1993; 151: 5699-703
• Display abstract

• We previously reported that a chimeric protein consisting of the human p55 TNF receptor covalently linked to a murine IgG1 Fc heavy chain acts as an efficient TNF inhibitor, as a result of its high binding affinity for native TNF trimers of both murine and human origin. A transgenic mouse line constitutively expressing the inhibitor from a cytomegalovirus promoter was established. All organs examined expressed the transgene. TNF inhibitory activity was easily detected in plasma of transgenic animals but not in plasma of nontransgenic littermates. This founder line was not found to have any obvious phenotype. Specifically, transgenic mice born to wild-type or transgenic females were indistinguishable from nontransgenic littermates with respect to their size at birth, at three months and at six months of age, with respect to the size and morphology of their lymphoid organs and with respect to their hematocrit, white blood cell count, and differential. Although TNF is known to be constitutively expressed by cells of the thymus and trophoblast, the lack of a clear phenotype in association with the constitutive expression of a TNF inhibitor suggests that TNF may be dispensable in early development.

• Tsantakis G, Schizas N, Havredaki M
• Interferon inhibitors and/or inactivators in sera from patients with neoplasias.
• Arch Immunol Ther Exp (Warsz). 1993; 41: 335-7
• Display abstract

• Measurements of interferons, their inhibitors and/or inactivators and tumor necrosis factor-like activities in sera from patients with neoplasias were performed. Interferon activity could not be detected in 96% of the sera samples examined. In contrast, both interferon inhibitors and/or inactivators and tumor necrosis factor-like activities were found in a high proportion (25-100%) of sera from cancer patients but not in sera from normal individuals.

• Ikegami H et al.
• Analysis of the site on a TNF-alpha molecule which affects type II TNF receptor binding in human cells.
• Lymphokine Cytokine Res. 1993; 12: 173-80
• Display abstract

• The epitope region on the TNF-alpha molecule recognized by monoclonal antibody (mAb) 3-D-6, which neutralizes the cytotoxic activity on murine LM cells, has been determined as Gly24-Gln-Leu-Gln-Trp-Leu-Asn-Arg31. To examine whether this region participates in TNF receptor binding in human cell lines, four kinds of TNF-alpha mutants (Gln25 --> Glu, Gln27 --> Glu, Leu29 --> Val, and Arg31 --> Ser) were prepared using site-directed mutagenesis. One mutant, mRS31, which has a nonconserative mutation at position 31 (Arg --> Ser), showed markedly reduced binding in U-937 cells and in HL-60 cells compared with the wild-type recombinant TNF-alpha (rTNF-alpha). These two cell lines have been reported to have both type I and type II TNF receptors. mRS31 also showed reduced cytotoxicity on U-937 cells. Another mutant, mLV29, which has a conservative mutation at position 29 (Leu --> Val), showed, to a lesser extent, reduced binding in U-937 cells and HL-60 cells and reduced cytotoxic activity in U-937 cells. However, all four TNF-alpha mutants showed a similar binding in HEp-2 cells and in HeLa cells, which have been reported to have only the type I TNF receptor. These results suggest that Leu29 may be involved in direct contact with the type II receptor and that the nonconservative mutation at position 31 may induce a local conformational change in the site involved in type II TNF receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

• Stull RA, Zon G, Szoka FC Jr
• Single-stranded phosphodiester and phosphorothioate oligonucleotides bind actinomycin D and interfere with tumor necrosis factor-induced lysis in the L929 cytotoxicity assay.
• Antisense Res Dev. 1993; 3: 295-300
• Display abstract

• Antisense oligonucleotides may prove useful tools to elucidate the biological functions of cytokines and to address regulatory control of cytokine expression. The functional activity of cytokines generally is determined by biological assays, and a standard biological assay for TNF activity measures the cytotoxic effect of TNF on actinomycin D-sensitized L929 cells (Matthews and Neale, 1987). We observed that phosphorothioate and phosphodiester oligonucleotides, at concentrations > 100 nM in supernatants tested in this bioassay, prevented TNF-induced lysis of L929 cells. This "protective" effect was due to an interaction of the single-stranded oligonucleotides with actinomycin D as demonstrated by UV spectra of an actinomycin D-oligonucleotide solution. Substitution of cycloheximide for actinomycin D in the L929 assay eliminated the protective effect of the oligonucleotide. Our results reinforce the importance of controlling for nonspecific effects of oligonucleotides, particularly when a functional assay for protein activity is used to screen for antisense-mediated reductions in target protein expression.

• Matsunaga K, Mashiba H, Seo Y, Wada S, Hata K
• Augmentation of the radiation-induced antiproliferative effect in combined use of a derivative of nitrosourea, ACNU, with recombinant human tumor necrosis factor.
• Immunopharmacology. 1992; 23: 199-204
• Display abstract

• The effect of combined use of a derivative of nitrosourea, ACNU, with recombinant human tumor necrosis factor (rhTNF) on the radiation-induced antiproliferative effect was examined using Meth A tumor cells. The radiation-induced antiproliferative effect was slightly augmented by the simultaneous addition of ACNU at 10 micrograms/ml in 5 Gy and 15 Gy irradiated groups. The antiproliferative effect was augmented in parallel with the radiation dose by the addition of rhTNF. Further augmentation of the proliferation inhibition was observed when both of ACNU (10 micrograms/ml) and 10 U/ml of rhTNF were added in combination with irradiation. Similar augmentation was observed when the target cells were treated with ACNU prior to irradiation and the addition of rhTNF.

• Mullin JM, Laughlin KV, Marano CW, Russo LM, Soler AP
• Modulation of tumor necrosis factor-induced increase in renal (LLC-PK1) transepithelial permeability.
• Am J Physiol. 1992; 263: 91524-91524
• Display abstract

• Tumor necrosis factor-alpha (TNF) causes a spontaneously reversible increase in tight junction permeability. TNF was the only cytokine tested that produced this effect. The effect on transepithelial permeability proceeds in four distinct phases: 1) a 60- to 90-min delay from time of application of TNF, 2) a rapid decrease in transepithelial resistance, 3) a recovery of transepithelial resistance to control level within 1 h, and 4) a further increase of transepithelial resistance above control levels. The recovery of transepithelial resistance occurs with or without TNF in the culture medium. Different protein kinase inhibitors affected different phases of this overall process. The tyrosine kinase inhibitor genistein significantly blocked the TNF effect. Neither transcription nor protein synthesis was required for transepithelial permeability to increase, but were required for the recovery. After the tight junctions have opened at 2 h in response to TNF, a second application of TNF will not produce the effect again for at least 12 h. The tight junctions will, however, open in response to phorbol esters during this time frame. Electron microscopy studies using apically applied ruthenium red suggest that TNF action results in < 10% of the junctions having increased permeability at any given time during the resistance decrease. The role of epithelial barrier permeability changes in TNF action in vivo is discussed.

• Tsan MF, White JE, Wong GH
• D-factor and growth hormone enhance tumor necrosis factor-induced increase of Mn superoxide dismutase mRNA and oxygen tolerance.
• Cytokine. 1992; 4: 101-5
• Display abstract

• D-Factor (differentiation-inducing factor or leukemia inhibitory factor) and growth hormone are proteins that regulate growth and differentiation of cells. In this study, we demonstrated that recombinant human D-factor and growth hormone caused a slight but significant protection of adult rats against oxygen toxicity without affecting levels of pulmonary manganous superoxide dismutase (MnSOD) mRNA. D-Factor and growth hormone also markedly enhanced tumor necrosis factor (TNF)-induced oxygen tolerance. This latter effect was associated with a marked enhancement of TNF-mediated induction of pulmonary MnSOD mRNA.

• Opipari AW Jr, Hu HM, Yabkowitz R, Dixit VM
• The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.
• J Biol Chem. 1992; 267: 12424-7
• Display abstract

• Resistance against the cytotoxic actions of tumor necrosis factor alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.

• MacKay RJ
• Association between serum cytotoxicity and selected clinical variables in 240 horses admitted to a veterinary hospital.
• Am J Vet Res. 1992; 53: 748-52
• Display abstract

• A total of 378 serum samples from 240 hospitalized horses and 47 sera from healthy control horses were assayed for growth effects on actinomycin D-treated L929 cells. On average, patient and control sera stimulated cell growth; however, mean percentage of the relative growth index (RGI) of sera from clinical cases was significantly (P less than 0.001) lower than that of control sera. Approximately 35% of patient sera and 6% of control sera had tumor necrosis factor-like cytotoxic activity for L929 cells (ie, RGI less than 100%). Sera from horses with either peritoneal leakage of gastrointestinal tract contents or any bacterial infection were significantly (P less than 0.05) more cytotoxic than sera from horses that did not have these clinical factors. A clear tendency was evident for horses that had the highest serum cytotoxicity (RGI less than 75%) to also have clinical profiles suggestive of endotoxemia. Fever, leukopenia, diarrhea, and gastrointestinal tract leakage were significantly (P less than 0.05) overrepresented among these horses, compared with horses without serum cytotoxicity. Bacterial infections and abdominal surgeries were also increased in this group, but not significantly. Of the 14 horses with serum RGI less than 75%, 13 had some form of gastrointestinal tract disease and the other had gram-negative septicemia. Survival to discharge was significantly (P less than 0.05) lower among horses in the high-cytotoxicity group than among horses without serum cytotoxicity. Diarrhea and bacterial infections were the only clinical factors found more frequently in horses with low serum cytotoxicity than in horses without serum cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

• Weil D
• What's new about tumor necrosis factors? Report on the fourth TNF International Congress.
• Eur Cytokine Netw. 1992; 3: 347-51

• Tsuji Y et al.
• Endogenous tumor necrosis factor functions as a resistant factor against hyperthermic cytotoxicity.
• Cancer Res. 1992; 52: 6258-62
• Display abstract

• One of the mechanisms of cytotoxicity by tumor necrosis factor (TNF) is the induction of reactive oxygen molecules. Cells producing endogenous tumor necrosis factor (enTNF) show resistance to the cytotoxicity of exogenous TNF by scavenging the reactive oxygen molecules. The intracellular hydroxyl radical production is also known to be involved in the heat-induced cytotoxicity. In the present study, we therefore examined the possibility that enTNF may act as a protective protein against the heat-induced cytotoxicity in a manner similar to that of exogenous TNF. Heat-sensitive L-M (mouse tumorigenic fibroblast) cells, originally expressing no enTNF, were transfected with a human TNF expression vector to produce enTNF. The stable transfectants showed apparent resistance to heat treatment. Conversely, when HeLa (human uterine cervical cancer) cells, originally producing an appreciable amount of enTNF, were transfected with an antisense TNF mRNA expression vector to inhibit enTNF synthesis, their heat sensitivity was enhanced. Furthermore, L-M cells which were transfected with nonsecretory human TNF expression vector also acquired resistance to heat treatment. In these cells, heat resistance correlated well with expression of enTNF and intracellular levels of manganous superoxide dismutase. These results indicate that enTNF exerts its intracellular protective effect against the heat-induced cytotoxicity by scavenging reactive oxygen with induced manganous superoxide dismutase in a manner similar to that found in cells treated with exogenous TNF.

• Shau H, Roberts RL
• Tumor necrosis factor stimulation of neutrophils for antitumor activity.
• Immunol Ser. 1992; 57: 523-33

• Mencoboni M, Lerza R, Bogliolo G
• [Tumor necrosis factor: a cytokine with multiple actions]
• Recenti Prog Med. 1992; 83: 15-7
• Display abstract

• In the past few years tumor necrosis factor (TNF), also known as cachectin, has been isolated, cloned and now human recombinant TNF is available. This cytokine has numerous actions, which can be divided into four groups: 1) antitumor function; 2) immunomodulating activity and function in the inflammatory response; 3) effects on metabolism; 4) other functions. TNF was first identified for its anticancer activity; it is able to induce hemorrhagic necrosis in subcutaneously implanted tumors, due to the induction of free radicals in tumor cells and to vascular damage. It can also activate T-lymphocytes to become lymphokine-activated killer cells (LAK cells) against tumors. TNF also plays an important role in the inflammatory response: it mediates many of the immunologic features of T-cell function and of infection, and is essential in septic shock. TNF is a cause of the hypertriglyceridemia and the cachexia that characterize chronic infections and neoplasms. In vitro this cytokine causes growth of vascular endothelial cells; this observation suggests that it could have an atherogenic role. In in vivo experiments severe hepatic ischemia-reperfusion injury results in TNF release with subsequent local and systemic injury that was significantly reduced by anti-TNF antiserum pretreatment. Thus, TNF could be a cause of ischemic tissue damage. In conclusion, while TNF is known to play many roles, the intercellular network of the cytokines is not yet sufficiently understood and so we are only just beginning to comprehend the full implication of this important molecule in both histology and pathology.

• Malik ST, Balkwill FR
• Antiproliferative and antitumor activity of TNF in vitro and in vivo.
• Immunol Ser. 1992; 56: 239-68

• Brouckaert PG, Fiers W, Lejeune FJ
• Coley's vaccine and TNF therapy.
• Nature. 1992; 358: 630-630

• Chouaib S, Robinet E, Zyad A, Branellec D
• [Tumor necrosis factor: pleiotropic cytokine]
• Bull Cancer. 1992; 79: 935-49
• Display abstract

• Originally described for its capacity to induce hemorrhagic necrosis of transplantable tumors in mice, TNF-alpha also exerts cytotoxic effects against some tumor cell lines in vitro. It is now known that TNF is an essential mediator of cellular immunity and a wide variety of biological activities of TNF in vitro and in vivo has been reported. TNF is an important mediator of inflammation and is involved during the pathogenesis of several auto-immune, infectious or cancer diseases. While some immunomodulatory properties of TNF have at least been partially elucidated, the biochemical basis of TNF cytotoxic action remains largely unknown. Furthermore, the molecular mechanisms of TNF susceptibility have yet to be clarified. Clinical studies with recombinant TNF as an anticancer agent are encouraging. Multiple phase I and phase II trials have been carried out without major therapeutic effect. In fact, TNF resistance and TNF-induced systemic toxicity are two major limitations for the use of TNF as an antineoplastic agent. The clinical application of human TNF remains an area of active research and innovative approaches such as gene therapy need to be elaborated. The elucidation of the process of lysis and the modulation of TNF resistance are crucial to the future development of TNF and its use in antitumor therapy.

• Stalc A, Sentjurc M, Sersa G, Novakovic S
• The influence of TNF on the membrane fluidity of tumor cells.
• Cancer Lett. 1992; 65: 183-7
• Display abstract

• Possible TNF (tumor necrosis factor) effects on the membrane fluidity of tumor cells were investigated. Viable tumor cells, TNF sensitive, were obtained from the ascitic form of the SA-1 tumor bearing mice. The influence of in vitro and in vivo treatment of cells with the TNF analog was investigated by EPR (electron paramagnetic resonance). SA-1 cells were spin labeled with the methylester of 5-doxylpalmitate, which primarily dissolves in the membranes. The maximal hyperfine splitting was determined and the empirical correlation time calculated. The results show that TNF significantly decreases the correlation time, i.e. it increases the fluidity of SA-1 cell membranes. Such alteration could contribute to the cytotoxicity of TNF.

• Seckinger P, Dayer JM
• Natural inhibitors of TNF.
• Immunol Ser. 1992; 56: 217-36

• Hofsli E, Waage A
• Effect of pyridoxine on tumor necrosis factor activities in vitro.
• Biotherapy. 1992; 5: 285-90
• Display abstract

• Clinical trials with tumor necrosis factor (TNF) as an antitumor agent have so far given rather disappointing results. In this study we show that the naturally occurring vitamin B6 compound, pyridoxine, enhances TNF-induced cytolysis of three subclones of a mouse fibrosarcoma cell line (WEHI 164). The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to be dependent on the cells sensitivity to TNF, as the enhancement was much more pronounced in the relatively TNF resistant subclone act-R(cl.12)-WEHI 164, than in the very TNF sensitive subclone WEHI 164 clone 13. Furthermore, our study shows that pyridoxine, in contrast to its enhancing effect on TNF-induced cytotoxicity, rather inhibits TNF-induced growth of human FS-4 fibroblasts. Pyridoxine also enhances lymphotoxin (LT)-induced tumor cell killing and inhibits LT-induced fibroblast growth. Pyridoxine is a relatively non-toxic agent in vivo. Our results suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone, in the treatment of cancer patients.

• Gordon HM, Kucera G, Salvo R, Boss JM
• Tumor necrosis factor induces genes involved in inflammation, cellular and tissue repair, and metabolism in murine fibroblasts.
• J Immunol. 1992; 148: 4021-7
• Display abstract

• TNF-alpha is a primary mediator of the inflammatory response and has been ascribed a wide range of biologic activities including the cytolysis of some but not all transformed cell lines in vitro. Since most cells, normal and transformed, will also be lysed if they are concurrently treated with inhibitors of transcription or translation, the ability to resist TNF lysis depends on the de novo induction of specific gene products. To identify genes that might be involved in the ability to resist TNF-mediated cytolysis, cDNA libraries enriched for TNF-induced genes were constructed and screened. Twenty-one genes that are induced by TNF were isolated. Ten of the cDNA clones were identified by DNA sequence analysis and function in inflammatory responses, tissue or cellular repair, or cellular metabolism. Seven genes were not identified and are novel. The remaining four genes are encoded in the mitochondrial genome. Interestingly, not all the mitochondrial transcripts are induced. This may reflect a fine tuning of certain components of mitochondria that may be necessary for survival after TNF treatment.

• Dudrick PS, Bland KI, Souba WW
• Effects of tumor necrosis factor on system ASC-mediated glutamine transport by human fibroblasts.
• J Surg Res. 1992; 52: 347-52
• Display abstract

• The effects of tumor necrosis factor-alpha (TNF) on glutamine GLN transport by cultured human fibroblasts were studied. Uptake of 3H-GLN was assayed in both the presence and absence of sodium in order to differentiate Na(+)-dependent and Na(+)-independent transport systems. GLN transport was linear (r = 0.99) for at least 15 min and occurred predominantly via a single Na(+)-dependent pathway, consistent with System ASC. Incubation of fibroblasts with TNF (1000 units/ml) for 12 hr resulted in a significant decrease in system ASC-mediated glutamine transport activity. TNF did not alter cell morphology or protein content. Kinetic studies indicated that the decrease in carrier-mediated Na(+)-dependent GLN transport was not due to a change in transporter affinity (Km = 117 +/- 23 microM in controls vs 86 +/- 23 microM in TNF, P = NS), but instead to a 45% decrease in maximal transport rate (Vmax = 4088 +/- 354 pmole/mg protein/30 sec in controls vs 2230 +/- 510 in TNF, P less than 0.05). TNF also decreased Na(+)-independent transport by 50% (mean uptake of 50 microM GLN = 94 +/- 13 pmole/mg protein/30 sec in controls vs 46 +/- 6 in TNF, P less than 0.02). In human fibroblasts, the activity of System ASC, which has generally been viewed as a hormonally unresponsive carrier, is decreased by TNF. This impairment in glutamine transport may result in inadequate amounts of intracellular glutamine to support fibroblast metabolism and possibly function.

• Branellec D, Chouaib S
• [TNF: antitumoral agent at the border lines of immunity and inflammation]
• Pathol Biol (Paris). 1991; 39: 230-9
• Display abstract

• Tumor Necrosis Factor-alpha (TNF) is mainly produced by activated monocytes and exerts pleiotropic biological effects on a wide variety of both normal and transformed cells. Originally described for its capacity to induce hemorrhagic necrosis of transplantable tumors in vivo and cytolysis of some tumor cells in vitro, TNF has also been shown to play an essential role during the inflammatory response, exerting dual, both beneficial and deleterious, effects. TNF, via a local production appears to be a key cytokine involved in antiviral, antibacterial and antiparasitic host defense mechanisms. Contreversely, deregulation of the inflammatory and immune reactions can be associated with a systemic TNF production, leading to toxic secondary effects. Recent cloning of the TNF receptors has provided additional insights in the complex physiology of TNF. It is now clearly established that both type I and type II TNF receptors can be cleaved and released as TNF binding proteins. The soluble fragments of TNF receptors can inhibit TNF-mediated tumor cell lysis in vitro and might therefore serve as regulators of TNF action. The antitumor potency of TNF also reflects the pleiotropic aspect of TNF. TNF-induced tumor regression, observed in various preclinical studies, appears to result from at least three distinct biological effects: mainly hemorrhagic necrosis via TNF action on tumor endothelium, TNF immunomodulatory activity on immune effector cells, and presumably a direct TNF-mediated cytotoxic effect against tumor cells. From the clinical trials performed with distinct recombinant materials a consensus has emerged about the disappopinting anticancer efficacy of TNF used in systemic administration. Further studies, aimed at better understanding the complex action of TNF, are required to possibly enhance its therapeutic index and subsequently to assess whether TNF still remains a promising therapeutic agent.

• Kull FC Jr, Besterman JM
• Drug-induced alterations of tumor necrosis factor-mediated cytotoxicity: discrimination of early versus late stage action.
• J Cell Biochem. 1990; 42: 1-12
• Display abstract

• The killing of L-M cells by murine tumor necrosis factor (mTNF) was investigated by a combination of drug, antiserum neutralization, and kinetic studies. Kinetic studies with 125I-mTNF showed that the bulk of association of ligand with L-M cells peaked within 2 hr and the ligand was not degraded. Cell surface receptors were depleted (down regulated) by 6 hr when death commenced. The off-rates of acid-dissociable (surface bound) and acid-indissociable (internalized) compartments were found to be 9 min and 35 hr, respectively. Nevertheless, complete cell killing required the persistent presence of mTNF for up to 20 hr. This requirement was ablated by the concomitant addition of cycloheximide. Antiserum completely inhibited cytotoxicity when it was applied up to 4 hr after mTNF, but antiserum added 1 hr after mTNF was not neutralizing in the presence of cycloheximide. Thus, the inclusion of cycloheximide temporally dissociated early events (internalization and signal transduction) from lysis. Other drugs with and without cycloheximide were found to preferentially affect either early or later aspects of cell death. Phorbol myristate acetate and the ionophore A23187 were potent inhibitors of cytotoxicity, and staurosporine was a potent enhancer. These agents were more effective when added 1 hr before mTNF and cycloheximide than when added 1 hr after and likely affected early events in the cytolytic program. In contrast, chloroquine and cAMP likely affect more terminal aspects of cytotoxicity. Dibutyrylcyclic AMP, cholera, and pertussis toxins enhanced cytotoxicity. They were equipotent when added either before or after mTNF regardless of the presence of cycloheximide. Likewise, chloroquine was an equipotent inhibitor when added either before or after mTNF regardless of the presence of cycloheximide. Agents that primarily affect association events may be more likely to impinge on other TNF-mediated activities than agents that primarily affect lysis.

• Scheringa M, Marquet RL
• TNF: a brief review with emphasis on its antitumor activity.
• Biotherapy. 1990; 2: 275-81

• Mege JL
• [Importance and limits of assay of TNF in pathology]
• Allerg Immunol (Paris). 1990; 22: 287-287

• Espevik T, Shalaby R, Liabakk NB, Waage A, Sundan A
• The role of TNF in regulation of cell function.
• Scand J Clin Lab Invest Suppl. 1990; 202: 160-160

• Iwamoto S, Takeda K
• [Possible cytotoxic mechanisms of TNF in vitro]
• Hum Cell. 1990; 3: 107-12
• Display abstract

• TNF is a protein originally isolated by its ability to induce hemorrhagic necrosis of some tumors in vivo, and cytotoxic effects against certain tumor cells, but not against normal cells in vitro. Though mechanisms of the cytotoxicity and selectivity of TNF action in vitro are not clear, there is much evidence that free radicals such as superoxide and hydroxyl radicals, mediated by TNF, are correlated with TNF cytotoxicity. Recently, manganous superoxide dismutase (MnSOD) in mitochondria has been demonstrated to be a rescue protein against TNF cytotoxicity and to be induced when the cells are exposed to TNF in the resistant cells. Two steroid hormones, glucocorticoid and 1,25-dihydroxyvitamin D3, block the cytotoxicity of TNF. Glucocorticoid may reduce TNF cytotoxicity by inhibiting phospholipase A2 and 1,25-dihydroxyvitamin D3 by inducing MnSOD in combination with TNF.

• Hosoi T, Takeda K, Konno K
• Synergism of prostaglandin E2 plus TNF in induction of differentiation of human monocytoid leukemic U-937 cells.
• Anticancer Res. 1989; 9: 615-8
• Display abstract

• Prostaglandin E2 (PGE2) induced differentiation of human monoblastic leukemia U-937 cells into cells with macrophage characteristics. PGE2 synergistically increased the differentiation, of U-937, ML-1 and HL-60 cells when combined with TNF. PGE1 and PGF2 also induced differentiation of U-937 cells; however, PGD2, deoxy-delta 9,12-13, 14-dihydro-PGD2 (delta 12-PGJ2), and PGI2 did not induce differentiation, either alone or in combination with TNF. PGE2 changed the dissociation constant of TNF for its receptors on U-937 cells only marginally, but it approximately doubled the number of binding sites.

• Beutler B, Cerami A
• The biology of cachectin/TNF--a primary mediator of the host response.
• Annu Rev Immunol. 1989; 7: 625-55

• Matache C, Negut E, Szegli G
• [Tumor necrosis factor]
• Rev Ig Bacteriol Virusol Parazitol Epidemiol Pneumoftiziol Bacteriol. 1989; 34: 289-99

• Wallach D et al.
• Mechanisms which take part in regulation of the response to tumor necrosis factor.
• Lymphokine Res. 1989; 8: 359-63

• Warren JS, Ward PA, Johnson KJ
• Tumor necrosis factor: a plurifunctional mediator of acute inflammation.
• Mod Pathol. 1988; 1: 242-7
• Display abstract

• In recent years intense research activity has been directed toward characterizing polypeptide mediators that regulate growth, differentiation, and function of cells involved in inflammation, immunity, and hematopoiesis. Tumor necrosis factor (TNF), or cachectin, is one of these cytokines. This review will summarize important observations that have led to our current definition of TNF. The relationship of TNF to other cytokines and basic TNF biology are discussed. Particular attention is directed toward the evidence which implicates TNF as an important mediator of acute inflammation.

• Clark IA, Chaudhri G
• The balance of useful and harmful effects of TNF, with special reference to malaria.
• Ann Inst Pasteur Immunol. 1988; 139: 305-6

• Wallach D, Holtmann H, Aderka D, Hahn T, Engelmann H, Nophar Y
• Sensitizing effects and mechanisms of desensitization in regulation of the in vivo response to TNF.
• Ann Inst Pasteur Immunol. 1988; 139: 323-7

• Bonard EC
• [Cachectin]
• Rev Med Suisse Romande. 1988; 108: 635-6

• Vilcek J et al.
• Mechanisms and significance of the mitogenic and antiviral actions of TNF.
• Ann Inst Pasteur Immunol. 1988; 139: 307-11

• Geigert J et al.
• Parameters for the evaluation of long-term stability of tumour necrosis factor preparations.
• Dev Biol Stand. 1988; 69: 129-38
• Display abstract

• Escherichia coli-derived Tumour Necrosis Factor (TNF) was formulated in the absence of a protein-carrier, both as a solution and as a lyophilized preparation. By means of three stability-indicating test methods (bioassay, SDS-PAGE, IEF), the long-term stability of these TNF preparations was evaluated. Both preparations showed no change upon storage for 9 months at -70 degrees C and -20 degrees C. Depending upon the test method, different rates of change were detected at elevated temperature. The analysis presented will assist in the design of future TNF reference standards.

• Mizuno D, Soma GI
• Endogenous and exogenous TNF therapy (EET therapy): conceptual and experimental grounds.
• Ann Inst Pasteur Immunol. 1988; 139: 282-5

• Niitsu Y, Watanabe N
• [Cytokines and receptors--their functions, structures and cloning of code genes. Tumor necrosis factor]
• Nippon Rinsho. 1988; 46: 1041-9

• Coffman FD, Green LM, Ware CF
• The relationship of receptor occupancy to the kinetics of cell death mediated by tumor necrosis factor.
• Lymphokine Res. 1988; 7: 371-83
• Display abstract

• An analysis of the relationship between TNF receptor occupancy and the kinetics of cell death has been conducted in the sensitive L929.10 murine fibrosarcoma cell line. The receptor affinity and number, measured by competitive and saturation binding assays with human 125I-TNF, is consistent with a single class of binding sites having a Kd = 1.5 nM and 7,000 sites/cell. Human lymphotoxin competed equally well with TNF for binding to the receptor. Determination of the kinetics of L929.10 cell death as a function of TNF concentration revealed three sequential phases to the cytolytic process: An induction phase, that lasted 4 to 9 hrs after the addition of TNF, in which no cell death occurred; the burst phase in which cells died at a rate of 12 to 14% per hr; and the third phase, termed the slow lytic phase, was characterized by a rate of lysis of 1 to 2% per hr. Two parameters, the length of the induction phase and the percentage of cells killed in the burst phase, were dependent on TNF receptor occupancy. Both of these receptor dependent processes were maximized at 10% receptor occupancy. Half maximal lysis of the L929.10 clone was achieved at 10 pM or 0.67% receptor occupancy. In contrast, the rates of cell death during the burst phase and the slow lytic phase were independent of the concentration of TNF. Mitomycin C, an antitumor drug that alkylates and crosslinks DNA, enhanced the sensitivity of the L929.10 cells to TNF mediated lysis by modulating both of the TNF receptor dependent processes and by doubling the (apparent) rate of cell death (22 to 25%/hr) during the burst phase. These results suggest a direct relationship exists between the strength of the TNF-receptor signal and nuclear events, perhaps those associated with processes involved in DNA replication or cell division, during the cytotoxic reaction induced by these inflammatory cytokines.

• Watanabe N et al.
• [Analysis of the signalling pathway of TNF in normal cells and tumor cells]
• Gan To Kagaku Ryoho. 1987; 14: 611-7
• Display abstract

• The internalization process and intracellular distribution of 125I-labeled TNF, in L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and in HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells bearing TNF receptor, were elucidated by pulse-chasing and by Percoll density gradient centrifugation. Effect of TNF treatment on the RNA and protein synthesis of target cells was also studied using 3H-UDR and 35S-methionine incorporation. In both L-M and HEL cells, receptor-bound 125I-TNF was rapidly internalized and delivered to lysosomes within 15-30 min, followed by degradation and release into the culture medium. RNA synthesis and protein synthesis were not affected by TNF treatment in HEL cells, but marked stimulation (3.5 times and 4.2 times, respectively) was observed in L-M cells.

• Sappino AP, Alberto P
• [Tumor necrosis factor and cachectin. The same hormone with multiple effects]
• Ann Pathol. 1987; 7: 239-41
• Display abstract

• Tumor necrosis factor (= TNF) is a polypeptide hormone produced mainly by macrophages and so named according to its cytostatic and cytocidal properties. Its recent structural and functional characterization has led to the recognition of its multiple effects: TNF administration elicits a state of shock with multi-organ lesions, reminiscent of endotoxinemia, and induces wasting similar to the one associated with chronic debilitating diseases, justifying its denomination as cachectin. At a cellular level, TNF exerts a wide range of biological activities, many of them also ascribed to another macrophage-associated cytokine, namely interleukin-1. Its pleotropic effects suggest it might be the mediator of many pathological phenomenons and therefore, attempts at modulating its production might have greater clinical impacts than its use as an antineoplastic agent.

• Tsujimoto M
• [Tumor necrosis factor--recent advances]
• Tanpakushitsu Kakusan Koso. 1987; 32: 386-95

• Ishida N, Yoshida T
• [The role of cytokines in cancer therapy]
• Gan To Kagaku Ryoho. 1987; 14: 1187-93
• Display abstract

• A variety of normal tissue or malignant cells can produce and/or release various biologically active substances (hormone-like mediators) now collectively called cytokines. Because immunological and non-immunological responses of malignant cells were modified by many of them, some cytokines have been employed as so-called Biological Response Modifiers (BRM) in the treatment of cancers in animals and humans. This overview discussed a few of the difficulties, probably inherent in cytokine therapy, that have already been encountered in early clinical trials as well as some of those that can be anticipated in future work. These include unexpected and undesirable reactions due to the systemic administration in relatively large amounts of a cytokine that is, under physiological conditions, supposed to act as a paracrine and/or autocrine among cells located within a limited distance. Even a pure recombinant preparation of a cytokine is now known to affect multiple target cells if they are accessible to it. Furthermore, this kind of therapy may sometimes be little more than a shot in the dark, since the physiological balance (homeostasis) among many of the cytokines present or produced in a host receiving a large quantity of exogenous cytokines is not well understood. Making the situation still more complicated, many types of tumor cells are known to release some of these cytokines spontaneously. Many challenging problems remain to be solved before we can confidently prescribe a cocktail of cytokines precisely suitable for a given patient according to the individual's in vivo cytokine profile. Nevertheless, in spite of all these reservations, cytokine therapy has been too frequently beneficial to be allowed to be discouraged. "Out of this nettle, danger, we pluck this flower, safety".

• Kawakami M
• [Is cachectin TNF?]
• Seikagaku. 1987; 59: 1244-7

• Watanabe N, Niitsu Y
• [Anti-tumor effect and the mechanism of action of human recombinant TNF]
• Gan To Kagaku Ryoho. 1986; 13: 1322-8
• Display abstract

• TNF is cytokine derived from macrophages and holds strong promise for application to cancer therapy because of its marked antitumor effects and its high specificity to tumors. The clinical application (Phase I-II) of TNF has been started because human recombinant TNF (rH-TNF) can been produced on a large scale. In spite of notable antitumor effects, little is known concerning the mechanism of action of its cytotoxic activity. In this article, the antitumor effects of rH-TNF against human and murine tumors, the mechanism of its action and the synergistic effects of rH-TNF in combination with IFN-gamma or with cyclophosphamide are reviewed.

• Watanabe N et al.
• [Analysis of TNF receptor by binding assay]
• Gan To Kagaku Ryoho. 1986; 13: 2630-6
• Display abstract

• The existence of a TNF receptor on TNF-sensitive tumor cells and on certain normal cells was elucidated by specific binding assay. A close correspondence (r = 0.855) was shown between the receptor number and the sensitivity of the tumor cells. However, for normal cells, despite the existence of TNF receptors, no cytotoxic effect was observed. Furthermore, certain normal diploid cells underwent proliferation as a result of TNF stimulation. It was therefore concluded that the existence of TNF receptor is essential but not sufficient in itself for TNF-induced cytotoxicity.

• Kildahl-Andersen O, Lamvik J
• [Tumor necrosis factor. A new principle in the fight against cancer?]
• Tidsskr Nor Laegeforen. 1986; 106: 1789-90

• Ito H et al.
• Molecular cloning of the gene encoding rabbit tumor necrosis factor.
• DNA. 1986; 5: 157-65
• Display abstract

• A rabbit genomic DNA library was screened using a cloned cDNA encoding rabbit tumor necrosis factor (TNF) as a probe. The entire gene sequence appears to be contained within a 3.2-kb Eco RI fragment. Comparison of the nucleotide sequence of the gene with that of the cDNA showed that the gene consists of four exons. The nucleotide sequence of the rabbit TNF gene is very homologous to that of the human TNF gene. Southern hybridization of the genomic DNAs showed that there is likely only a single TNF gene in each of the rabbit and human genomes.

• Granger GA, Orr SL, Yamamoto RS
• Lymphotoxins, macrophage cytotoxins, and tumor necrosis factors: an interrelated family of antitumor effector molecules.
• J Clin Immunol. 1985; 5: 217-9

• Takahashi K, Kisugi J, Gatanaga T, Yamazaki M, Mizuno D, Abe S
• [Induction of cytotoxic activity in sera by immunomodulators]
• Yakugaku Zasshi. 1985; 105: 862-5

• Orita K
• [The possible modes of action of TNF (tumor necrosis factor)]
• Gan To Kagaku Ryoho. 1984; 11: 1379-86
• Display abstract

• TNF has been characterized by the fact that it not only induces hemorrhagic necrosis in various established tumors including Meth. A in vivo but also shows antitumor activity against several transformed cell lines in vitro. However, most studies on TNF have been carried out not with purified Preparations but with mouse or rabbit serum containing other cytokines like INF or with so-called tumor necrosis serum (TNS). Therefore, it is somewhat difficult and rather early to review the action mechanisms of TNF. According to a few reports which have used TNS to study the node of he action of TNF in vitro, its activity against transformed cell lines can be tentatively summarized as follows. First, the TNF molecule binds to the corresponding receptor on the cell surface, and then is transferred to the lysosome in a similar manner to the internalization of growth hormone and insulin. The TNF on the lysosomal membrane their induces injury to the membrane as a labilizer, finally causing cell destruction. TNF seems to be a cytotoxic rather than cytolytic substance. However, this recently described mechanism in vitro is not able to explain how TNF causes the central hemorrhagic necrosis of established solid tumors in vivo. Many unknown action mechanisms of TNF still remain to be clarified.

• Haranaka K, Satomi N, Sakurai A, Kunii O
• [Studies on tumor necrosis factor (TNF)]
• Nippon Rinsho. 1982; 40: 1872-9

• Kull FC Jr, Cuatrecasas P
• Possible requirement of internalization in the mechanism of in vitro cytotoxicity in tumor necrosis serum.
• Cancer Res. 1981; 41: 4885-90
• Display abstract

• The mechanism of in vitro cytotoxicity by tumor necrosis serum (TNS) and a purified fraction was examined using sensitive L-M cells. Cell death was assessed by uptake of the dye trypan blue and/or by release of radiolabeled chromium. Cell killing was time and dose dependent. Cell survival was assessed by counting the number of survivors via their ability to internalize the dye neutral red and/or by adherent cell protein. The cytotoxin was not cytostatic. Survival was found to be inversely proportional to cell death. The change in survival was used to estimate the number of cells killed. The number of cells killed was logarithmically related to the amount of toxin. The ability to kill a fixed number of cells was inversely related to the number of cells in the assay well. It was estimated that, at ideal cell seed numbers, 1 microgram of TNS protein per 250 microliter killed about 50,000 cells in a 20-hr period. Sensitivity was equated with the amount of TNS required to kill 35,000 cells in 20 hr. Inhibitors of RNA and protein synthesis and also elevated temperatures enhanced sensitivity. The combined treatment of 1 microM actinomycin D and 40 degrees enhanced sensitivity by 15-fold. Resistant normal and tumorigenic cell lines (including human) were rendered sensitive by concomitantly treating them with TNS and cycloheximide or actinomycin D. Cytoskeletal-disrupting agents (colchicine, Colcemid, and cytochalasin B), inhibitors of lysosome activity (chloroquine, methylamine, and leupeptin), and 32 degrees all depressed sensitivity. Sensitivity was nearly equivalent in calcium-free medium. Results substituting partially purified cytotoxin were similar. The results suggest that a toxic factor(s) may need to be internalized and that lysosomal activity may be necessary for cell killing. Comparisons with lymphotoxin are discussed.

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