Localization of the genes for tumor necrosis factor and lymphotoxin between the HLA class I and III regions by field inversion gel electrophoresis.

Medizinische Klinik, University of Tübingen, Federal Republic of Germany.
Immunogenetics (Impact Factor: 2.49). 02/1988; 27(1):66-9. DOI: 10.1007/BF00404447
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Available from: Elisabeth H Weiss, Jun 29, 2015
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    ABSTRACT: Summary Sincea dysregulatedsynthesisoftumor necrosisfactora (TNF-a) may be involvedin the pathogenesisofautoimmune diseases, itwas ofinterest toprecisely locatetherecentlyreported Ncol restriction fragmentlengthpolymorphism (RFLP) oftheTNF-a region.However,by mapping of56.8kb ofoverlappingcosmidclonesanddirectsequencing,we couldlocalize the polymorphicNcol restriction sitewithinthefirstintronoftheTNF-0 gene and notinthe TNF-a gene.To studywhetherregulatorymechanismsareaffected bythispolymorphism,we analyzedtheTNF-a/TNF-0 productionof phytohemagglutinin-stimuated peripheralblood mononuclearcells ofindividuals homozygousfortheTNF-0 NcolRFLP byELISA andconcomitant Northern blotanalysis . On days2-4 afterstimulationwith mitogen,the TNFB*1 allele correspondingtoa5.3-kbNcol fragmentpresentedwithasignificantly higherTNF-0 response. A mRNA analysis demonstratedthathigherproteinlevels ofTNF-0 correlate alsowithincreased amountsofTNF-0 transcripts .No allelic association wasfoundinrespecttoTNF-a production. Tofurtherinvestigate apossible allelic influence on transcription, we determinedtheDNA sequence of2kb ofthe5'portionofourclonedTNFB*2 allele andcompareditwiththeavailable TNF-0 sequences .By computer-aided recognition motifsearchofDNA bindingfactors, we reportputative bindingsitesconservedbetweenmouse and man inthe5'flankingregionaswellasinintron 1oftheTNF-0 gene,foundalsoinothercytokinepromotersequences .Inaddition, bypolymerase chainreactionamplification andsequencingof740by ofthe5'partofTNF-0 ofindividuals typedhomozygouslyfortheNcol RFLP, we couldshow thataminoacidposition26isconserved asasparagineintheTNFB *1andasthreonineintheTNFB *2sequence.A previouslyreported, EcoRI RFLP inthe3'untranslated regionofTNF-0 doesnotsegregatewitheitherofthetwo alleles .Thus,fourTNFB alleles cande definedattheDNA level .
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    ABSTRACT: We studied the effect of the different types of interferons on the production of cytotoxin by human peripheral blood mononuclear cells (PBMCs) stimulated with the mitogen phytohemagglutinin (PHA). Maximum secreted levels of cytotoxin were observed at day 3 in cul- ture and consisted of both tumor necrosis factor � (TNF- a) and lymphotoxin as determined by specifIc antibodies. Type I interferons (IFN-a and IFN-f3) consistently sup- pressed cytotoxin production. Both TNF-a and lym- photoxin were significantly suppressed. Mean suppres- sion by IFN-a and IFN-f3 (1000 U/mi) was 56 and 66%, respectively, in PBMCs from 18 different donors. The suppressive effects of IFN-a and IFN-f.3 on cytotoxin production were dose responsive over a range of 10 to 1000 U/mi. Type II interferon (IFN-'y) did not have con- sistent significant effects. Pretreatment with IFN-a or IFN-/3 for 24 or 48 h prior to PHA stimulation also resulted in significant suppression. Supplementation with interleukin-2 (10 U/ml) or IFN-'y (1000 U/mi) did not overcome cytotoxin suppression by IFN-a or IFN-f3. Cytotoxin suppression by IFN-a and IFN-f3 together ap- peared to be noninteractive. Suppression appeared not to � be due to blockade of the cytotoxin release, since both cell-associated cytotoxin and secreted cytotoxin were sup- pressed to the same level. These results demonstrated that cytotoxin and lymphotoxin production by PHA- stimulated PBMCs could be down-regulated by type I in- terferons and that there is a substantial difference be- tween the action of type I interferons and type II interfe- rons (IFN-y) in modulating the biosynthesis of cytotoxins.J. Lcukoc. Biol. 52: 165-172; 1992.
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    ABSTRACT: The production of tumour necrosis factor (TNF) and interleukin 1 (IL-1) by lipopolysaccharide-activated mononuclear cells from 39 healthy donors was studied in vitro by bioassay and ELISA. The donors were typed for HLA-A, -B, -C, -DR, and -DP antigens. There was no detectable production of TNF beta (lymphotoxin). The intracellular levels of bioactive TNF alpha were minimal or undetectable in all cases. Cells from HLA-DR2+ individuals secreted significantly lower amounts of TNF alpha than cells from HLA-DR2- donors [2 ng/ml (1.5-4.4) and 7.5 ng/ml (3.9-8.3) respectively (medians 25-75%); P less than 0.01]. The difference disappeared if the cells were preactivated for 2 days with 1000 U/ml of recombinant gamma interferon (rIFN-gamma). In some individuals, the TNF alpha response increased considerably after IFN-gamma priming, in particular in those possessing the HLA-DR2 antigen. In contrast, there was no detectable difference in the production of IL-1 beta between the donors, and the IL-1 beta response decreased significantly after rIFN-gamma priming in HLA-DR2+ individuals [2.3 ng/ml (1.1-8.4) versus 7.2 ng/ml (5-7.9); P less than 0.05] and in HLA-DR2- individuals [3 ng/ml (1.1-5.3) versus 5.7 ng/ml (3.9-7.5); P less than 0.01]. There was no correlation between the TNF alpha and IL-1 responses and any of the other HLA-DR, -DP, or -B antigens. There was a significant positive correlation between the levels of TNF alpha measured by ELISA and by cytotoxicity assay. However, the TNF alpha-containing supernatants from 9 out of 37 individuals appeared to contain inhibitor(s) of the biological activity of TNF alpha. The presence of inhibitor(s) was not associated with any HLA antigens.
    Scandinavian Journal of Immunology 12/1988; 28(5):599-606. DOI:10.1111/j.1365-3083.1988.tb01492.x · 1.88 Impact Factor