# Association of Single Nucleotide Polymorphisms in the IL-18 Gene with Production of IL-18 Protein by Mononuclear Cells from Healthy Donors

Authors: Khripko Olga Pavlovna; Sennikova Nadegda Sergeevna; Lopatnikova Julia Anatolievna; Khripko Jurii Ivanovich; Filipenko Maxim Leonidovich; Khrapov Evgenii Alexandrovich; Gelfgat Evgenii L'vovich; Yakushenko Elena Vladimirovna; Kozlov Vladimir Alexandrovich; Sennikov Sergei Vitalievich
Journal: Mediators of Inflammation
 (2008)

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## Abstract

IL-18 has proinflammatory effects and participates in both innate and adaptive cellular and humoral immunity. A number of SNPs that influence IL-18 production are found in the gene promoter region. We investigated the association of SNPs in the IL-18 promoter at−607 and −137 with the level of IL-18 protein production by PBMC from healthy donors from Southwestern Siberia. The genetic distribution of these SNPs in the promoter site was established by PCR. IL-18 protein production was determined by ELISA. Our results showed that PBMC from donors carrying allele 137C have lower levels of both spontaneous and LPS-stimulated IL-18 production. In contrast, PBMC from donors carrying allele 607A showed significant increases in spontaneous and stimulated IL-18 production compared to wild type. Our study suggests that the SNPs −607 and −137 in the promoter region of the IL-18 gene influence the level of IL-18 protein production by PBMC from healthy donors in Southwestern Siberia.


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## Body

## 1. Introduction

IL-18 is a pleiotropic proinflammatory cytokine
that stimulates production of IFN-γ, TNF-α, IL-1, IL-2, adhesion molecules and apoptosis
factors, increasing T-lymphocyte proliferation, and enhancing the lytic
activity of NK-cells. It participates in the cellular and humoral immune
response [1], both innate and adaptive [2]. It has been shown that IL-18 is
involved in the pathogenesis of various diseases including type-I diabetes [3],
rheumatoid arthritis [4], Crohn's disease [5], and liver cirrhosis [6]. Allelic
variants of cytokine genes associated with promoter gene region polymorphisms
do not influence the protein amino acid sequence but can result in changes in
cytokine production. In consequence, they may alter the immune responses mediated
by the cytokine in question and could be associated with various immunological
diseases. Three single nucleotide polymorphisms (SNPs) are found in the IL-18
gene promoter in positions −656 G→T, −607 C→A, and −137 G→C. Two of these, −607 C→A and −137 G→C, are located at the binding sites for CREB
transcriptional factors (cAMP response-element binding proteins) and the H4TF-1 nuclear factor,
respectively, therefore mutation at these two sites could influence IL-18
expression and change the production of the cytokine [7]. Recent studies have
investigated the relationship between IL-18 gene polymorphisms and
predispositions to various diseases. It has been shown that individuals
carrying the −607C −137C haplotype
are prevalent in rheumatoid arthritis cases among the populations of Germany and Scotland. Women carrying the
C-allele at −137 have a
heightened risk of developing ovarian carcinoma [8]. The presence of such a
genotype in patients with nasopharyngeal carcinoma of an undifferentiated type
worsens the prognosis for the disease and is associated with earlier metastasis
[9]. The G-allele at −137 is associated
with the development of bronchial asthma and dermal allergic reactions [10]. In
patients with hepatitis B from the Chinese population, the −137GG and −607CA allele
variants of the IL-18 gene promoter are consistently more frequent; conversely,
hepatitis B is encountered consistently more rarely in persons with the
C-allele at −137 [11].
Nevertheless, the impact of one or other IL-18 allele variant on the level of
protein production has not been adequately studied.The aim of our study was therefore
as follows: to investigate the distribution of IL-18 allele variants at
positions −607C→A and −137G→C within a population of healthy donors from
Southwestern Siberia, and the influence of these allele variants on the level
of IL-18 production by their peripheral blood mononuclear cells (MNCs).


## 2. Materials and Methods

### 2.1. Object of Research

DNA was obtained from PBMC from adult (20–50 years of age)
healthy donors (n=146) to study polymorphisms in the promoter region of the
IL-18 gene and the production IL-18 protein. All subjects gave written informed
consent for enrolment in the study, which was approved by the local Ethics
Committee.


### 2.2. Definition of Genotypes IL-18

Genomic DNA was extracted from PBMC by a set
Test-NA (DNA-technology, Russia). Allele-specific
amplification was assessed by fluorescent detection of the products of real-time
PCR. Four primers were used for amplification, two specific for each polymorphism
at positions −607 and −137.
Amplification was controled by paired primers specific to the particular
genomic site. Products were detected using the intercalating dye SYBR-green I.
PCR was performed using an iQ4 Cycler (Bio-Rad, USA). The reaction
mixture (15μl final volume) contained 0.3 μM specific and control primers; PCR
buffer (16 mM Tris-HCl, pH 8.9, 2.4 mM MgCl2, 65 mM (NH4)2SO4);
0.2 mM dNTP; 1× SYBR-green I; 1 unit Taq-polymerase; and 5 ng DNA. The PCR
conditions were as follows: denaturation (95°C, 3 minutes) followed
by 5 cycles of amplification at 95°C for 10 seconds, 57°C
for 10 seconds, and 72°C for 10 seconds; then 30 cycles at 95°C
for 3 seconds, 59°C for 3 seconds, 72°C for 3 seconds, 78°C
for 10 seconds (plate read), and 82°C for 10 seconds (plate read). A
melting curve analysis was then performed: 60 cycles starting at 65°C
with temperature increments of 0.5°C, the plate being read at each
cycle. The data obtained were interpreted on the basis with the increase in
fluorescence, and the melting curve was used to assess specificity. The
specific primers used were
137 C5′-TAATGTAATATCACTATTTTCATGAAATC-3′137 G5′-AATGTAATATCACTATTTTCATGAAATG-3′607 C5′-GTTGCAGAAAGTGTAAAAATTATTAC-3′607 A5′-GTTGCAGAAAGTGTAAAAATTATTAA-3′.The internal control primers wereLTM 1 tgggtgctagaggtataatcgLTM 2 ttagaggaagctgggtaagag.As we used two pairs of allele-specific primers, we studied the
reactions in four test tubes simultaneously for each sample to detect any of
the four possible haplotypes.


### 2.3. Preparation of Conditioned Medium from PBMC

PBMC were isolated from heparinized blood
using Ficoll-Urografin density gradients and were cultured (1×106 cells/mL) for 48 hours in a 48-well plate (Beckton
Dikkinson, USA)
in the presence or absence of LPS (055/B5, Sigma, USA)
at a final concentration of 10 μg/mL.


### 2.4. Definition of IL-18 Level

The level of IL-18 protein in the conditioned
medium was determined by ELISA (Bender Med Systems, Austria).


### 2.5. Statistical Analysis

All SNPs were in Hardy-Weinberg equilibrium
in investigated population.For statistical analysis of the data, we used the
nonparametric Mann-Uitni's test employing the Statistica 6.0 program. To
identify quantitative differences in IL-18 production levels, we used quantile
rank classification according to the Mosteller-Tewkee algorithm [12]. To
analyze the IL-18 concentrations, we
determined the probability limits of
the empirical distribution quantiles—(X(qi)). A number
of quantile ranges (K), into which the total amplitude of variation of the
IL-18 level was split, were calculated starting from a minimal sample size
(Nmin) by Sturges's formula. χ2-criterion (Pχ) and Fisher's precise method for small samples
(Ptmf) were used to judge the reliability ofoccurrence for qualitative
features and frequencies of events within different ranges of IL-18 variation
[13].


## 2.1. Object of Research

DNA was obtained from PBMC from adult (20–50 years of age)
healthy donors (n=146) to study polymorphisms in the promoter region of the
IL-18 gene and the production IL-18 protein. All subjects gave written informed
consent for enrolment in the study, which was approved by the local Ethics
Committee.


## 2.2. Definition of Genotypes IL-18

Genomic DNA was extracted from PBMC by a set
Test-NA (DNA-technology, Russia). Allele-specific
amplification was assessed by fluorescent detection of the products of real-time
PCR. Four primers were used for amplification, two specific for each polymorphism
at positions −607 and −137.
Amplification was controled by paired primers specific to the particular
genomic site. Products were detected using the intercalating dye SYBR-green I.
PCR was performed using an iQ4 Cycler (Bio-Rad, USA). The reaction
mixture (15μl final volume) contained 0.3 μM specific and control primers; PCR
buffer (16 mM Tris-HCl, pH 8.9, 2.4 mM MgCl2, 65 mM (NH4)2SO4);
0.2 mM dNTP; 1× SYBR-green I; 1 unit Taq-polymerase; and 5 ng DNA. The PCR
conditions were as follows: denaturation (95°C, 3 minutes) followed
by 5 cycles of amplification at 95°C for 10 seconds, 57°C
for 10 seconds, and 72°C for 10 seconds; then 30 cycles at 95°C
for 3 seconds, 59°C for 3 seconds, 72°C for 3 seconds, 78°C
for 10 seconds (plate read), and 82°C for 10 seconds (plate read). A
melting curve analysis was then performed: 60 cycles starting at 65°C
with temperature increments of 0.5°C, the plate being read at each
cycle. The data obtained were interpreted on the basis with the increase in
fluorescence, and the melting curve was used to assess specificity. The
specific primers used were
137 C5′-TAATGTAATATCACTATTTTCATGAAATC-3′137 G5′-AATGTAATATCACTATTTTCATGAAATG-3′607 C5′-GTTGCAGAAAGTGTAAAAATTATTAC-3′607 A5′-GTTGCAGAAAGTGTAAAAATTATTAA-3′.The internal control primers wereLTM 1 tgggtgctagaggtataatcgLTM 2 ttagaggaagctgggtaagag.As we used two pairs of allele-specific primers, we studied the
reactions in four test tubes simultaneously for each sample to detect any of
the four possible haplotypes.


## 2.3. Preparation of Conditioned Medium from PBMC

PBMC were isolated from heparinized blood
using Ficoll-Urografin density gradients and were cultured (1×106 cells/mL) for 48 hours in a 48-well plate (Beckton
Dikkinson, USA)
in the presence or absence of LPS (055/B5, Sigma, USA)
at a final concentration of 10 μg/mL.


## 2.4. Definition of IL-18 Level

The level of IL-18 protein in the conditioned
medium was determined by ELISA (Bender Med Systems, Austria).


## 2.5. Statistical Analysis

All SNPs were in Hardy-Weinberg equilibrium
in investigated population.For statistical analysis of the data, we used the
nonparametric Mann-Uitni's test employing the Statistica 6.0 program. To
identify quantitative differences in IL-18 production levels, we used quantile
rank classification according to the Mosteller-Tewkee algorithm [12]. To
analyze the IL-18 concentrations, we
determined the probability limits of
the empirical distribution quantiles—(X(qi)). A number
of quantile ranges (K), into which the total amplitude of variation of the
IL-18 level was split, were calculated starting from a minimal sample size
(Nmin) by Sturges's formula. χ2-criterion (Pχ) and Fisher's precise method for small samples
(Ptmf) were used to judge the reliability ofoccurrence for qualitative
features and frequencies of events within different ranges of IL-18 variation
[13].


## 3. Results

### 3.1. IL-18 Gene Promoter Genotype Distribution

The distribution of the
susceptibility/resistance gene alleles responsible for resistance to or risk of
developing immunopathological diseases is unique to each population. This could
be one reason for the diversity of immune responses to an antigen. This study
of two SNPs in the IL-18 gene was conducted on a group of healthy persons
(n=146). The relative frequencies of the C/C, C/A, and A/A variants in the 607C→A polymorphism were 39.2%, 44.7%, and 16.1%,
respectively. The C and A alleles occurred at frequencies of 64.5% and 35.5%,
respectively. Analysis of the IL-18 gene polymorphism at the −137→G position gave the following results: the
frequency of the homozygous variant (G/G) was 45.5%, and that of the
heterozygous variant G/C was 42.7%; the C/C genotype was found in 11.8% of
cases, and the G and C allele frequencies were 66.8% and 33.2%, respectively.
The frequencies of the AC ( −607/−137) haplotype were 22%,
AG 14.6%, CG 55.9%, and CC 7.5%.


### 3.2. IL-18 Production Related to the −607C→ A Polymorphism

We next investigated the IL-18 concentration in
media conditioned by the PBMC from healthy donors. The median levels of IL-18
protein were 21.86 pg/mL [9.83; 57.60 (quartiles)] for spontaneous production and 72.44 pg/mL [32.96; 103.07
(quartiles)] for LPS-stimulated production. We further studied the correlation
between IL-18 production and the polymorphic variants −607C→A in the IL-18 gene promoter (see Figure 2).IL-18 production by LPS-stimulated PBMC was
significantly greater in healthy donors carrying the CA genotype than in those
with the CC genotype at the −607 position of
the promoter. We analyzed the relationship between genotype frequency
distributions and IL-18 production, dividing the cytokine concentrations into
“low,” “medium,” and “high” classes. Concentrations in the 15–85% quantile
range (3.5–93.5 pg/mL) for
spontaneous production were classed as “medium.” Production levels outside
these limits were classed as “low” or “high” (see Table1). We found a higher
frequency of A-allele carriers among persons with a high level of spontaneous
IL-18 production.Table 1
Distribution of frequencies of IL-18 gene promoter genotypes at the −607 A→C position in relation to the level of
spontaneous IL-18 secretion.
Quantiles of level of spontaneousIL-18 level−607CC−607AN(pemf)pχ2IL-18 secretionpg/mLnnLow (0–15%)0–3.47110.2031>0.1Medium (15–85%)3.5–95.34457High (85–100%)95.4–1385190.0239<0.05All5687


### 3.3. IL-18 Production Related to the −137G→ C Polymorphism

We studied the correlation between IL-18 production
by PBMC from healthy donors and variants of the IL-18 genotype −137 at the
position, and we obtained the following data (see Figure1).Figure 1
Effect of polymorphic variants in the
IL-18 gene promoter region at  −137 G→C on IL-18 protein production by PBMC (data
are represented as median and 25 and 75 percentiles) (↔pMann-Uitni=0.0494).Figure 2
Effect of polymorphic variants in the
promoter region of the IL-18 gene at the −607 A→C position on IL-18 production by PBMC (data
are represented as median and 25 and 75 percentiles) (↔pMann-Uitni=0.045).IL-18 production by LPS-stimulated
PBMC was significantly less in persons carrying the CC genotype than in those
with the GG genotype at the −137 position. To
investigate this further, we analyzed the relationship between the genotype
frequency distribution and levels of IL-18 production. The limits of three
primary ranges of variation in IL-18 production levels were determined by quantile
rank analysis which is tolerant of random statistical “surges” and critical
deviations of a sign from normal distribution. In accordance with common
practice, all empirically obtained concentrations of this cytokine were
classified as “low,” “medium,” or “high.” Concentrations in the range 15–85% (25.6–114.3 pg/mL) for
LPS-stimulated production were classed as “medium.” Concentrations outside the
limits of these quantiles, that is, less than 25.6 pg/mL and more than 114.3 pg/mL, were classed as “low” and “high,” respectively.We evaluated the allele variant
frequencies among producers of different levels of IL-18. The results are shown
in Table2.Table 2
Distribution of frequencies of IL-18 gene promoter genotypes at the −137 G→C position in relation to the level of
LPS-stimulated IL-18 secretion.
Quantiles of level of LPS-IL-18 level−137GG−137CN(pemf)pχ2stimulated IL-18 secretionpg/mLnnLow (0–15%)0–25.63140.0098<0.05Medium (15–85%)25.7–114.24850High (85–100%)114.3–298.414140.1445>0.1All6578The frequency of the C allele at
the −137 position was
significantly higher in donors with low LPS-stimulated IL-18 production than in
those with medium or high production. Thus, LPS-stimulated production of IL-18
is lower in persons with the CC genotype than in donors with the GG genotype at
the −137 position of
the promoter. The C allele frequency is greater in the group with a low level
of stimulated IL-18 production.


### 3.4. Capacity to Produce IL-18 by Mononuclear Cells is Associated with IL-18 Polymorphism

As these two polymorphisms, −137 G/C
and −607 C/A, were in strong linkage disequilibrium [14], all possible
combinations of alleles −607 C→A and −137 G→C have been investigated. There has been shown
increased spontaneous production of IL-18 associated with haplogenotypes −607CN/−137GN in comparison with −607AA/−137GN and −607CN/−137CC. Thus alleles
−607C and −137G are associated with increase of spontaneous production of
IL-18. Increased LPS-stimulated production of IL-18 is associated with a haplogenotype −607CN/−137GN in comparison with −607CN/−137CC (see Figure 3).Figure 3
Capacity to produce IL-18 by
mononuclear cells is associated with −607/−137 IL-18 polymorphism (data are
represented as median and 25 and 75 percentiles) (↔pMann-Uitni<0.05).This study showed that the
frequencies of IL-18 genotypes in healthy donors from Novosibirsk city
(Southwestern Siberia) were as follows: −607CC, 32.2%;
−607AC, 44.7%; −607AA, 16.1%; −137GG, 45.5%; −137GC, 42.7%; and −137CC, 11.8%. For
the haplotypes, the values were as follows: AC (−607/−137), 22%; AG,
14.6%; CG, 55.9%; and CC, 7.5%. LPS-stimulated production of IL-18 was lower in
persons with the CC genotype than in donors with the GG genotype at the −137 position of
the gene promoter. IL-18 production by LPS-stimulated PBMC from healthy donors
carrying the CA genotype was significantly higher than from those carrying the
CC genotype at the −607 position.
Alleles −607C and −137G are associated with increase of spontaneous production
of IL-18, allele −137G increases spontaneous and
stimulated production.


## 3.1. IL-18 Gene Promoter Genotype Distribution

The distribution of the
susceptibility/resistance gene alleles responsible for resistance to or risk of
developing immunopathological diseases is unique to each population. This could
be one reason for the diversity of immune responses to an antigen. This study
of two SNPs in the IL-18 gene was conducted on a group of healthy persons
(n=146). The relative frequencies of the C/C, C/A, and A/A variants in the 607C→A polymorphism were 39.2%, 44.7%, and 16.1%,
respectively. The C and A alleles occurred at frequencies of 64.5% and 35.5%,
respectively. Analysis of the IL-18 gene polymorphism at the −137→G position gave the following results: the
frequency of the homozygous variant (G/G) was 45.5%, and that of the
heterozygous variant G/C was 42.7%; the C/C genotype was found in 11.8% of
cases, and the G and C allele frequencies were 66.8% and 33.2%, respectively.
The frequencies of the AC ( −607/−137) haplotype were 22%,
AG 14.6%, CG 55.9%, and CC 7.5%.


## 3.2. IL-18 Production Related to the −607C→ A Polymorphism

We next investigated the IL-18 concentration in
media conditioned by the PBMC from healthy donors. The median levels of IL-18
protein were 21.86 pg/mL [9.83; 57.60 (quartiles)] for spontaneous production and 72.44 pg/mL [32.96; 103.07
(quartiles)] for LPS-stimulated production. We further studied the correlation
between IL-18 production and the polymorphic variants −607C→A in the IL-18 gene promoter (see Figure 2).IL-18 production by LPS-stimulated PBMC was
significantly greater in healthy donors carrying the CA genotype than in those
with the CC genotype at the −607 position of
the promoter. We analyzed the relationship between genotype frequency
distributions and IL-18 production, dividing the cytokine concentrations into
“low,” “medium,” and “high” classes. Concentrations in the 15–85% quantile
range (3.5–93.5 pg/mL) for
spontaneous production were classed as “medium.” Production levels outside
these limits were classed as “low” or “high” (see Table1). We found a higher
frequency of A-allele carriers among persons with a high level of spontaneous
IL-18 production.Table 1
Distribution of frequencies of IL-18 gene promoter genotypes at the −607 A→C position in relation to the level of
spontaneous IL-18 secretion.
Quantiles of level of spontaneousIL-18 level−607CC−607AN(pemf)pχ2IL-18 secretionpg/mLnnLow (0–15%)0–3.47110.2031>0.1Medium (15–85%)3.5–95.34457High (85–100%)95.4–1385190.0239<0.05All5687


## 3.3. IL-18 Production Related to the −137G→ C Polymorphism

We studied the correlation between IL-18 production
by PBMC from healthy donors and variants of the IL-18 genotype −137 at the
position, and we obtained the following data (see Figure1).Figure 1
Effect of polymorphic variants in the
IL-18 gene promoter region at  −137 G→C on IL-18 protein production by PBMC (data
are represented as median and 25 and 75 percentiles) (↔pMann-Uitni=0.0494).Figure 2
Effect of polymorphic variants in the
promoter region of the IL-18 gene at the −607 A→C position on IL-18 production by PBMC (data
are represented as median and 25 and 75 percentiles) (↔pMann-Uitni=0.045).IL-18 production by LPS-stimulated
PBMC was significantly less in persons carrying the CC genotype than in those
with the GG genotype at the −137 position. To
investigate this further, we analyzed the relationship between the genotype
frequency distribution and levels of IL-18 production. The limits of three
primary ranges of variation in IL-18 production levels were determined by quantile
rank analysis which is tolerant of random statistical “surges” and critical
deviations of a sign from normal distribution. In accordance with common
practice, all empirically obtained concentrations of this cytokine were
classified as “low,” “medium,” or “high.” Concentrations in the range 15–85% (25.6–114.3 pg/mL) for
LPS-stimulated production were classed as “medium.” Concentrations outside the
limits of these quantiles, that is, less than 25.6 pg/mL and more than 114.3 pg/mL, were classed as “low” and “high,” respectively.We evaluated the allele variant
frequencies among producers of different levels of IL-18. The results are shown
in Table2.Table 2
Distribution of frequencies of IL-18 gene promoter genotypes at the −137 G→C position in relation to the level of
LPS-stimulated IL-18 secretion.
Quantiles of level of LPS-IL-18 level−137GG−137CN(pemf)pχ2stimulated IL-18 secretionpg/mLnnLow (0–15%)0–25.63140.0098<0.05Medium (15–85%)25.7–114.24850High (85–100%)114.3–298.414140.1445>0.1All6578The frequency of the C allele at
the −137 position was
significantly higher in donors with low LPS-stimulated IL-18 production than in
those with medium or high production. Thus, LPS-stimulated production of IL-18
is lower in persons with the CC genotype than in donors with the GG genotype at
the −137 position of
the promoter. The C allele frequency is greater in the group with a low level
of stimulated IL-18 production.


## 3.4. Capacity to Produce IL-18 by Mononuclear Cells is Associated with IL-18 Polymorphism

As these two polymorphisms, −137 G/C
and −607 C/A, were in strong linkage disequilibrium [14], all possible
combinations of alleles −607 C→A and −137 G→C have been investigated. There has been shown
increased spontaneous production of IL-18 associated with haplogenotypes −607CN/−137GN in comparison with −607AA/−137GN and −607CN/−137CC. Thus alleles
−607C and −137G are associated with increase of spontaneous production of
IL-18. Increased LPS-stimulated production of IL-18 is associated with a haplogenotype −607CN/−137GN in comparison with −607CN/−137CC (see Figure 3).Figure 3
Capacity to produce IL-18 by
mononuclear cells is associated with −607/−137 IL-18 polymorphism (data are
represented as median and 25 and 75 percentiles) (↔pMann-Uitni<0.05).This study showed that the
frequencies of IL-18 genotypes in healthy donors from Novosibirsk city
(Southwestern Siberia) were as follows: −607CC, 32.2%;
−607AC, 44.7%; −607AA, 16.1%; −137GG, 45.5%; −137GC, 42.7%; and −137CC, 11.8%. For
the haplotypes, the values were as follows: AC (−607/−137), 22%; AG,
14.6%; CG, 55.9%; and CC, 7.5%. LPS-stimulated production of IL-18 was lower in
persons with the CC genotype than in donors with the GG genotype at the −137 position of
the gene promoter. IL-18 production by LPS-stimulated PBMC from healthy donors
carrying the CA genotype was significantly higher than from those carrying the
CC genotype at the −607 position.
Alleles −607C and −137G are associated with increase of spontaneous production
of IL-18, allele −137G increases spontaneous and
stimulated production.


## 4. Discussion

Genetic factors
are important in the pathogenesis of all major human diseases and it is known
that human populations vary genetically because of polymorphisms, which can
affect aspects of the immune response. Diverse polymorphic variants of cytokine
genes may induce high or low levels of protein production. Single nucleotide
polymorphisms in the IL-18 gene promoter at −607 C→A and −137 G→C affect functionally active parts of this
promoter, that is, binding sites for CREB transcriptional factors and the
H4TF-1 nuclear factor, respectively. Therefore, mutation at these two sites
could influence IL-18 expression and alter the level of IL-18 production [7].Considering the distributions of
IL-18 gene alleles in healthy donors from different populations, we found
various frequency rates. In the Japanese population, the genotypes GG, GC, and
CC were found at the −137 position of
the promoter with frequencies of 75.4%, 23.1%, and 1.2%, respectively; the
genotypes AA, AC, and CC at the −607 position had
the frequencies 43.9%, 39.2%, and 16.9%, respectively [15]. Genotype
frequencies in the Chinese population were −137GG, 67.3%; −137GC, 30%; −137CC, 2.7%; −607AA, 24.7%; −607AC, 53.3%; and −607CC, 22% [11].
For European populations from Switzerland [10] and
Italy [9], the characteristic frequencies of the genotypes −137GG, −137GC, and −137CC were 54.1%
and 48.3%, 38.7% and 43.8%, and 7.2% and 7.9%, respectively. IL-18 genotypes at
the −607 position of
the promoter were found in the Italian population at the following rates: AA,
23.6%; AC, 47.2%; CC, 29.2% [9].We found the following IL-18
genotype frequencies in healthy donors from Novosibirsk (Southwestern Siberia): GG, GC, and CC at the −137 position of
the promoter were found in 45.5%, 42.7%, and 11.8% of cases, respectively; AA,
AC, and CC at the −607 position were
found in 16.1%, 44.7%, and 39.2%, respectively. Thus, the genotype frequencies
within the population of Southwestern Siberia are similar to those of European
countries (Italy and Switzerland).
There are, however, some differences in genotype distribution between the
inhabitants of Southwestern Siberia and the populations of China and Japan.It is well known that gene promoter polymorphisms can affect the level
of protein production. Our study demonstrated a relationship between two IL-18
gene promoter polymorphisms and the level of LPS-stimulated IL-18 production.
PBMC from persons carrying the C allele at the −137 position of
the promoter show low LPS-stimulated production of the cytokine. Also, the
level of IL-18 production by PBMC from donors with the −607AC genotype is
higher than that from donors with the −607CC genotype.
The A allele is likewise associated with greater LPS-stimulated IL-18
production. There appears little dose effect of the
allele A of −607 SNP and we could not detect any difference of IL-18 production
when heterozygote for −137 SNP was considered as a whole. However, after
splitting heterozygote for one SNP according to their genotype at other SNP, we
were able to demonstrate differential haplogenotypic IL-18 production. The
−607CN/−137GN haplogenotype was associated with a higher spontaneous IL-18
production. Increased LPS-stimulated production of IL-18 was associated with a
haplogenotype −607CN/−137GN in comparison with −607CN/−137CC. Thus alleles −607C and −137G are associated
with increase of spontaneous production of IL-18,
allele −137G increases spontaneous and stimulated production. It is possible to assume that
only in case of presence of both alleles conducts to increase production of
IL-18 protein. The increased frequency of allele −607A with high
production IL-18 is connected with heterozygotes −607CA (16 donors from 19).Arimitsu et al. [16] showed that a
polymorphic variant of the IL-18 gene influenced IL-18 production by monocytes.
In particular, they showed that spontaneous and LPS-stimulated production of
IL-18 in volunteers with the −137GG genotype
was higher than in those with the −137GC genotype,
which is in agreement with our results.IL-18 is a pleiotropic
proinflammatory cytokine that predominantly influences the differentiation of
type 1 T-helper cells, thus participating in the
establishment of the cellular immune response and inflammatory reactions. IL-18
has been shown to have antitumor activity. A study of the relationship between
IL-18 gene promoter polymorphisms and the likelihood of developing oncological
diseases showed that women carrying the C allele in the −137 position of
the promoter are at greater risk of developing ovarian carcinoma [8]. On the
basis of our results and data from the literature, one could suppose that the
presence of the C allele at the −137 position in
the IL-18 gene promoter in such patients results in a less efficient antitumor
immune response because IL-18 production by immunocompetent cells is
diminished.The serum level of the
proinflammatory factor IL-18 is known to be significantly elevated in patients
with atopic asthma during the acute phase [17]. Studies of the association
between IL-18 polymorphism and atopic asthma have revealed that the G allele at
the −137 position
leads to an elevated risk of disease development; moreover, none of the 74
patients examined had the CC genotype [10], which according to our data is
associated with reduced IL-18 production. Moreover, the linkage disequilibrium
was observed between the −137 and 105 polymorphisms of the IL-18 gene and the
105A allele of the IL-18 gene which may be associated with the pathogenesis of asthma [18].Premature infants with reduced birth weight are at
risk of developing necrotizing enterocolitis and IL-18 that is also involved in the pathogenesis
of this disease. Analysis of the association between disease severity and
genotype frequencies at the −607 position of
the IL-18 gene showed that the AA genotype frequency is significantly higher in
patients with the third and most severe stage of necrotizing enterocolitis,
when the intestinal wall is perforated [19].On the basis of present knowledge,
one may assume that allelic variants of the IL-18 gene promoter at the −607C→A and −137G→C positions, which, according to our results,
influence the level of protein production, could result in conditions of which
the pathogenesis involves a significant role for IL-18.In conclusion, this study shows
that the IL-18 genotype frequencies among healthy donors from Novosibirsk city
(Southwestern Siberia) are as follows: −607CC, 39.2%; −607AC, 44.7%;
−607AA, 16.1%; −137GG, 45.5%; −137GC, 42.7%; and −137CC, 11.8%.
This is consistent with the distributions within European populations (Italy and Switzerland). Allelic variants in
the IL-18 gene promoter at the −607C→A and −137G→C positions influence the level of production
of the mediator by immunocompetent cells. LPS-stimulated production of IL-18 in
persons with the CC genotype is lower than that in donors with the GG genotype
at the −137 position of
the promoter. The C allele frequency is significantly higher in the group with
low LPS-stimulated IL-18 production. LPS-stimulated production of IL-18 by PBMC
from healthy donors is significantly greater in those carrying CA genotype at
the −607 position. Thus alleles −607C and −137G are associated with the increase
of spontaneous production of IL-18 and allele −137G
increases spontaneous and stimulated production. It is possible to assume that only in case of presence of
both alleles conducts to increase production of IL-18 protein.



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*Source: 309721-2008-10-20.xml*