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Effects of a Polymorphism in the Human Tumor Necrosis Factor

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Effects of a Polymorphism in the Human Tumor Necrosis Factor Proc. Natl. Acad. Sci. USA Vol. 94, pp. 3195–3199, April 1997 Immunology Effects of a polymorphism in the human tumor necrosis factor a promoter on transcriptional activation (geneticsymajor histocompatibility complexycytokineygene regulationyautoimmune diseases) ANTHONY G. WILSON†,JULIAN A. SYMONS†,TARRA L. MCDOWELL†,HUGH O. MCDEVITT‡, AND GORDON W. DUFF†§ †Section of Molecular Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom; and ‡Departments of Microbiology and Immunology, and Medicine, Stanford University, Stanford, CA 94305 Contributed by Hugh O. McDevitt, December 23, 1996 ABSTRACT Tumor necrosis factor a (TNFa) is a potent of TNFA in different strains of mice. Further, variation in the immunomodulator and proinflammatory cytokine that has production of TNFa from macrophages has been shown to been implicated in the pathogenesis of autoimmune and vary between strains (10). Several of these polymorphisms infectious diseases. For example, plasma levels of TNFa are have been correlated with TNFa production and with suscep- positively correlated with severity and mortality in malaria tibility to, or severity of, several diseases. The (NZB 3 and leishmaniasis. We have previously described a polymor- NZW)F1 mouse develops a severe autoimmune disease that is phism at 2308 in the TNFa promoter and shown that the rare very similar to systemic lupus erythematosus (SLE) in humans. allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3- A restriction fragment length polymorphism in the TNFA gene DQ2, which is associated with autoimmunity and high TNFa is correlated with low production of TNFa and with the production. Homozygosity for TNF2 carries a sevenfold in- development of lupus nephritis (11), and replacement therapy creased risk of death from cerebral malaria. Here we dem- with recombinant TNFa delays the onset of nephritis with onstrate, with reporter genes under the control of the two increased survival rate (12). Correlation of TNFa polymor- allelic TNF promoters, that TNF2 is a much stronger tran- phism with production of TNFa mRNA and resistance to scriptional activator than the common allele (TNF1) in a development of murine Toxoplasma gondii encephalitis has human B cell line. Footprint analysis using DNase I and B cell also been demonstrated in the BALByc strain (13). A study of nuclear extract showed the generation of a hypersensitive site Th2 cell-mediated local inflammatory responses has shown at 2308 and an adjacent area of protection. There was no that the TNF dependence of this phenomenon is related to difference in affinity of the DNA-binding protein(s) between H2D haplotypes and corresponding TNFa production pheno- the two alleles. These results show that this polymorphism has types, suggesting, at least in mice, that differential expression direct effects on TNFa gene regulation and may be responsible of TNFa from distinct alleles may influence the nature of an for the association of TNF2 with high TNFa phenotype and immune response (14). more severe disease in infections such as malaria and leish- TNF has potent biological actions, and control of its pro- maniasis. duction is tightly regulated, occurring both at the transcrip- tional and posttranscriptional levels (15). In response to lipo- Tumor necrosis factor a (TNFa) is a potent cytokine with a polysaccharide stimulation of macrophages, TNF transcription wide range of proinflammatory activities (1). It is classically increases 3-fold, TNF mRNA increases 50- to 100-fold, and produced by monocytesymacrophages, although other cell protein secretion increases by a factor of '10,000-fold (16). types, such as T and B cells, also produce significant amounts. Sequences within the 1100-bp stretch of DNA between the 39 The TNFA gene lies in the class III region of the major end of the lymphotoxin alpha gene and the 59 end of TNFA histocompatibility complex (MHC), '250 kilobases centro- have been shown to be central to the control of transcription meric of the HLA-B locus and 850 kilobases telomeric of (17, 18). HLA-DR. In view of its biological effects and gene location it Recently we and others have described two polymorphisms has been speculated that polymorphism within this locus might in the human TNFA promoter at 2308 (19) and 2238 (20), contribute to MHC associations with autoimmune and infec- both involving the substitution of guanine by adenosine in the tious diseases (2), particularly those in which TNFa has been uncommon alleles. We showed that the rare allele at 2308 implicated in initiating or sustaining the inflammatory re- (TNF2) is part of an extended MHC haplotype HLA-A1-B8- sponse, such as rheumatoid arthritis (3), or where increasing DR3-DQ2 (21), which is associated with high TNFa produc- blood levels have been shown to be predictive of poorer tion (5, 6). Studies in large populations have indicated that outcome, such as malaria (4). This has been supported by the carriage of TNF2 is associated with a worse outcome in association of specific MHC haplotypes with different TNFa phenotypes: DR3 and DR4 haplotypes produce higher levels cerebral malaria (22) and in leishmaniasis (23). of TNFa (5, 6) while DR2 haplotypes are associated with low To test whether the 2308 polymorphism has a functional production (5, 7), suggesting that functional polymorphism significance, we have investigated its effects on transcription might exist within regions that regulate the TNFA gene. using reporter gene assays. Our results show that the TNF2 Studies in mice have implicated the TNF locus with disease allele is a much more powerful transcriptional activator than phenotype. Polymorphisms have been described in the pro- the common allele. Although we can demonstrate specific moter region (8), the first intron, and 39 untranslated region (9) binding of a nuclear protein and DNase I hypersensitivity at the polymorphic site, there was no obvious difference in The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: TNFa, tumor necrosis factor a; TNFA, gene for TNFa; accordance with 18 U.S.C. §1734 solely to indicate this fact. MHC, major histocompatibility complex; SLE, systemic lupus ery- thematosus; CAT, chloramphenicol acetyltransferase; EMSA, elec- Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA trophoretic mobility-shift assay; IL, interleukin; PMA, phorbol 12- 0027-8424y97y943195-5$2.00y0 myristate 13-acetate. PNAS is available online at http:yywww.pnas.org. §To whom reprint requests should be addressed. 3195 Downloaded by guest on September 30, 2021 3196 Immunology: Wilson et al. Proc. Natl. Acad. Sci. USA 94 (1997) affinity of the protein(s) for the two alleles. These results Extraction of DNA-Binding Proteins from Raji Cells. Nu- indicate that this polymorphism may have a direct effect on clear proteins were extracted from Raji cells as described (27). transcriptional activity and may underlie the association of the Prior to use, the protein concentration was determined. HLA-A1-B8-DR3 haplotype with high TNFa production and Generation of Radiolabeled TNFA Promoter Fragment. A be directly responsible for the poorer outcome reported in 119-bp fragment (2345 to 2226) of the TNFa promoter was malaria and leishmaniasis with carriage of the TNF2 allele. amplified by PCR from TNF1 and TNF2 homozygous indi- viduals using primers 59-CAAAAGAAATGGAGGCAAT-39 and 59-TCCTCCCTGCTCCGATTCCG-39. The two allelic MATERIALS AND METHODS fragments were then cloned into the TA vector, and the Generation and Cloning of TNFa Promoter Fragments. A sequences were confirmed as above. Probe was prepared by fragment of 691 bp (2585 to 1106) of the TNFA gene was restriction digestion using NsiI and HindIII, [a-32P]dCTP amplified by PCR using primers 59-GCTTGTCCCTGCTAC- end-labeled using the Klenow fragment (Promega), and then CCGC-39 and 59-GTCAGGGGATGTGGCGTCT-39, and cy- purified with a Sephadex G-50 NICK column (Pharmacia). cles as described (19). The fragments were cloned into the TA The specific activity of the probe was measured with a vector and used to transform Escherichia coli (strain INVF9a) b-counter. (Invitrogen, United States Biochemical). Following selection Electrophoretic Mobility Shift Assay (EMSA). Nuclear pro- and propagation, pure plasmid DNA was prepared by standard tein extract (10 mg) was incubated with 2 mg of poly(dI-dC) methods (24). The TNF promoter alleles were removed from (Pharmacia) in 25 ml of buffer composed of 10 mM TriszHCl the TA vector by restriction enzyme digestion with HindIII and (pH 7.5), 75 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, XbaI (Promega) to allow for directional cloning into the 12.5% glycerol, and 0.1% (volyvol) Triton X-100 at room pBLCAT3 expression vector (25). The sequences of the inserts temperature for 30 min. After the addition of 2.5 ng of labeled were checked by the dideoxy chain termination method using probe and incubation for a further 30 min, 2.5 ml of loading Sequenase (United States Biochemical). buffer consisting of 250 mM TriszHCl (pH 7.5), 0.2% bromo- Transfection of Human B Cells. Experiments were per- phenol blue, 0.2% xylene cyanol, and 40% glycerol was added formed using the human Raji B cell line. Cells were cultured and the samples were electrophoresed in a 0.25 3 TBE (90 mM in 13 RPMI 1640 medium adjusted to pH 7.4 with 1 M NaOH, Trisy64.6 mM boric acidy2.5 mM EDTA, pH 8.3)y4% non- buffered with 7.5% (volyvol) sodium bicarbonate, and supple- denaturing polyacrylamide gel. Visualization of bands was by autoradiography. Quantification of competitor DNA concen- mented with 5% (volyvol) fetal calf serum (GIBCOyBRL), trations was by measurement of optical density at 260 nm. This penicillin (100 units per ml), streptomycin (100 mgyml), and glutamine (2 mM) (Northumbria Biologicals, Northumber- was checked by comparing band intensities following agarose land, England).
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