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tumor necrosis factor-alpha.
CF (cytotoxic factor);
DIF (differentiation inducing factor);
EP (endogenous pyrogens);
Macrophage-derived cytotoxic factor;
J774-derived cytotoxic factor;
MCF (macrophage cytotoxic factor);
MCT (macrophage cytotoxin);
MD-FGF [monocyte-derived fibroblast growth factor]
PCF (peritoneal cytotoxic factor);
RCF (Released cytotoxic factor).
See also: individual entries for further information.
The new nomenclature is TNFSF2 [TNF ligand superfamily member 2], based on homology with other members of the TNF ligand superfamily of proteins.
TNF is secreted by macrophages, monocytes, neutrophils, T-cells, NK-cells following their stimulation by bacterial lipopolysaccharides. Cells expressing CD4 secrete TNF-alpha while CD8(+) cells secrete little or no TNF-alpha. Stimulated peripheral neutrophilic granulocytes but also unstimulated cells and also a number of transformed cell lines, astrocytes, microglial cells, smooth muscle cells, and fibroblasts also secrete TNF. Human milk also contains this factor (see: MGF, milk growth factor).
The synthesis of TNF-alpha is induced by many different stimuli including interferons (see: IFN), IL2, GM-CSF, SP (substance P; see also: Tachykinins), Bradykinin, Immune complexes, inhibitors of cyclooxygenase and PAF (platelet activating factor).
The release of TNF-alpha from isolated rat macrophages in culture is stimulated by Histogranin.
The production of TNF is inhibited by IL6, TGF-beta, vitamin D3, prostaglandin E2, dexamethasone, CsA (Cyclosporin A), and antagonists of PAF (platelet activating factor).
Human TNF-alpha is a non-glycosylated protein of 17 kDa and a length of 157 amino acids. Murine TNF-alpha is N-glycosylated. Homology with TNF-beta is approximately 30 %. TNF-alpha forms dimers and trimers.
The 17 kDa form of the factor is produced by processing of a precursor protein of 233 amino acids. A TNF-alpha converting enzyme (see: TACA) has been shown to mediate this conversion.
A transmembrane form of 26 kDa has been described also.
TNF-alpha contains a single disulfide bond that can be destroyed without altering the biological activity of the factor. Mutations Ala84 to Val and Val91 to Ala reduce the cytotoxic activity of the factor almost completely. These sites are involved in receptor binding. The deletion of 7 N-terminal amino acids and the replacement of Pro8Ser9Asp10 by ArgLysArg yields a mutated factor with an approximately 10-fold enhanced antitumor activity and increased receptor binding, as demonstrated by the L-M cell assay, while at the same time reducing the toxicity. For other genetically engineered variants see: TNF-alpha muteins.
The gene has a length of approximately 3.6 kb and contains four exons. The primary transcript has a length of 2762 nucleotides and encodes a precursor protein of 233 amino acids. The aminoterminal 78 amino acids function as a presequence (see also: gene expression).
The human gene maps to chromosome 6p23-6q12. It is located between class 1 HLA region for HLA-B and the gene encoding complement factor C. The gene encoding TNF-beta is approximately 1.2 kb downstream of the TNF-alpha gene. However, both genes are regulated independently. The two genes also lie close to each other on murine chromosome 17.
Two receptors of 55-60 kDa and 75-80 kDa have been described.
The 55-60 kDa has been given the designation CD120a in the nomenclature of CD antigens and is also referred to as TNFRSF1A [TNF receptor superfamily member 1A].
Various designations have been used for this receptor:
p55 TNF receptor
p60 TNF receptor
TBP1 [TNF binding protein-1]
TNFAR (TNF-alpha receptor alpha subunit)
TNFR [TNF receptor]
TNFR1 [TNF-alpha receptor type 1, TNF receptor 1]
TNFR55 [TNF-alpha receptor 55 kDa, 55 kDa TNF-alpha receptor]
TNFR60 [TNF-alpha receptor 60 kDa, 60 kDa TNF-alpha receptor]
TNF receptor 1.
The 75-80 kDa receptor has been given the designation CD120b in the nomenclature of CD antigens and is also referred to as TNFRSF1B [TNF receptor superfamily member 1B]. Various designations have been used for this receptor:
TBP-2 [tumor necrosis factor binding protein-1]
TNFR2 [TNF receptor type 2; TNF receptor 2]
TNFR75 [TNF-alpha receptor 75 kDa]
TNFR80 [TNF-alpha receptor 80 kDa]
Approximately 500-10000 high-affinity receptors (Ka = 2.5x 1010**-9 M) for TNF-alpha are expressed on all somatic cell types with the exception of erythrocytes.
One receptor is a glycosylated protein of 455 amino acids that contains an extracellular domain of 171 and a cytoplasmic domain of 221 amino acids. Sequence homologies in the cysteine-rich domains of the extracellular portion reveal that the receptor is related to the low affinity receptor of NGF and to human cell surface antigen CD40.
Deletion analysis in the C-terminal intracellular region of the 55 kDa receptor, TNFR1 has revealed the existence of a so-called death domain, which is involved in signaling processes leading to programmed cell death. The death domain of TNFR1 interacts with a variety of other signaling adaptor molecules, including TRADD, and RIP.
The two known receptors bind both TNF-alpha and TNF-beta. p55 is expressed particularly on cells susceptible to the cytotoxic action of TNF. p75 is also present on many cell types, especially those of myeloid origin (for a virus-encoded homolog of the receptor subunit see also: EBI-6: EBV induced gene-6). It is expressed strongly on stimulated T-cells and B-lymphocytes. The differential activities of TNF on various cell types, i.e., growth-promoting and growth-inhibiting activities, are probably mediated by the differential expression and/or regulation of multiple receptors in combination with other distinct receptor-associated proteins. p55 appears to play a critical role in host defenses against micro-organisms and their pathogenic factors (see subentry Transgenic /Knock-out/Antisense studies).
A third receptor subtype is expressed in normal human liver. It binds TNF-alpha but not TNF-beta. Some viruses contain genes encoding secreted proteins with TNF binding properties that are closely homologous to the p55 and p75 TNF receptors (see: Viroceptor).
Differential effects of the two receptor subtypes have been found also in TNF mediated adhesion of leukocytes to the endothelium. It appears that engagement of the p55 receptor specifically leads to the induction of the cellular adhesion molecules ICAM-1 (CD54), E-selectin, VCAM-1, and CD44, while engagement of both the p55 and the p75 receptor induces expression of integrin-alpha-2.
Truncated soluble forms of the receptor have been found also. The soluble forms, in particular the soluble extracellular domain of the p60 receptor, block the antiproliferative effects of TNF and, therefore, may modulate the harmful effects of TNF.
Apart from the membrane-bound receptors several soluble proteins that bind TNF have been described. These proteins of approximately 30 kDa, called TBP-1 and TBP-2 (tumor necrosis factor binding proteins; see: TNFBP), are derived from the TNF-binding domain of the membrane receptor. They can be isolated from urine and serum and probably function as physiological regulators of TNF activities by inhibiting binding of TNF to its receptor (see: TNF-BF, TNF blocking factor).
Receptor densities are reduced by IL1 and tumor promoters such as Phorbol esters. The expression of TNF-alpha receptor density is induced by IFN-alpha, IFN-beta, and IFN-gamma.
For signal transducers that associate with the cytoplasmic domains of members of the TNF receptor superfamily see: TRAF (Tumor necrosis factor receptor-associated factors).
Human TNF-alpha is active on murine cells with a slightly reduced specific activity. In general, TNF-alpha and TNF-beta display similar spectra of biological activities in in vitro systems, although TNF-beta is often less potent or displays apparent partial agonist activity.
TNF-alpha shows a wide spectrum of biological activities. It causes cytolysis and cytostasis of many tumor cell lines in vitro. Sensitive cells die within hours after exposure to picomolar concentrations of the factor and this involves, at least in part, mitochondria-derived second messenger molecules serving as common mediators of TNF cytotoxic and gene-regulatory signaling pathways. The factor induces hemorrhagic necrosis of transplanted tumors. Within hours after injection TNF-alpha leads to the destruction of small blood vessels within malignant tumors. The factor also enhances phagocytosis and cytotoxicity in neutrophilic granulocytes and also modulates the expression of many other proteins, including fos, myc, IL1 and IL6 (see: TIS genes).
The 26 kDa form of TNF is found predominantly on monocytes and T-cells after cell activation. It is also biologically active and mediates cell destruction by direct cell-to-cell contacts (see: juxtacrine).
In vivo TNF-alpha in combination with IL1 is responsible for many alterations of the endothelium. It inhibits anticoagulatory mechanisms and promotes thrombotic processes and therefore plays an important role in pathological processes such as venous thromboses, arteriosclerosis, vasculitis, and disseminated intravasal coagulation (see also: Systemic inflammatory response syndrome). The expression of membrane thrombomodulin is decreased by TNF-alpha. TNF-alpha is a potent chemoattractant for neutrophils (see also: Chemotaxis) and also increases their adherence to the endothelium (see also subentry: receptors). The chemotactic properties of fMLP (Formyl-Met-Leu-Phe) for neutrophils are enhanced by TNF-alpha. TNF-alpha induces the synthesis of a number of chemoattractant cytokines (see also: Chemotaxis), including IP-10, JE, KC, in a cell-type and tissue-specific manner.
Although TNF inhibits the growth of endothelial cells in vitro it is a potent promoter of angiogenesis in vivo. The angiogenic activity of TNF is significantly inhibited by IFN-gamma.
TNF-alpha is a growth factor for normal human diploid fibroblasts. It promotes the synthesis of collagenase and prostaglandin E2 in fibroblasts. It may function also as an autocrine growth modulator for human chronic lymphocytic leukemia cells in vivo and has been described to be an autocrine growth modulator for neuroblastoma cells. The autocrine growth-promoting activity is inhibited by IL4.
In resting macrophages TNF induces the synthesis of IL1 and prostaglandin E2. It also stimulates phagocytosis and the synthesis of superoxide dismutase in macrophages. TNF activates osteoclasts and thus induces bone resorption.
TNF-alpha inhibits the synthesis of lipoprotein lipase and thus suppresses lipogenetic metabolism in adipocytes.
In progenitors of leukocytes and lymphocytes TNF stimulates the expression of class 1 and II HLA and differentiation antigens, and the production of IL1, colony stimulating factors (see: CSF), IFN-gamma, arachidonic acid metabolism. It also stimulates the biosynthesis of collagenases in endothelial cells and synovial cells.
IL6 suppresses the synthesis of IL1 induced by bacterial endotoxins and TNF, and the synthesis of TNF induced by endotoxins.
The neurotransmitter SP (substance P; see also: Tachykinins) induces the synthesis of TNF and IL1 in macrophages. IL1, like IL6, stimulates the synthesis of ACTH (corticotropin) in the pituitary. Glucocorticoids synthesized in response to ACTH in turn inhibit the synthesis of IL6, IL1 and TNF in vivo, thus establishing a negative feedback loop between the immune system and neuroendocrine functions.
TNF promotes the proliferation of astroglial cells and microglial cells and therefore may be involved in pathological processes such as astrogliosis and demyelinisation.
TNF-alpha enhances the proliferation of T-cells induced by various stimuli in the absence of IL2. Some subpopulations of T-cells only respond to IL2 in the presence of TNF-alpha. In The presence of IL2 TNF-alpha promotes the proliferation and differentiation of B-cells.
The functional capacities of skin Langerhans cells are also influenced by TNF-alpha. These cells are not capable of initiating primary immune responses such as contact sensibilisation. They are converted into immunostimulatory dendritic cells by GM-CSF and also IL1. These cells therefore are a reservoir for immunologically immature lymphoid dendritic cells. The enhanced ability of maturated Langerhans cells to process antigens is significantly reduced by TNF-alpha.
Although TNF-alpha is required also for normal immune responses the overexpression has severe pathological consequences. TNF-alpha is the major mediator of cachexia observed in tumor patients (hence its name: Cachectin). TNF is also responsible for some of the severe effects during Gram-negative sepsis (see: Systemic inflammatory response syndrome).
TNF mediates part of the cell mediated immunity against obligate and facultative bacteria and parasites. It confers protection against Listeria monocytogenes infections, and anti-TNF antibodies weaken the ability of mice to cope with these infections.
TRANSGENIC ANIMALS, KNOCK-OUT, AND ANTISENSE STUDIES
The consequences of a deregulated expression of TNF-alpha have been studied in transgenic mice expressing the human factor. Overexpression of TNF-alpha precipitates chronic inflammatory polyarthritis, which can be prevented completely by treatment of the animals with monoclonal antibodies directed against the human factor. Transgenic mice constitutively overexpressing human TNF-alpha in their T-cell compartment under the control of human CD2 gene regulatory signals have been shown to develop marked histologic and cellular changes locally in their lymphoid organ and a lethal wasting syndrome associated with widespread vascular thrombosis and tissue necrosis. These changes could be neutralized also by the administration of monoclonal antibodies specific for human TNF-alpha. In another TNF-alpha transgenic mouse model the expression of the transgene in murine pancreatic beta-cells results in severe and permanent insulitis without evolution towards diabetes.
Transgenic mouse line bearing a reporter gene construct have been constructed in which chloramphenicol acetyl transferase coding sequences are expressed under the control of TNF regulatory sequences. In these animals expression of the transferase within tissues reflects TNF production. Analysis of these animals shows that the transferase is expressed constitutively in both the fetal and maternal thymuses, and in the placenta, but in no other tissues. Crosses between transgenic and non-transgenic mice indicate that trophoblasts, rather than the decidua or uterus, is the source of transferase activity. A soluble TNF receptor/IgG heavy chain chimeric protein, which strongly inhibits TNF activity in vitro and in vivo, can cross the placenta, but does not interrupt pregnancy, and has no obvious effect on fetal development. These findings suggest that TNF may not be required for completion of a normal gestation.
Recent studies with mice deficient in p55 receptor expression (generated by targeted homologous recombination in embryonic stem cells) demonstrate that the development of populations of thymocytes and lymphocytes are unaltered and that clonal deletion of potentially self-reactive T-cells is not impaired. The loss of p55 functions in these mice, which still express the p75 TNF receptor, renders mice resistant to lethal dosages of either bacterial lipopolysaccharides or enterotoxins (see also: Systemic inflammatory response syndrome). These mice, however, readily succumb to infections with L. monocytogenes.
DETECTION AND ASSAY METHODS
TNF-alpha can be detected in bioassays involving cell lines that respond to it (see: BT-20, CT6, EL4; KYM-1D4, PK15; L929; L-M; MO7E; T1165; WEHI-3B). TNF-alpha can be detected also by a sensitive sandwich enzyme immunoassay, ELISA, an immunoradiometric assay (IRMA), and by an assay designated RELAY (receptor mediated label-transfer assay). Intracellular factor is detected by two color immunofluorescence flow cytometry. Higuchi et al (1995) have described an assay based on the release of tritiated thymidine from cells undergoing apoptosis after treatment with either TNF-alpha or TNF-beta. IFN-alpha, IFN-beta, IFN-gamma, TGF-beta, IL4, LIF and GM-CSF have been shown not to interfere with this assay. Turpeinen and Stenman have developed a sandwich-type time-resolved immunofluorometric assay (IFMA) for TNF-alpha. The assay also measures TNF-alpha complexed with soluble receptors. An alternative and entirely different detection method is RT-PCR quantitation of cytokines. For further information see also subentry "Assays" in the reference section. For further information on assays for cytokines see also: bioassays, cytokine assays.
CLINICAL USE AND SIGNIFICANCE
In contrast to chemotherapeutic drugs TNF specifically attacks malignant cells. Extensive preclinical studies have documented a direct cytostatic and cytotoxic effect of TNF-alpha against subcutaneous human xenografts and lymph node metastases in nude mice (see also: Immunodeficient mice), as well as a variety of immunomodulatory effects on various immune effector cells, including neutrophils, macrophages, and T-cells. Single- and multiple-dose phase I studies have confirmed that TNF can be administered safely to patients with advanced malignancies in a dose range associated with anticancer effect without concomitant serious toxicities such as shock and cachexia. However, clinical trials on the whole have unfortunately so far failed to demonstrate significant improvements in cancer treatment, with TNF resistance and TNF induced systemic toxicity being two major limitations for the use of TNF as an antineoplastic agent in most cases. The combined use of TNF and cytotoxic or immune modulatory agents, particularly IFN-gamma and possibly IL2, may be of advantage in the treatment of some tumors. In some cases intratumoral application of TNF has been found to be of advantage in tumor control.
Some mutant forms of TNF-beta with selective activity on the p55 receptor have been described recently. It has been shown that activation of the p55 receptor is sufficient to trigger cytotoxic activity towards transformed cells. Some of these mutants have been described to retain their antitumor activity in nude mice (see also: Immunodeficient mice) carrying transplanted human tumors. It is hoped that such mutant forms may induce less systemic toxicity in man.
TNF can be used to increase the aggressiveness of lymphokine-activated killer cells (see: LAK cells).
There are some indications that inhibitors of TNF may be of advantage. Since TNF is found in the synovial fluid of patients suffering from arthritis, these inhibitors may be helpful in ameliorating the disease and this has been shown to be the case in animal models of severe collagen induced arthritis. Inhibitors may ameliorate also the severe consequences of Systemic inflammatory response syndrome.
TNF-alpha appears to be an important autocrine modulator promoting the survival of hairy cell leukemia cells. It may be important, therefore, in the pathogenesis of this disease.
Studies with an experimental fibrosarcoma metastasis model have shown that TNF induces significant enhancement of the number of metastases in the lung. It has been suggested that low doses of endogenous TNF or administration of TNF during cytokine therapy may enhance the metastatic potential of circulating tumor cells. The transduction of murine tumor cells with a functional TNF-alpha gene has been shown to lead to the rejection of the genetically modified cells by syngeneic hosts (for cancer vaccines see also: Cytokine gene transfer).
TNF-alpha has been shown also to protect hematopoietic progenitors (see also: hematopoiesis) against irradiation and cytotoxic agents, suggesting that it may have some potential therapeutic applications in aplasia induced by chemotherapy or bone marrow transplantation.
One case of severe therapy-resistant Morbus Crohn has been treated with monoclonal antibodies directed against TNF-alpha. Treatment has been reported to have resulted in a complete remission lasting for three months.
See REFERENCES for entry TNF-alpha.
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