Cloning and mutation analysis of the human IL-18 promoter: a possible role of polymorphisms in expression regulation

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Abstract

Interleukin-18 is a proinflammatory cytokine, which strongly induces IFN-γ production. We have cloned the human IL-18 promoter and screened for possible polymorphisms. Three single nucleotide polymorphisms were detected in the promoter and two polymorphisms in the 5′-nontranslated region of the gene. Three combinations of these polymorphisms were observed in the Swedish population. All IL-18 promoter alleles were found to have clear promoter activity when inserted into a luciferase reporter vector. There were no significant differences in promoter activity between alleles without stimulation, but after stimulation with PMA/ionomicin one of the alleles had clearly lower activity than the other (P<0.01). Measurement of IL-18 and IFN-γ production in 48 multiple sclerosis patients by RT-PCR showed slightly higher expression of IL-18 in individuals homozygous for the most frequent haplotype. Two IL-18 promoter polymorphisms were analyzed by sequence specific PCR in 208 multiple sclerosis patients and 139 healthy controls, however, no significant differences were found. In summary, our data indicate that common IL-18 promoter polymorphisms influence the expression of IL-18 and potentially also of IFN-γ.

Introduction

Interleukin-18 (IL-18) is a proinflammatory cytokine initially isolated from liver cells (Okamura et al., 1995, Ushio et al., 1996). It shares structural features with interleukin-1β (IL-1β). Both cytokines are produced as inactive precursor molecules, which are then processed by caspase-1 whereby biologically active molecules are released (Ghayur et al., 1997). Recently, the IL-18 receptor was identified. It was found that IL-18 binds to the molecule formerly known as the IL-1 receptor related protein, which is highly similar to the IL-1 receptor (Torigoe et al., 1997). IL-18 is mainly produced by activated macrophages and, like interleukin-12 (IL-12), is able to induce interferon-γ (IFN-γ) (Okamura et al., 1995). In fact, IL-12 and IL-18 exhibit synergism in interferon-γ induction. This effect is based on the ability of IL-12 to upregulate IL-18 receptor expression on target cells (Yoshimoto et al., 1998). IL-18 also enhances expression of granulocyte macrophage colony stimulating factor (Ushio et al., 1996, Horwood et al., 1998) and Fas (Dao et al., 1996) ligand and decreases the production of interleukin-10 (Ushio et al., 1996). Primary targets for IL-18 are T lymphocytes and NK cells (Takeda et al., 1998, Garcia et al., 1999).

Being involved in the proinflammatory cytokine network, IL-18 could have an important role in the development of inflammatory diseases. Increased production of IL-18 was observed in the acute phase of experimental autoimmune encephalomyelitis (EAE) (Jander and Stoll, 1998) and antibodies to IL-18 were able to prevent EAE in Lewis rats (Wildbaum et al., 1998). A rise in IL-18 also preceded development of the acute phase of diabetes in the NOD mouse (Rothe et al., 1997). In humans, upregulation of IL-18 production was shown in Crohn’s disease (Monteleone et al., 1999). In multiple sclerosis (MS), IL-18 was found expressed in demyelinating brain lesions (Balashov et al., 1999).

Regulation of IL-18 expression has been extensively studied by several research groups and its promoter has been cloned in the mouse (Tone et al., 1997). Recently sequences upstream of the human IL-18 cDNA were published and studied for promoter activity (Takeuchi et al., 1999). However, the detailed structure and sequence variations of the human IL-18 promoter has remained uncharacterized. Here, we present functional properties of the human IL-18 promoter as well as the presence of a number of polymorphisms and their distribution in MS.

Section snippets

IL-18 promoter cloning

The sequence upstream of the published IL-18 mRNA (Ushio et al., 1996) sequence was cloned using the GenomeWalker kit (Clontech). Briefly, the GenomeWalker kit contains five libraries of uncloned, adaptor-ligated genomic DNA fragments. Fragments for each library are prepared by cutting genomic DNA with one of five restriction enzymes (EcoRV, ScaI, DraI, PvuII, SspI). A gene of interest is amplified from these libraries by two consecutive PCR reactions. For IL-18 promoter amplification, we use

Cloning and characterization of IL-18 promoter region

Amplification of the sequences upstream of the published IL-18 mRNA sequence (GeneBank accession no. D49950) from genomic libraries resulted in three different fragments (from DraI, PvuII and SspI genomic libraries, 604-bp, 1500-bp and 586-bp, respectively). Sequences of the fragments perfectly matched each other. In this way, 1378 base pairs of sequence upstream from the known human IL-18 mRNA sequence were obtained (Fig. 1).

By 5′-RACE analysis, two major transcription start sites were

Discussion

In this article we describe the cloning and functional analysis of the sequence upstream from the human IL-18 first exon. This sequence has clear promoter activity, which can be significantly enhanced by stimulation with PMA/ionomicin. Studies of the mouse IL-18 promoter have shown similar increase of activity under stimulation. In the mouse, two alternative promoters were found (Tone et al., 1997), whereas two promoters may also be present in humans (Takeuchi et al., 1999). However, a more

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