Genes and Immunity (2001) 2, 181–190  2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene Structural characterisation of the distal 5' flanking region of the human interleukin-10 gene D Kube 1 , H Rieth 1 , J Eskdale 2 , PG Kremsner 1 and G Gallagher 2 1 Eberhard-Karls-Universita ¨t Universita ¨tsklinikum, Institut fu ¨ r Tropenmedizin Sektion Humanparasitologie, D-72074 Tu ¨ bingen, Germany; 2 University of Glasgow Department of Surgery, Queen Elizabeth Building, Glasgow Royal Infirmary, Glasgow G31 2ER Scotland, UK Interleukin-10 (IL-10) is an important immunoregulatory cytokine. The recent characterisation of the proximal 5' flanking region of IL-10 led to the identification of the promoter region. Two polymorphic dinucleotide repeats and 10 single nucleotide polymorphisms (SNPs) have been identified and suggested to be useful genetic markers in several diseases. We have sequenced a further 5275 bp from -9296 to -4021 of the distal part of the 5' flanking region of the human IL-10 gene from the cosmid clone pWE15-4/11. Our sequence analysis reveals a high density of Alu-repeats within the IL-10 gene locus, including three novel, related structures which we term Alu-IL10 (A-C). Using three overlapping PCR products spanning 5110 bp of this distal part of the IL-10 gene the following single base pair substitutions were identified: at -8571 C/T , -8531 G/A , -6752 A/T , -6208 G/C, -5402 C/G. In addition a heterozygous three base pair deletion at -7400 was observed. The SNPs at -8571 C/T and -8531 G/A are contained within an Alu-repeat. These data should further the understanding of how the IL-10 gene is controlled in man and how its function may vary between individuals. Genes and Immunity (2001) 2, 181–190. Keywords: interleukin-10; distal 5' flanking region; polymorphisms Introduction Recently we isolated the proximal 5'-flanking region of interleukin-10 (IL-10), characterized the promoter region and described a 4200-bp fragment. 1–4 IL-10 is an important multifunctional cytokine and a key regulatory component of many aspects of the immune response. 5 IL- 10 exerts a wide spectrum of biological activities in vitro and in vivo and is implicated in the regulation of the inflammatory and immune responses. 5–7 The ability of IL- 10 to block activation of cytokine synthesis and several accessory cell functions renders this cytokine a potent suppressor of the effector functions of macrophages, T cells and NK-cells. 8–10 Among the different cell types affected by IL-10, monocytes/macrophages and lympho- cytes appear to be particularly modified with regard to their function, morphology, and phenotype. The influence of IL-10 in the basic biology of the human immune system is reflected in its involvement with a range of autoimmune and malignant diseases. 6,7 In particular, IL-10 may contribute to the development and progression of diseases which involve the prolifer- Correspondence: D Kube, Eberhard-Karls-Universita ¨t, Institut fu ¨ r Trop- enmedizin Sektion Humanparasitologie, Wilhelmstraβe 27 D-72074 Tu ¨b- ingen, Germany. E-mail: Grant@laboratory.gg G Gallagher, University of Glasgow Department of Surgery, Queen Eliza- beth Building, Glasgow Royal Infirmary, Glasgow G31 2ER Scotland, UK. E-mail: Grant@laboratory.gg The sequence data reported in this paper have been submitted to the human genome database and have been assigned the accession number (update to X78437). Received 3 January 2001; revised 15 February 2001; accepted 20 February 2001 ation and differentiation of B cells, or the growth of human B-cell lymphomas, where its expression can be profoundly dysregulated 11,12 IL-10 is able to promote the proliferation and differentiation of B cells and the pro- duction of autoantibodies. 11,13 Indeed, elevated levels of IL-10 are found in the serum of systemic lupus ery- thematosus (SLE) patients and the number of IL-10 secreting cells is also increased. 14,15 Recent reports indi- cate that this cytokine may contribute to Epstein–Barr virus (EBV)-associated transformation. 16–20 It has been shown that IL-10 is an autocrine growth factor for AIDS- associated lymphoma cells in vitro and that IL-10 is a pathogenic factor for lymphoma development in huS- CID-mice. 18,21 Increased levels of IL-10 were detected in sera of patients with non-Hodgkin’s lymphomas as well as in Hodgkin’s disease. 22–25 High IL-10 levels were also associated with poor prognosis in acute infectious dis- eases, particularly meningococcal meningitis. 26,27 The expression of IL-10 is tightly regulated and the lev- els of constitutive expression in normal leukocytes are extremely low. In contrast, IL-10 is expressed in a range of activated cell-types, including monocytes, T cells and B cells. In addition, IL-10 is apparently constitutively secreted by EBV-immortalised B cells and EBV-positive Burkitt’s lymphoma (BL) cells. 16,17,19,28 However, the mechanism of the IL-10 induction in B cells is still unclear. Levels of IL-10 secretion are variable between individ- uals and almost 70% of such intra-individual variation is heritable. 29 It is reasonable therefore to examine the human IL-10 gene for polymorphic elements associated with such variation. We isolated the proximal 5'-flanking region of IL-10, characterised the promoter region and