ORIGINAL ARTICLE

Association of interleukin-4 gene polymorphisms with ischemic heart failure

Mohammad Jafar Mahmoudi1, Mona Hedayat2, Mohammad Taghvaei3, Ebrahim Nematipour4, Elham Farhadi5, Nilufar Esfahanian3, Maryam Sadr3, Maryam Mahmoudi6, Keramat Nourijelyani7, Ali Akbar Amirzargar3, 8, Nima Rezaei3, 8, 9

1Division of Cardiology, Department of Internal Medicine, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
3Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran
4Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran
5Hematology Department, School of Allied Medical Science, Tehran University of Medical Sciences, Tehran, Iran
6School of Nutrition and Dietetics, Tehran University of Medical Sciences, Tehran, Iran
7Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
8Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
9Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran

Address for correspondence: Nima Rezaei, MD, PhD, Children’s Medical Center Hospital, Dr Gharib St, Keshavarz Blvd, Tehran, Iran, e-mail: rezaei_nima@tums.ac.ir

Received: 24.02.2013 Accepted: 06.04.2013

Introduction

Chronic heart failure (HF) is a clinical syndrome characterized by progressive ventricular dilation, depressed contractile function, and increased morbidity and mortality [1]. Despite our increasing knowledge of the molecular, genetic, and biochemical pathways involved in the pathogenesis of chronic HF, much is left to be understood.

Increased circulating and intracardiac levels of proinflammatory cytokines have been reported repeatedly in patients with chronic HF [2–5], and more important, their contributions in mediating cardiac adaptive and maladaptive responses have been a topic of intensive research [6, 7].

Given the potential immunomodulatory effects of interleukin-4 (IL-4) and its importance in inhibiting the production of proinflammatory cytokines by monocytes and activated T cells [8, 9], IL-4 gene polymorphisms altering cytokine production might affect individual susceptibility to ischemic heart failure (IHF). The association of IL-4 gene polymorphisms and a number of diseases with possible underlying immune disturbances have already been investigated [10–14]. In the present study, 3 cytokine single nucleotide polymorphisms situated at positions –1098 (G/T), –590 (C/T), and –33 (C/T) in the promoter region of the IL-4 gene were investigated in Iranian patients with IHF.

Methods

Subjects

In the present study, a total of 43 Iranian patients with end-stage IHF (mean age 60.05 ± 11.97; 34 men, 9 women) with angiographically significant coronary artery disease, defined as ≥ 50% diameter stenosis in at least one of the major coronary arteries, were enrolled. The diagnosis of end-stage HF was based on impaired left ventricular (LV) systolic function (LV ejection fraction ≤ 40%) and LV dilation (LV end-diastolic diameter > 5.5 cm) on echocardiography. All patients underwent transthoracic echocardiography and cardiac catheterization. Subjects with chronic lung disease, malignancies, recent myocardial infarction, and acute decompensated HF within 3 months before recruitment were excluded. Eligible patients were in stable clinical condition and received conventional medical therapy for at least 3 months.

One hundred and thirty nine control subjects (mean age 45.63 ± 10.84; 101 men, 39 women) were randomly selected from healthy volunteers, as previously described [15]. Written informed consent was obtained from all participants prior to blood sampling. This study was approved by the Ethical Committee of Tehran University of Medical Sciences.

Genotyping

Cytokine typing was performed on genomic DNA by polymerase chain reaction with sequence-specific primers (PCR-SSP) assay (PCR-SSP kit, Heidelberg University, Heidelberg, Germany), as previously described in detail [15]. Briefly, amplification was carried out using a thermal cycler Techne Flexigene apparatus (Rosche, Cambridge, UK). The presence or absence of PCR products was visualized by 2% agarose gel electrophoresis. All individuals were genotyped for 3 polymorphic sites in IL-4 gene: –1098 T/G, –590 C/T, and –33 C/T.

Statistical analysis

Statistical analyses were performed with Graph­- Pad Prism 5.00 for Windows (Graphpad Software). Allele, genotype, and haplotype frequencies for all cytokine gene polymorphisms were calculated by direct counting. Frequencies of alleles, genotypes, and haplotypes were compared between the patient and control groups using the Fisher’s exact test. The odds ratio and 95% confidence intervals were calculated.

Results

Alleles and genotype frequencies

Interlekin-4 allelic and genotype frequencies in patients with IHF and healthy controls are presented in Table 1. The frequency of the IL-4 –590/T allele in the patient group was significantly higher than in the control group (73.3% in patients vs. 46.4% in controls, p < 0.0001). The most frequent genotypes in patients with IHF were IL-4 CC genotype at position –590 (51.1% in patients vs. 7.2% in controls, p < 0.0001), IL-4 CC genotype at position –33 (65% in patients vs. 43.9% in controls, p = 0.021), and IL-4 TT genotype at position –33 (15% in patients vs. 0% in controls, p < 0.0001). In contrast the frequency of the following genotypes in the patient group was significantly lower than in the control group: IL-4 TG at position –1098 (39.6% in patients vs. 59% in controls, p = 0.035), IL-4 TC at position –590 (44.2% in patients vs. 92.8% in controls, p < 0.0001), and IL-4 TC at position –33 (20% in patients vs. 56.1% in controls, p < 0.0001).

Table 1. Comparisons of allele, genotype and haplotype frequencies between patients with ischemic heart failure and controls.

Haplotype frequencies

The frequency of haplotypes of IL-4 (–1098, –590, –33) in patients with IHF and healthy controls are shown in Table 1. The most frequent haplotype in our patients was IL-4 TCC which was significantly higher than in the control group (47.5% in patients vs. 23.4% in controls, p < 0.0001). In addition IL-4 TCT (5% in patients vs. 0.7% in controls, p = 0.0242) and GCT (3.75% in patients vs. 0% in controls, p = 0.0108) haplotypes were significantly more common in the patient group compared to the controls. In contrast, the frequencies of the following haplotypes in the patient group were significantly lower than in the controls: IL-4 GCC (17.5% in patients vs. 30% in controls, p = 0.032), TTT (15% in patients vs. 27.3% in controls, p = 0.0268), and TTC (8.75% in patients vs. 18.3% in controls, p = 0.0399).

Discussion

In the present study, the frequency of the IL-4/C allele at position –590 was decreased significantly in patients with IHF; however, the frequency of the CC genotype at the same position was significantly overrepresented in patients compared to controls. In addition, while less than half of patients had the TC genotype at position –590, it was found in the majority of the healthy controls. It has been shown that the C to T exchange at position –590 of the IL-4 gene is associated with increased IL-4 production, with the CC, TC, and TT genotypes being associated with low, intermediate, and high IL-4 production, respectively [16]. With regard to IL-4 –590 C/T polymorphism, Bijlsma et al. [17] reported no correlation between IL-4 promoter gene polymorphism at position –590 and HF, and allograft rejection after heart transplantation. However, in patients with the CC genotype (low IL-4 production), receiving a heart from a T-positive donor reduced the incidence of rejection substantially. It has been concluded that high IL-4 production within –590/T-positive donor hearts protects against rejection.

For IL-4 at position –33, there was no significant difference in allele frequencies between our patients and controls. However, we found significant positive associations with the CC and TT genotypes in our patients compared to controls; while the TC genotype at the same position was decreased significantly in patients but not in controls. It has been shown that IL-4 promoters harboring –33/C allele play a significant role in the activation of IL-4 transcription, and that the homozygous variant of IL-4 (–33) CC is associated with increased IL-4 production [18]. In this study, the CC genotype at position –33 was the most frequent genotype in both groups of patients and controls; however, while none of the healthy controls was found to have the TT genotype, it was significantly overrepresented in patients with IHF.

The IL-4 (–1098, –590, –33) haplotypes GCC, TTT, TCC, and TTC are the most common haplotypes in normal Iranian population [15]. In the present study, the TCC haplotype was the most frequent haplotype in patients with IHF and was significantly overrepresented in patients compared to controls. However, the GCC, TTT, and TTC haplotypes in the patient group were significantly less frequent than in the healthy controls. On the other hand, the IL-4 TCT and GCT haplotypes were significantly overrepresented in patients than in controls. Given the fact that IL-4 (–1098) T/G polymorphism was found to have no correlation with serum IL-4 level [19], and that the IL-4 –590/C and –33/T alleles are associated with decreased IL-4 production [17, 18], it could be suggested that there is a tendency towards lowered IL-4 production in at least a subset of patients with IHF. However, given the small number of patients in our study, we must view these results with caution.

Interleukin-4 is an important mediator of T-helper 2 (Th2) immune responses by inducing differentiation of naive CD4+ T cells to Th2 cells [20], regulating humoral immune responses [21], and inhibiting the production of Th1 cytokines by monocytes and activated T cells [8, 9]. Moreover, IL-4 stimulates fibroblast proliferation and collagen synthesis, and thus has been implicated in the progression of fibrosis [22]. Increased urinary excretion of circulating procollagen type-III amino-terminal propeptide (PIIINP) has been known as a marker of ongoing fibrotic processes [23, 24]. Likewise, increased serum PIIINP levels have also been demonstrated in patients with ischemic and dilated cardiomyopathy [22, 25]. It has been demonstrated that IL-4, most likely produced by mast cells in the heart during pressure overload, significantly contributes to cardiac fibrosis [26]. In patients with HF, the urinary IL-4 level correlated with LV end-systolic volume index, LV end-diastolic volume index, and with PIIINP [27]. The correlation between urinary IL-4 level and cardiac fibrosis and remodeling was found to be stronger in patients with hypertensive cardiomyopathy [27]. At the present time, the pathologic significance of IL-4 in the development and progression of chronic HF is not well understood. The majority of studies failed to demonstrate any significant difference in IL-4 producing peripheral CD4+ T cells and IL-4 levels between the two groups of patients and controls [28–30].

Conclusions

To the best of our knowledge, we demonstrated for the first time the association between certain allele, genotype, and haplotype frequencies in IL-4 gene with IHF. Further studies will provide additional understanding of the possible role of cytokine gene polymorphisms in the development and progression of IHF.

Acknowledgements

This study was supported by grant from Tehran University of Medical Sciences and Health Services (87-04-93-9584).

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