The genes for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are tandemly arranged on chromosome 17 of the mouse

We have isolated clones containing the gene for tumor necrosis factor (TNF-alpha) from a mouse genomic library. Four out of five clones containing the TNF-alpha gene also hybridized to a human lymphotoxin (TNF-beta) probe. We constructed a restriction enzyme cleavage map of a 6.4 kb region from one of the genomic clones. From partial sequencing data and hybridizations with exon-specific oligonucleotide probes, we conclude that this region contains the mouse TNF-alpha and TNF-beta genes in a tandem arrangement, that they are separated by only about 1100 bases, and that their intron-exon structure is very similar to that seen in man. We probed genomic blots of DNA from human/mouse hybrids containing single mouse chromosomes for the presence of the mouse TNF genes. The results show that the genes are located on mouse chromosome 17, which also contains the major histocompatibility complex. Therefore, both the mouse and the human TNF genes are tandemly arranged and located on the same chromosome as the MHC.     

Production of tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) by murine pre-B and B cell lymphomas

Media from murine pre-B and B lymphoma cell cultures, but not from myeloma cell cultures, was cytotoxic to WEHI 164 cells, causing these TNF-sensitive targets to release 51Cr. The cytotoxic activity in the culture medium reached maximum levels approximately 4 days after the cell culture was initiated. The constitutive production of the factors was not influenced by depletion of serum from the medium or by the addition of either phorbol ester or bacterial endotoxin. The factor has a Mr greater than 10 kDa, and its cytotoxicity was abolished by anti-serum against murine TNF. Northern blot analysis with the use of cDNA probes to murine tumor necrosis factor (TNF-alpha) and lymphotoxin (LT, TNF-beta) showed high levels of TNF-mRNA in the pre-B cell lines, lower levels in the mature B cell lines and no TNF-mRNA in the myeloma cell lines. LT mRNA was present in pre-B cell lines, at a much lower concentration in only one of the B cell lines, and was not present in three other B lymphomas or in the myelomas tested. The results show a positive correlation between the presence of TNF and/or LT mRNA and the 51Cr-releasing activity present in the cell culture medium. Our data indicate that TNF and LT can be produced by murine B cells and that the synthesis of these cytokines may be restricted to certain differentiation stages of the B cell lineage.     

DNA sequence polymorphism at the human tumor necrosis factor (TNF) locus. Numerous TNF/lymphotoxin alleles tagged by two closely linked microsatellites in the upstream region of the lymphotoxin (TNF-beta) gene.

TNF-alpha and lymphotoxin (LT, TNF-beta) genes are tandemly arranged and map within the MHC centromeric to HLA-B and telomeric to the class III genes. Both cytokines encoded by these genes are potent immunomodulators. On the other hand, some MHC-linked autoimmune diseases are characterized by abnormal levels of their expression or inducibility. A search for the putative disease-associated TNF/LT alleles depends on the informative genetic markers at the TNF locus. Previously, a low degree of genetic polymorphism at the human TNF locus has been reported, mostly bi-allelic RFLP. To localize and define additional polymorphic markers, we probed the collection of genomic clones with synthetic tandemly repeated dinucleotides, corresponding to the sequences known as microsatellites. We mapped and characterized three (TC/GA) and one (AC/GT) repeats within cloned 40-kb DNA comprising the human TNF locus. Using a polymerase chain reaction-based technique, we analyzed three of these four microsatellites and observed their length of polymorphism. Using DNA samples from blood donors, two families, and three human cell lines, we detected 13 distinct alleles of the AC/GT microsatellite neighboring human TNF genes. The variability was further increased by simultaneous analysis of the second linked microsatellite. This linked TC/GA repeat showed at least five alleles, whereas the least polymorphic TC/GA repeat located in the first intron of LT (TNF-beta) gene had two alleles. TNF alleles defined by microsatellites were stably inherited and segregated in the Mendelian way. Therefore, we describe thus far the most informative level of DNA sequence polymorphism in this part of human MHC. We propose a nomenclature for microsatellite tagged LT/TNF alleles based on their size and variability, which could also be extended to include RFLP and other not yet identified polymorphic markers. Microsatellite tagged polymorphism described here can be used in systematic linkage studies of HLA-associated diseases.     

Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice

Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.     

Mouse lymphotoxin and tumor necrosis factor: structural analysis of the cloned genes, physical linkage, and chromosomal position

Lymphotoxin (LT) and tumor necrosis factor (TNF) are cytotoxic and immunoregulatory lymphokines which have similar activities but are produced by different cell types. We have cloned the murine LT and TNF genes from a lambda:mouse DNA recombinant library, using as probes synthetic oligonucleotides defined by portions of the human LT or TNF cDNA sequences. Analysis of the genomic clones indicates that the LT and TNF genes are physically linked, i.e., approximately 1.2 kb separates the 3' end of LT from the 5' end of TNF genes. By using, first, a series of recombinant inbred lines, and second, a series of H-2-recombinant congenic strains, we determined that the LT/TNF gene cluster lies on chromosome 17, closely linked to the H-2D end of the murine H-2 complex. Comparison of the primary sequence of murine and human LT revealed that the intron-exon structure of murine LT is similar in these two species. Comparison of the predicted amino acid sequences of murine and human LT indicates that the proteins are about 72% homologous with much greater sequence conservation in regions encoding the COOH-terminal portion. Comparison of the 5' flanking sequence of LT to a number of genes that are specifically expressed in activated T cells reveals a number of conserved sequences that may play a role in control of these genes.     

Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization.

Human Tumor Necrosis Factor and Lymphotoxin are cytotoxic proteins which have similar biological activities and share 30 percent amino acid homology. The single copy genes which encode these proteins share several structural features: each gene is approximately three kilobase pairs in length and is interrupted by three introns. In addition, these genes are closely linked and have been mapped to human chromosome 6. However, only the last exons of both genes, which code for more than 80 percent of each secreted protein, are significantly homologous (56 percent).     

Cloning and characterization of gene TNF alpha encoding equine tumor necrosis factor alpha.

We report the molecular cloning and nucleotide sequence of the equine gene encoding tumor necrosis factor alpha. The 2610-bp genomic sequence was derived from three overlapping polymerase chain reaction products.     

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

Cytolysis of tumor necrosis factor (TNF)-resistant tumor targets. Differential cytotoxicity of monocytes activated by the interferons, IL-2, and TNF

Herein we demonstrate that IFN-alpha, IFN-gamma, and IL-2 can induce human peripheral blood monocyte-mediated lysis of tumor cells that are resistant to both the direct effects of TNF and to monocytes activated by TNF. Monocytes activated by TNF kill only TNF-sensitive tumor targets, whereas those activated by IFN and IL-2 can lyse both TNF-sensitive and TNF-resistant tumor targets. Monocyte cytotoxicity against TNF-sensitive lines induced by the IFN, IL-2, or TNF can be completely abrogated by the addition of anti-TNF antibodies. In contrast, anti-TNF antibodies have no effect on IFN- or IL-2-induced monocyte cytotoxicity against TNF resistant targets, confirming non-TNF-mediated lysis induced by lymphokine-activated monocytes. Neither induction of TNF receptors by IFN-gamma nor inhibition of RNA synthesis by actinomycin D increased the susceptibility of TNF-resistant tumor targets to TNF-mediated monocyte cytotoxicity. Thus, non-TNF-mediated modes of monocyte cytotoxicity are induced by IFN and IL-2, but not by TNF, indicating that different cytotoxic mechanisms are responsible for the lysis of TNF-sensitive and TNF-resistant tumor cells. In addition, these findings also suggest that TNF-sensitive lines are susceptible only to TNF-mediated killing and apparently insensitive to non-TNF-mediated monocyte cytotoxicity.     

Alleles and haplotypes of tumor necrosis factor (TNF) alpha and beta genes in three ethnic populations of Sulawesi Indonesia

Polymorphic variation in two cytokine genes, tumor necrosis factor (TNF) -alpha and -beta, was examined in three ethnic groups, the Bugis, the Makassans, and the Torajans, who inhabit Sulawesi, a large island in the Indonesian archipelago, and formerly a Dutch colony. TNF-alpha and -beta are key molecules in immune responses to infection, and both have been implicated in the pathogenesis and clinical manifestations of parasitic diseases. Several polymorphic variants with the potential to affect cytokine levels in autoimmune diseases and parasitic and bacterial infection have been reported. Two loci in the promoter region of TNF-alpha and two sites in the first intron of TNF-beta were scored in a maximum of 150 Bugis, 168 Makassans, and 58 Torajans. Genotypes at the two TNF-alpha loci are not in Hardy-Weinberg equilibrium because of a deficit of heterozygotes (p < 0.05). However, genotypes at the TNF-beta loci exhibit Hardy-Weinberg equilibrium. A comparison of allelic and genotypic frequencies at all TNF loci across the ethnic groups reveals that the differences are significant for TNFalpha(308) (p < 0.01) and for TNFbeta(NcoI) (p < 0.05). Overall, the distribution of the alleles differs from that seen in the few Asian populations for which data are available (p < 0.05). Construction of 4-locus haplotypes showed that, in addition to the five previously reported, four novel haplotypes were present in Sulawesi. These novel haplotypes were in low frequency, and two were seen only in Bugis (haplotypes F and J) and one (haplotype K) only in Makassans. The other, haplotype D, was present in Makassans and Torajans. Preliminary sampling of other ethnic groups suggests that three of these haplotypes (D, F, and J) may be restricted to Asian or Asian-derived populations. The frequency of the common TNF haplotypes differed between Dutch and Sulawesi populations, and these data also indicated that haplotype E, which has a relatively high frequency in the Dutch (25%), may be a useful marker of Dutch/European admixture in Indonesian populations, in which it is either rare (1%) or absent. The results suggest that unique allelic combinations with potential to influence cytokine secretion are present in Sulawesi, possibly as a consequence of parasite-driven selection, and argue for more extensive investigation of haplotype distribution in parasite-endemic areas.     

Effects of tumor necrosis factor (TNF) on lipolytic activities of rat heart

Summary Tumor Necrosis Factor (TNF) inhibits lipoprotein lipase activity in cultured myocytes and in the Langendorff rat heart after 3 h perfusion with TNF of glucocorticoid-pretreated rats. TNF acutely stimulates glyc(ogen)olysis and concomitantly endogenous lipolysis. The latter was significantly increased only when rats had been pretreated with glucocorticoid or fed a trierucate-rich diet. Under these conditions, contractile activity of the Langendorff hearts was acutely increased by TNF The mechanism of the actue increase of contractile function and the accompanying increased glycolytic and lipolytic activities, by TNF, may be explained by increased cytosolic Ca²⁺ and cAMP levels.