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Cytokine (Tnfα, Ltα and IL-10) Polymorphisms in Inflammatory

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Genes and Immunity (2000) 1, 185–190 2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00 www.nature.com/gene Cytokine (TNF␣,LT␣ and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies K Koss1,2,3, J Satsangi1, GC Fanning3, KI Welsh3 and DP Jewell1 1Gastroenterology Unit, Radcliffe Infirmary, Oxford, UK; 2Gastroenterology Department, Medical University of Gdansk, Poland; 3Transplant Immunology, Nuffield Department of Surgery, Churchill Hospital, Oxford, UK The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNF␣), lymphotoxin-alpha (LT␣) and interleukin-10 (IL-10) genes on stimulated TNF␣ and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn’s disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNF␣,LT␣ and IL-10 genes. Production of the TNF␣ and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNF␣ gene, three haplotypes of LT␣ and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LT␣-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LT␣-2 haplotypes were associated with higher TNF␣ production in CD patients, and the TNF-4 haplotype was associated with lower TNF␣ production in UC patients. The A allele in the IL-10 promoter region at position −1082 was associated with decreased IL- 10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNF␣,LT␣ and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls. Genes and Immunity (2000) 1, 185–190. Keywords: TNF␣;LT␣; IL-10; Crohn’s disease; ulcerative colitis; polymerase chain reaction Introduction important producers of TNF and IL-10 but not LT␣, are known to be in a state of activation. Indeed the pro- ␣ Tumour necrosis factor-alpha (TNF ) is a potent pro- duction of TNF␣ has led to clinical strategies for its block- inflammatory and immunoregulatory cytokine that plays ade by anti-TNF␣ antibodies in Crohn’s disease (CD).8 a key role in the initiation, regulation and perpetuation IL-10 also has proven relevance to IBD since knockout of of the inflammatory response, whereas interleukin-10 (IL- the IL-10 gene induces chronic enterocolitis in mice.9 The 10) inhibits the production of the pro-inflammatory cyto- concentration of IL-10 has been reported to be increased ␣ ␥ kines (IL-1, TNF , IFN- and IL-6) through its action on in patients with both active ulcerative colitis (UC) and 1–3 Th1 cells and macrophages. The close interrelation- active CD when compared to healthy controls.10 This ␣ ships between TNF and IL-10 have been confirmed in study tests the hypothesis that the cytokine profile may 4,5 several studies. Another possible interacting cytokine is be imbalanced in IBD due to both monocyte activation ␣ lymphotoxin-alpha (LT ), which shares the same recep- and genetic polymorphism. tors with TNF␣ and also maps in the HLA class III region on the short arm of chromosome 6. Stable TNF␣ pro- duction, varying less than 15%, has been described, when Results single individuals are sampled over time.6 This makes comparisons between individuals more meaningful. TNF␣ and LT␣ polymorphisms Studies on monozygotic twins and their first-degree rela- Four haplotypes of the three polymorphisms in the TNF␣ tives, using ex vivo endotoxin stimulated whole blood gene and three haplotypes of the three polymorphisms in ␣ samples, provide evidence that 60% of TNF and 75% the LT␣ gene were identified in the examined population of IL-10 variation in production capacity appears to be (Table 1). There were no significant differences in fre- 7 genetically determined. quencies of the biallelic polymorphisms or whole haplo- In inflammatory bowel disease (IBD), monocytes, types between controls, CD and UC patients (Table 2). When subgroups of patients were analysed, haplotype TNF-2 was more frequent in women with extensive com- Correspondence: Dr Konrad Koss, House 29F, New Staff Residence, City pared to distal colitis (31% vs 12%, P = 0.028). When ana- General Hospital, Stoke on Trent ST4 6QG, UK. ␣ E-mail: konradkossȰyahoo.com lysed for the combined haplotype TNF-2-LT -2, the Received 21 June 1999; revised 7 September 1999; accepted 24 Sep- association became stronger (21% vs 56%, P = 0.0007). tember 1999 There was strong linkage disequilibrium between TNF-2 Genetics of TNF, LT and IL-10 in IBD K Koss et al 186 Table 1 Defined haplotypes of TNF␣,LT␣ and IL-10 gene polymorphisms observed in examined population of 435 subjects. Positions of biallelic polymorphisms (+488, −238, −308, +720, +365, +249, −1082, −819, −592) are calculated from the mRNA start codons TNF␣ +488 −238 −308 LT␣ +720 +365 +249 IL-10 −1082 −819 −592 (4538) (AA26) (AspHI) (NcoI) TNF-1 G G G LT␣−1 C C A IL10-GCC G C C TNF-2 G G A LT␣−2 A G G IL10-ACC A C C TNF-3 A G G LT␣-3 C G A IL10-ATA A T A TNF-4 G A G Table 2 TNF␣,LT␣ and IL-10 haplotypes frequencies in Crohn’s effect of steroids was observed in UC patients but the disease (CD), ulcerative colitis (UC) patients and controls. Haplo- difference was not significant, perhaps due to the fact types frequencies were calculated in the group of 236 healthy con- that all patients were receiving 5-ASA. trols, 111 UC, and 91 CD patients. No significant difference in geno- type or haplotype frequencies were observed in those groups examined Effect of CD, UC and smoking on TNF and IL-10 production ␣ Haplotypes Controls UC patients CD patients The LPS-stimulated TNF production was higher in con- (n = 236) (n = 111) (n = 91) trols than in the group of 47 CD or 49 UC patients with- out steroid treatment (Controls: 8958 ± 1978 pg/mL vs TNF-1 0.67 0.72 0.75 CD: 4791 ± 1819 pg/mL, P = 0.0045 or vs UC: 6028 ± 2393 TNF-2 0.2 0.17 0.15 pg/mL, P = 0.015). Also LPS-stimulated IL-10 production TNF-3 0.08 0.06 0.04 was higher in controls than in UC (P = 0.012) or CD TNF-4 0.06 0.05 0.06 patients (Controls: 235 ± 108 pg/mL vs UC: 164 ± 69 LT␣-1 0.38 0.37 0.36 = ± = LT␣-2 0.36 0.41 0.36 pg/mL; P 0.012 or vs CD: 123 43 pg/mL, P 0.023). LT␣-3 0.26 0.23 0.27 Smokers with CD showed higher stimulated IL-10 pro- TNF-2/LT␣-2 0.18 0.16 0.14 tein production in whole blood than non-smokers (208 ± IL10 GCC 0.52 0.55 0.58 76 pg/mL vs 105 ± 34 pg/mL, P = 0.011). This was not IL10 ACC 0.23 0.21 0.20 apparent in healthy controls (281 ± 111 pg/mL vs 220 ± IL10 ATA 0.25 0.24 0.22 78 pg/mL) and UC patients (176 ± 76 pg/mL vs 158 ± 68 pg/mL). There was a tendency towards lower LPS-stimulated TNF␣ production in UC patients with extensive colitis haplotype and LT␣-2 haplotype, which both together compared with distal colitis (4024 ± 1760 vs 6584 ± 1665 form the most frequent haplotype in healthy controls and pg/mL), and a tendency towards lower TNF␣ production IBD patients. in CD patients with colonic involvement compared with ileitis (3784 ± 1671 vs 5340 ± 1891 pg/mL). However, IL-10 polymorphisms these differences did not reach statistical significance. The three biallelic polymorphisms of the IL-10 gene occurred in only three haplotypes (Table 1). No signifi- TNF␣ and LT␣ polymorphisms and TNF␣ production cant differences in allele or haplotypes frequencies were Since steroids have been shown to suppress TNF␣ and observed between the patients and controls (Table 2). IL-10 release from circulating mononuclear cells, func- tional analysis of TNF␣,LT␣ and IL-10 polymorphisms Effect of medication on TNF␣ and IL-10 production were performed in patients who had not received steroid TNF␣ and IL-10 production was measured in lipopoly- treatment for at least a month prior to assay. saccharide (LPS)-stimulated blood taken from a ran- In CD patients, TNF-2 or LT␣-2 haplotypes were asso- domly selected group of 52 controls and 136 IBD patients. ciated with higher TNF␣ production (TNF-2: 6383 ± 828 Large intra-individual differences in LPS-stimulated vs 3890 ± 1544 pg/mL, P = 0.003; LT␣-2: 6039 ± 1322 vs whole blood production of cytokines were observed. No 3487 ± 2142 pg/mL, P = 0.002). However, in controls the effect on the number of white blood cells (WBC) in the LT␣-2 haplotype was associated with lower TNF␣ pro- blood samples and stimulated TNF␣ or IL-10 production duction (8458 ± 1810 vs 10731 ± 2044 pg/mL, P = 0.012). was observed. In UC patients a lower production of TNF␣ was seen in In IBD patients, steroid treatment significantly association with the TNF-4 haplotype (Table 3). decreased LPS-stimulated TNF␣ secretion (1418 ± 1799 vs 5612 ± 1972 pg/mL, P = 0.013). No significant effect on IL-10 polymorphism and IL-10 production LPS-stimulated TNF␣ secretion was observed in patients Due to the stimulating effect of 5-ASA, SLSZ and the sup- treated with 5-aminosalicylic acid (5-ASA) or sulphasala- pressing effect of prednisolone on IL-10 production, data zine.
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  • Blockade of LIGHT/Ltβ and CD40 Signaling Induces Allospecific T Cell Anergy, Preventing Graft-Versus-Host Disease

    Blockade of LIGHT/Ltβ and CD40 Signaling Induces Allospecific T Cell Anergy, Preventing Graft-Versus-Host Disease

    Blockade of LIGHT/LTβ and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease Koji Tamada, … , Bruce R. Blazar, Lieping Chen J Clin Invest. 2002;109(4):549-557. https://doi.org/10.1172/JCI13604. Article Previous studies have shown that blockade of LIGHT, a T cell costimulatory molecule belonging to the TNF superfamily, by soluble lymphotoxin β receptor–Ig (LTβR-Ig) inhibits the cytotoxic T lymphocyte (CTL) response to host antigenic disparities and ameliorates lethal graft-versus-host disease (GVHD) in a B6 to BDF1 mouse model. Here, we demonstrate that infusion of an mAb against CD40 ligand (CD40L) further increases the efficacy of LTβR-Ig, leading to complete prevention of GVHD. We further demonstrate that alloantigen-specific CTLs become anergic upon rapid expansion, and persist in the tolerized mice as a result of costimulatory blockade. Transfer of anergic CTLs to secondary F1 mice fails to induce GVHD despite the fact that anergic CTLs can be stimulated to proliferate in vitro by antigens and cytokines. Our study provides a potential new approach for the prevention of lethal GVHD. Find the latest version: https://jci.me/13604/pdf Blockade of LIGHT/LTβ and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease Koji Tamada,1 Hideto Tamura,1 Dallas Flies,1 Yang-Xin Fu,2 Esteban Celis,1 Larry R. Pease,1 Bruce R. Blazar,3 and Lieping Chen1 1Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA 2Department of Pathology, University of Chicago, Chicago, Illinois, USA 3Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA Address correspondence to: Lieping Chen, Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.