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.     

Expression profiling in knockout mice: lymphotoxin versus tumor necrosis factor in the maintenance of splenic microarchitecture

Expression profiling provides a powerful approach to define the underlying molecular mechanisms in disease. Several techniques referred collectively to as gene profiling may be also helpful in the analysis of the phenotype of mice with targeted mutations, especially if applied to distinct histological compartments, to specific cell types or to evaluate the effect of specific challenges, such as infection. Here we review several of the existing techniques applicable to genetic knockout studies, and share our experience from the study of mice with tumor necrosis factor (TNF) and lymphotoxin (LT) deficiencies, with specific emphasis on the distinction between TNF- and LT-mediated signalling pathways in vivo. Gene expression profiling analysis of TNF/LT-deficient mice supports the notion that TNF and LT, originally discovered as distinct biological activities, manifest both distinct and redundant functions in vivo.     

Novel lymphotoxin alpha (LTalpha) knockout mice with unperturbed tumor necrosis factor expression: reassessing LTalpha biological functions

Lymphotoxin alpha (LTalpha) can exist in soluble form and exert tumor necrosis factor (TNF)-like activity through TNF receptors. Based on the phenotypes of knockout (KO) mice, the physiological functions of LTalpha and TNF are considered partly redundant, in particular, in supporting the microarchitecture of the spleen and in host defense. We exploited Cre-LoxP technology to generate a novel neomycin resistance gene (neo) cassette-free LTalpha-deficient mouse strain (neo-free LTalpha KO [LTalphaDelta/Delta]). Unlike the "conventional" LTalpha-/- mice, new LTalphaDelta/Delta animals were capable of producing normal levels of systemic TNF upon lipopolysaccharide (LPS) challenge and were susceptible to LPS/D-galactosamine (D-GalN) toxicity. Activated neutrophils, monocytes, and macrophages from LTalphaDelta/Delta mice expressed TNF normally at both the mRNA and protein levels as opposed to conventional LTalpha KO mice, which showed substantial decreases in TNF. Additionally, the spleens of the neo-free LTalpha KO mice displayed several features resembling those of LTbeta KO mice rather than conventional LTalpha KO animals. The phenotype of the new LTalphaDelta/Delta mice indicates that LTalpha plays a smaller role in lymphoid organ maintenance than previously thought and has no direct role in the regulation of TNF expression.     

Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes. TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4+ T cell hybridoma

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

Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes

Reverse signaling of transmembrane TNF (mTNF) contributes to the versatility of this cytokine superfamily. Previously, we could demonstrate that mTNF acting as receptor confers resistance to bacterial lipopolysaccharide in monocytes and macrophages (MO/MPhi). Reverse signaling can be induced by incubation with the monoclonal anti-TNF antibody 195F and other TNF antagonists, such as the humanized monoclonal antibody infliximab and the humanized soluble TNF receptor construct etanercept, respectively, all in former or present clinical use. Here, we addressed the question whether there are differences in modulating the LPS response in MO/MPhi among these three antagonists. Whereas 195F and infliximab suppress both, the release of an LPS-induced endothelial cell apoptotic factor and proinflammatory cytokines, etanercept only protected against the LPS-triggered apoptosis activity, but left the LPS-induced cytokine release unchanged. These data could have clinical impact with regard to TNF neutralization strategies.     

Histamine antagonizes tumor necrosis factor (TNF) signaling by stimulating TNF receptor shedding from the cell surface and Golgi storage pool

Tumor necrosis factor (TNF) activates pro-inflammatory functions of vascular endothelial cells (EC) through binding to receptor type 1 (TNFR1) molecules expressed on the cell surface. The majority of TNFR1 molecules are localized to the Golgi apparatus. Soluble forms of TNFR1 (as well as of TNFR2) can be shed from the EC surface and inhibit TNF actions. The relationships among cell surface, Golgi-associated, and shed forms of TNFR1 are unclear. Here we report that histamine causes transient loss of surface TNFR1, TNFR1 shedding, and mobilization of TNFR1 molecules from the Golgi in cultured human EC. The Golgi pool of TNFR1 serves both to replenish cell surface receptors and as a source of shed receptor. Histamine-induced shedding is blocked by TNF-alpha protease inhibitor, an inhibitor of TNF-alpha-converting enzyme, and through the H1 receptor via a MEK-1/p42 and p44 mitogen-activated protein kinase pathway. Cultured EC with histamine-induced surface receptor loss become transiently refractory to TNF. Histamine injection into human skin engrafted on immunodeficient mice similarly caused shedding of TNFR1 and diminished TNF-mediated induction of endothelial adhesion molecules. These results both clarify relationships among TNFR1 populations and reveal a novel anti-inflammatory activity of histamine.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.     

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.     

Sequence of the tumor necrosis factor/cachectin (TNF) gene from Peromyscus leucopus (family Cricetidae)

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M59233.     

In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.     

Establishment of double grafted tumor model in mice and role of "tumor necrosis factor" (TNF)]

A double grafted tumor model (colon 26 and Meth-A) was established in BALB/c mice. The primary colon 26 tumor growth was inhibited by the secondary transplantation of Meth-A tumor cells into the same host, and the prolongation of mean survival time was also observed. To investigate the mechanism of the prolongation of survival, the role of tumor necrosis factor (TNF) was examined. The intraperitoneal inoculation of human TNF-alpha (1 x 10(4) units) for 5 days, as well as the secondary Meth-A tumor cell transplantation, resulted in the prolongation of survival. Moreover, the prolongation of survival disappeared by the inoculation of rabbit anti-TNF-alpha antibodies. These results suggest that TNF-alpha play an important role in the mechanism of a prolongation of survival by the secondary transplantation of the tumor cells.     

Chromosomal localization and molecular organization of human genomic fragment containing TNF/LT locus in transgenic mice

Molecular organization, copy number and chromosomal localization of human TNF/LT locus fragment were determined in genomes of two transgenic mouse lines. Genome of the first one contains two copies, organized in head-to-tail manner and determined on eighth chromosome by karyotyping; single transgene copy of the second line is observed on the fifth chromosome. These mice could serve as valuable model for studying both human tumor necrosis factor and lymphotoxin physiological functions.     

Chromosomal localization and molecular organization of the human genomic fragment containing the TNF/LT locus in transgenic mice

The molecular organization, copy number, and chromosome location of the human TNF/LT transgenes were studied in the genomes of two transgenic mouse strains. One strain proved to carry two transgene copies arranged head-to-tail and detected on chromosome 8 by karyotyping. The other strain had one transgene copy observed on chromosome 5. The strains provide a model for studying the physiological functions of the tumor necrosis factor and lymphotoxin.     

Glia-pinealocyte network: the paracrine modulation of melatonin synthesis by tumor necrosis factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.