Streszczenie

IL-18 jest plejotropową cytokiną odgrywającą istotną rolę w modulowaniu odpowiedzi immunolo­gicznej i w patogenezie wielu schorzeń o podłożu zapalnym. W pracy opisano związek między 7 jednonukleotydowymi polimorfizmami w genie IL18 a ekspresją IL18 na poziomie mRNA i wy­dzielaniem tej cytokiny przez stymulowane jednojądrzaste komórki krwi obwodowej (PBMC). PBMC izolowano z krwi obwodowej 29 zdrowych ochotników, u których oznaczono polimor­fizmy IL18: rs1946518: A>C, rs187238: G>C, rs360718: A>C, rs360722: C>T, rs360721: C>G, rs549908: T>G i rs5744292: A>G. Stężenia IL-18 oraz poziomy mRNA analizowano po 48-go­dzinnej inkubacji z różnymi stymulatorami (PHA, LPS, przeciwciałami anty-CD3/CD28). Po za­stosowaniu LPS i przeciwciał anty-CD3/CD28 stężenia IL-18 były istotnie statystycznie mniej­sze u homozygot AA rs1946518 w porównaniu z nosicielami allela C. Gdy analizowano poziomy mRNA IL18 między komórkami niestymulowanymi oraz stymulowanymi PHA i LPS istotnie ob­niżoną ekspresję genu stwierdzono u nosicieli genotypu AA rs1946518 w porównaniu do nosi­cieli allela C. Podobne zmiany obserwowano w odniesieniu do polimorfizmu rs187238, jednakże istotność statystyczną stwierdzono tylko dla komórek stymulowanych PHA. Wyniki naszej pra­cy potwierdzają rolę polimorfizmów rs1946518 (-607A>C) i rs187238 (-137G>C) jako genetycz­nych determinant wpływających na ekspresję IL18.

Słowa kluczowe:polimorfizm genetyczny • interleukina 18 • jednojądrzaste komórki krwi obwodowej • stymulatory

Summary

Introduction: Interleukin-18 (IL-18) is a pleiotropic cytokine playing an important role as a modulator of im­mune responses, found to play a role in pathogenesis of numerous inflammatory-associated di­sorders. In the present study a potential association between 7 common single-nucleotide poly­morphisms (SNPs) spanning the whole IL18 gene, gene expression and the release of IL-18 from the stimulated peripheral blood mononuclear cells (PBMCs) was investigated.
Materials/Methods: PBMCs were isolated from peripheral blood of 29 healthy volunteers, genotyped for the presen­ce of IL18 SNPs: rs1946518: A>C, rs187238: G>C, rs360718: A>C, rs360722: C>T, rs360721: C>G, rs549908: T>G, and rs5744292: A>G. IL-18 concentration and IL18 mRNA levels were in­vestigated after incubation of cells for 48 h with different stimulants (PHA, LPS, and anti-CD3/CD28 antibodies).
Results: After treatment with LPS and antibodies IL-18 concentrations were significantly lower in rs1946518AA homozygotes than in C allele carriers. When differences in IL18 mRNA levels be­tween non-stimulated and stimulated cells were analyzed, significantly decreased gene expres­sion was noted in rs1946518 AA homozygotes (as compared with C allele carriers) in samples treated with PHA and LPS. Similar trends were observed in the case of rs187238 SNP; however, the differences reached statistical significance only after PHA treatment.
Conclusions: Our study supports the role of rs1946518 (-607A>C) and rs187238 (-137G>C) SNPs as genetic determinants of the observed variability in IL18 expression.

Key words:genetic polymorphism • interleukin-18 • peripheral blood mononuclear cells • stimulants

Introduction

Interleukin-18 (IL-18) is a pleiotropic cytokine playing an important role as a modulator of immune responses. IL-18 was first discovered as a potent IFN-inducing factor, pro­duced by macrophages and dendritic cells. IL-18 was the first cytokine found to activate Th1 cells to produce abun­dant IFN-γ (interferon-gamma) without T cell receptor en­gagement [8,9,13]. Constitutively produced as an inactive precursor (Pro-IL-18) by several cell types, IL-18 is acti­vated in response to inflammatory and infectious stimuli. Pro-IL-18 expression is widespread, including monocytes, macrophages, dendritic cells, keratinocytes, articular chon­drocytes, synovial fibroblasts and osteoclasts [2]. In addi­tion to its effects on Th1 cells, IL-18 is a strong stimulator of the activity of natural killer cells and CD8+ lymphocy­tes. Together with IL-2, IL-18 can also stimulate the pro­duction of IL-13 and other Th2 cytokines [1]. IL-18 was found to play a role in pathogenesis of numerous inflam­matory-associated disorders, including infections, autoim­mune diseases, cancer, as well as metabolic syndrome and atherosclerosis [2,3,15].

Although the IL18 gene was resequenced in different po­pulations, only a few missense and mRNA splicing inter­fering single-nucleotide polymorphisms (SNPs) have been found. However, some variation within the 5′ untranslated region (UTR) and 3′ UTR was observed, which may cause differences in translation rate and mRNA stability, as well as variation within the proximal promoter that may cause alterations in transcription rate [11]. Two single-nucleoti­de polymorphisms at position -607 (rs1946518: C>A) and -137 (rs187238: G>C) in the promoter region were initial­ly found to be associated with IL18 gene promoter trans­criptional activity [5]. In the most thorough investigation into the link between IL18 variation and IL-18 serum le­vels, two of five investigated SNPs were significantly as­sociated with circulating IL-18 levels. IL18 polymorphism was also found to increase the risk of some diseases of in­flammatory background, including asthma, rheumatoid ar­thritis, atherosclerosis, Crohn’s disease, multiple sclero­sis and type I diabetes [11]. In the present study we tried to investigate a potential association between 7 common SNPs spanning the whole IL18 gene, gene expression and the release of IL-18 from the stimulated peripheral blood mononuclear cells (PBMCs).

Materials and Methods

Twenty-nine healthy volunteers (17 females and 12 males) were enrolled in the study after submission of their writ­ten informed consent. All subjects were Caucasian from the Pomeranian region of Poland. The study was approved by the local ethics committee.

Genomic DNA was extracted from whole blood samples using GeneMATRIX Quick Blood DNA Purification Kit (EURx, Poland). The subjects were genotyped for the presence of 7 common SNPs spanning the whole IL18 gene (rs1946518: A>C and rs187238: G>C in the promo­ter region, rs360718: A>C in 5′-UTR, in exon 1, rs360722: C>T and rs360721: C>G in intron 1, rs549908: T>G sy­nonymous SNP in exon 4, and rs5744292: A>G in exon 6 (3′-UTR)), using allele-specific amplification or PCR-RFLP assays, as described by us in detail elsewhere [10].

PBMCs were isolated from peripheral blood of study sub­jects using the Ficoll Paque procedure. After centrifuga­tion (30 min., 400 × g, at room temp.) the mononuclear fraction was collected, washed twice in PBS, suspended in RPMI enriched with 10% fetal bovine serum and incuba­ted on a tissue culture plate (for 1 hour at 37°C, 5% CO₂) to get rid of adherent cells (monocytes). Non-adherent cells were then collected, counted and seeded at a density of 2×10⁶/2 mL in 24-well plates. Stimulation of cells was performed by adding phytohaemagglutinin (PHA) at the concentration of 20 µg/mL or lipopolysaccharide (LPS) at 40 ng/mL or 40 ng/mL anti-CD3+ 10 µg/mL anti-CD28 antibodies for 48 h incubation periods at 37°C, 5% CO₂. Secretion of IL-18 by PBMC was determined in superna­tants by the MBL human IL-18 immunoassay (ELISA) (Medical & Biological Laboratories Co., LTD, Japan) ac­cording to the manufacturer’s protocol.

Total RNA was extracted from PBMCs collected after 48 h of incubation, using the RNeasy kit (Qiagen, USA). Reverse transcription was performed with the RETROscript First Strand Synthesis Kit (Ambion, USA), using Moloney murine leukemia virus (M-MLV) reverse transcriptase and random decamers in a total volume of 20 µl. Subsequently, cDNA probes were used as a template for a real-time quantitative PCR (QPCR) analysis. QPCR was performed in duplicate in the 7500 Fast Real Time PCR System (Applied Biosystems, USA), using a commercially available validated TaqMan assay for the human IL18 gene (assay ID: Hs99999040_m1, Applied Biosystems, USA) and an endogenous control – ACTB. Calculations were performed using the ΔΔCt rela­tive quantification method. All Ct values were normalized to the values obtained for the endogenous control for each sample. Fold change between groups was calculated from the means of the logarithmic expression values.

Since the distribution of IL-18 concentration and relati­ve mRNA expression values were different from normal (Shapiro-Wilk test), they were analyzed in relation to the IL18 genotype using the Mann-Whitney U-test. Differences in IL-18 concentration and IL18 mRNA levels between non­-stimulated and stimulated PBMCs were analyzed by me­ans of the Wilcoxon test. All calculations were performed using Statistica 8.0 software (StatSoft, Warsaw, Poland).

Results

Concentration of interleukin-18 collected from superna­tant after 48 h of incubation was similar in non-stimulated (mean ±SD: 25.0±15.6) and stimulated PBMCs (25.5±18.7 for PHA, and 26.9±14.5 for LPS). Only in the case of an­ti-CD3/CD28 stimulation did the values differ significan­tly from the non-stimulated control (30.0±18.10, p=0.026, Wilcoxon test). In contrast, IL18 relative mRNA levels measured after 48 h were significantly decreased in cells treated with all stimulants (12.6% of relative expression detected in control probes for PHA, p=0.00001; 52.2% for LPS, p=0.007; 36.2% for anti-CD3/CD28, p= 0.011).

As rs360718 was fully linked to rs187238, and rs549908 to rs5744292 (100% concordance of obtained genotypes), those two SNPs were excluded from the subsequent analy­sis. IL18 genotype had no influence on IL-18 release from non-stimulated PBMCs. However, significant differences were noted in IL-18 concentration between cells from pa­tients stratified by rs1946518 genotype, treated with LPS and antibodies, i.e. mean IL-18 concentrations were signi­ficantly lower in AA homozygotes than in C allele carriers (Table 1). In the case of PHA treatment, rs360722 SNP was associated with IL-18 levels (lower in CC homozygo­tes vs. T allele carriers). No other associations were ob­served. In the case of IL18 relative mRNA expression, no genotype-dependent differences were noted in non-stimu­lated or stimulated PBMCs (Table 2). However, when diffe­rences in IL18 relative expression between non-stimulated and stimulated cells were analyzed for each sample inste­ad of raw relative quantity values, significantly decreased gene expression was noted in rs1946518 AA homozygo­tes (as compared with C allele carriers) in samples treated with PHA and LPS (Table 3). Similar trends were obse­rved in the case of rs187238 SNP; however, the differences reached statistical significance only after PHA treatment.

Table 1. Interleukin-18 release from stimulated PBMCs in relation to IL18 genotype

Table 2. IL18 mRNA expression in stimulated PBMCs in relation to IL18 genotype

Table 3. Changes in IL18 mRNA expression in stimulated PBMCs in relation to IL18 genotype

Discussion

In the present study we examined the potential effect of common SNPs spanning the IL18 gene on gene expression and the release of IL-18 by in vitro stimulated peripheral blood mononuclear cells. The cells were treated with three different stimulators: phytohaemagglutinin, lipopolysac­charide and anti-CD3/CD28 antibodies.

To date, two SNPs, rs1946518: C>A and rs187238: G>C (at positions -607 and -137), in the promoter region of IL18 have been most thoroughly studied. In the initial study by Giedraitis et al. [5], lower promoter activity was observed for rs1946518 A and rs187238 C alleles and they were con­sequently suggested to be ‘low activity’ alleles in contrast to rs1946518: A>C and rs187238 G – ‘high activity’ al­leles. Giedraitis et al. [5] studied the transcriptional acti­vity of IL18 in HeLa 229 cells, transfected with the pro­moter region of the human IL18 gene, containing SNPs rs1946518 and rs187238. The authors did not find signifi­cant differences in promoter activity between alleles witho­ut stimulation, but after stimulation with PMA/ionomycin they observed increased transcription activity associated with rs1946518 C and rs187238 G alleles. In that study the expression of IL18 in PBMCs of multiple sclerosis patients was also studied, revealing slightly (non-significantly) in­creased mRNA levels in subjects carrying rs1946518 C and rs187238 G alleles. These observations are in concordan­ce with our study, in which higher IL-18 release and IL18 mRNA expression were associated with rs1946518 C and rs187238 G alleles, reaching statistical significance in the case of rs1946518 SNP in IL-18 release after stimulation with LPS and anti-CD3/CD28 antibodies (Tables 1, 2).

Similar results were obtained by Khripko et al. [6], who me­asured IL-18 concentrations in PBMCs after 48 h of LPS stimulation. IL-18 production by LPS-stimulated PBMC was significantly greater in healthy donors carrying the rs1946518 CA genotype than in those with the CC genotype, and the ‘low activity’ rs187238 C allele frequency was gre­ater in the group with a low level of IL-18 production [6].

The carriers of rs360722 T allele showed increased rele­ase of IL-18 after stimulation with PHA when compared to CC homozygotes. However, this observation is like­ly to be accidental, as that observation was not observed in the case of other stimulants or in mRNA level analysis. Moreover, no other SNPs (e.g. rs360718: A>C, rs360721: C>G, rs549908: T>G, and rs5744292: A>G) were found to be associated with IL-18 release or transcription rate.

As found by Marshall et al. [7] using semi-quantitative rever­se transcription PCR, IL18 gene expression may be elevated only in a short period (4-6 h) after stimulation of PBMCs with LPS or other antigens, and is then depressed below the con­stitutive level found in non-treated cells. In our study mRNA levels of IL18 transcripts after 48 h of incubation were also significantly lower in stimulated cells when compared with non-stimulated controls. Although IL18 expression was not significantly correlated with analyzed SNPs, a comparison between stimulated PBMCs and non-stimulated controls re­vealed that IL18 transcription was significantly more depres­sed in rs1946518 AA homozygotes when compared with C allele carriers (Table 3). A similar association was observed in the case of rs187238 C ‘low activity’ allele, reaching sta­tistical significance in the case of PHA stimulation.

In the largest study on IL18 variation and in vivo IL-18 levels, five tag SNPs (rs1946519: G>T, rs360717: C>T, rs549908: A>C, rs5744292: A>G, rs4937100: T>C) were analyzed in 1288 patients with coronary artery disease (rs1946518 and rs187238 were not studied). Although two SNPs were significantly associated with circulating IL-18 levels, genetic polymorphism explained only 1.8% of the variability of IL-18 levels [12]. In the study of Zhou et al. [14], the polymorphisms rs1946519: G>T and rs1946518: C>A were significantly associated with serum levels of IL-18 in patients with sarcoidosis, but not in controls. In another study, Evans et al. [4] investigated the association between circulating IL-18 serum levels and rs187238: G>C SNP (described there as -137G>C) within the IL18 gene in black South African women with cardiovascular disease, and revealed that IL-18 levels were not associated with IL18 genotype. However, IL18 gene polymorphism was also fo­und to significantly increase the risk of some diseases of in­flammatory background, including asthma, rheumatoid ar­thritis, atherosclerosis, Crohn’s disease, multiple sclerosis and type I diabetes [11]. Our observation that IL-18 levels in PBMCs were genotype-dependent only after stimulation (in contrast to non-stimulated PBMCs) could be joined with the results of previous investigations, leading to a hypothe­sis that the impact of IL18 polymorphism is only pronoun­ced in particular conditions, after application of some sti­mulatory factors, in vitro as well as in vivo.

Our study supports the role of rs1946518 and rs187238 SNPs as the main genetic determinants of IL18 expression varia­bility and their greater significance compared to other SNPs within the IL18 gene. However, genetic polymorphism is probably responsible only for a part of the observed diffe­rences in IL-18 production, and its diagnostic application seems to be limited. Each subject is the carrier of vario­us polymorphisms and haplotypes in the IL18 gene, often exerting opposite effects on IL-18 protein production. IL-18 production is the outcome of the influence of various poly­morphisms and haplotypes. Therefore the functional signifi­cance of these polymorphisms in vivo may not be consistent with experimental studies due to different combinations of particular SNPs and haplotypes in various subjects.

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The authors have no potential conflicts of interest to declare.

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