Interleukin-10 (IL-10)

1.2.2.1 The IL-10 Gene

The gene for IL-10 is located on chromosome 1 and was mapped to a region between 1q31 and 1q32 (Eskdale et al., 1997). The gene spans 4.7 kb and consists of 5 exons separated by 4 introns. The transcriptional control of the gene has not been fully elucidated. Within the gene, there are several non-coding sequences that include TATA and CAT boxes, AP- 1 and NF-kB binding sites, a ORE, a glucocorticoid responsive element (GRE), and many NF-IL6 consensus binding sites (de Waal Malefyt, 2001). An ARE is also present in the 3’ UTR (Le et al., 1997). Transcription of IL-10 is known to be influenced by other cytokines such as TNF, IFNy and IL-12 (Kube etal., 2001)

1.2.2.2 The IL-10 Molecule

The IL-10 gene encodes for a protein of 178 amino acids. The first 18 amino acids encode the signal sequence, so that the mature molecule is 160 amino acids long and is about 17KDa. The molecule is biologically active as a non-covalently linked homodimer (de Waal Malefyt, 2001). IL- 10 shares common sequences with BGRF1, an open reading frame of the Epstein-Barr virus (EBV). BCRF1 is now considered the viral form of IL- 10 (vlL-10). It appears that human IL-10 is more closely related to BCRF- 1/vlL-10 (84% amino acid identity) than it is to its murine counterpart (73% amino acid identity). A single amino acid, at position 87, determines the

presence of immunostimulatory properties of IL-10 (Ding et al., 2000). vlL-10 lacks several of the immunostimulatory properties conferred to its human homologue, as it has a different amino acid at this critical position. It is likely that this amino acid interacts with an essential element of the IL- 10 receptor complex.

1.2.2.3 Functions

IL-10 was originally identified as a factor generated by mouse T-helper 2 (Th2) cells, which inhibits the synthesis of cytokines from T h i cells (See Section 1.3.1). For this reason it was known as cytokine synthesis inhibitory factor (CSIF) (Fiorentino etal., 1989).

In humans however, IL-10 cannot be considered a Th2 specific cytokine since its expression more closely resembles that of IL-6 than that of IL-4. Several cell types have been identified as sources of IL-10, these include CD4+ and CD8+ T lymphocytes, monocytes/macrophages, mast cells, eosinophils, kératinocytes and various tumour cells (Pretolani etal., 1999). Monocytes are usually the major source. The stimuli that promote IL-10 production include any of the microbial pathogens that are able to activate monocytes/macrophages, I cells or B cells.

IL-10 is thought of as an anti-inflammatory cytokine as it is able to inhibit the production of a wide range of pro-inflammatory cytokines. Upon activation, human monocytes produce large amounts of IL-10, the production of which usually follows that of the pro-inflammatory cytokines.

IL-10 therefore contributes to the subsequent attenuation of the inflammatory response. In lipopolysaccharide (LPS)-activated human monocytes, IL-10 inhibits the production of IL-1 a, IL-1p, IL-3, IL-6, IL-8, TNF-a, G-CSF and GM-CSF (de Waal Malefyt et al., 1991). All of these cytokines play roles in the recruitment and activation of cells in the immune system. This inhibition of cytokine synthesis is mainly via transcriptional inhibition (Wang et a!., 1994) but also by promoting the degradation of cytokine mRNA. Through a negative feedback mechanism, IL-10 is also able to inhibit its own production. Unlike mouse IL-10, the human form is also able to inhibit cytokine production from both T hi and Th2 cells. The inhibitory effect on IFNy production is indirect and is mediated through the suppression of IL-12 synthesis.

Further anti-inflammatory properties of IL-10 are:

• It stimulates the production of IL-1 receptor antagonist from monocytes and neutrophils;

• It prevents free radical and prostaglandin release from macrophages; • It targets neutrophils, where production of cytokines and other

mediators is inhibited;

• It prevents antigen-specific T cell responses via the downregulation of MHC class II, B7 and ICAM-1 on antigen presenting cells (APOs) (Pretolani etal., 1999).

IL-10 also has a number of immunostimulatory functions. For instance, IL- 10 augments the proliferation of mature and immature T cells in response

to IL-2 and IL-4 (MacNeil et al., 1990). Furthermore, Thompson-Snipes et ai. (1991) have shown that it can enhance the growth of mast cells and their progenitors when combined with IL-3 or IL-4. IL-10 also stimulates B cells activated by their antigen receptors or through CD40, where it has a co-stimulatory effect on proliferation. Moreover, it induces B cells to secrete large amounts of IgG, IgA and IgM (Rousset etal., 1992).

1.2.2.4 IL-10 Polymorphisms

In the IL-10 gene, three SNPs have been described: -1082 (G-^A), -819 (C->T) and -592 (G->A), with the -1082 polymorphism being associated with differential IL-10 production. Lymphocytes stimulated with concanavalin A were shown to produce significantly increased amounts of IL-10 if negative for the IL10 -1082A allele (Turner et al., 1997a). Similar results have also been obtained from LPS-stimulated whole blood (Koss

et al., 2000b). The transcriptional activity of the IL-10 promoter has also been found to be lower if the ATA haplotype is present (Crawley et al.,

1999). Recently, other SNPs have been described for example at -627 and -117 (Grove etal., 2000).

Two CA-repeat microsatellite polymorphisms have been described that are located nearby to the IL-10 gene. The first, IL-10G, is a CA-repeat that lies upstream of the gene between -1193 to -1151 and is very polymorphic having up to 16 alleles (Eskdale & Gallagher, 1995). The second, IL-1 OR lies between -4004 and -3978 in the 5’ flanking region. This CA-repeat is less polymorphic (Eskdale et al., 1996). As with many

of the other polymorphisms, these microsatellites have been associated with differential cytokine production. The IL-10.R2/IL-10.G14 haplotype has been associated with high IL-10 secretion whilst the IL-10.R3/IL- 10.G7 haplotype has been associated with low secretion (Eskdale et al.,

1998b).