Cardiology Journal 1 2014-5

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

Abstract

Background: As of 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, the IL-4 gene polymorphisms were investigated in a group of patients with chronic heart failure due to ischemic heart disease.

Methods: Forty three patients with ischemic heart failure (IHF) were enrolled in this study and compared with 139 healthy individuals. The allele and genotype frequency of 3 single nucleotide polymorphisms within the IL-4 gene were determined.

Results: The frequency of the IL-4 –590/T allele in the patient group was significantly higher than in the control group (p < 0.0001). The most frequent genotypes in patients with IHF were IL-4 (–590) CC (p < 0.0001), IL-4 (–33) CC (p = 0.021), and IL-4 (–33) TT (p < 0.0001). The frequency of the following genotypes was significantly lower in patients compared to controls: IL-4 (–1098) TG (p = 0.035), IL-4 (–590) TC (p < 0.0001), and IL-4 (–33) TC (p < 0.0001). The most frequent IL-4 haplotypes in the patient group, which were significantly higher than in the control group, were TCC (p < 0.0001), TCT (p = 0.0242), and GCT (p = 0.0108) haplotypes. In contrast, the frequencies of the following haplotypes in the patient group were significantly lower than in the controls: GCC (p = 0.032), TTT (p = 0.0268), and TTC (p = 0.0399).

Conclusions: Certain alleles, genotypes, and haplotypes in IL-4 gene were overrepresented in patients with IHF, which may, in turn, predispose individuals to this disease. (Cardiol J 2014; 21, 1: 24–28)

Key words: heart failure, gene polymorphisms, interleukin-4

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.

Cytokine

Position

Alleles/genotypes/

/haplotypes

Patients

(n = 43)

Controls

(n = 139)

P

OR (95% CI)

IL-4

–1098

G

T

GG

TG

TT

19 (22.1%)

67 (77.9%)

1 (2.3%)

17 (39.6%)

25 (58.1%)

84 (30.2)

194 (69.8%)

1 (0.7%)

82 (59%)

56 (40.3%)

0.171

0.171

0.418

0.035

0.053

0.65 (0.37–1.16)

1.53 (0.86–2.70)

3.29 (0.20–53.71)

0.45 (0.23–0.91)

2.06 (1.03–4.12)

–590

C

T

CC

TC

TT

23 (26.7%)

63 (73.3%)

22 (51.1%)

19 (44.2%)

2 (4.7%)

149 (53.6%)

129 (46.4%)

10 (7.2%)

129 (92.8%)

0 (0%)

< 0.0001

< 0.0001

< 0.0001

< 0.0001

0.055

0.32 (0.19–0.54)

3.16 (1.86–5.39)

13.51 (5.61–32.53)

0.06 (0.03–0.15)

16.81 (0.79–357.3)

–33

C

T

CC

TC

TT

60 (75%)

20 (25%)

26 (65%)

8 (20%)

6 (15%)

200 (71.9%)

78 (28.1%)

61 (43.9%)

78 (56.1%)

0 (0%)

0.670

0.670

0.021

< 0.0001

< 0.0001

1.17 (0.66–2.07)

0.86 (0.48–1.51)

2.38 (1.14–4.93)

0.20 (0.08–0.45)

52.57 (2.89–956.5)

IL-4

–1098,

–590,

–33

GCC

TTT

TCC

TTC

TCT

GTT

GCT

GTC

14 (17.5%)

12 (15%)

38 (47.5%)

7 (8.75%)

4 (5%)

1 (1.25%)

3 (3.75%)

1 (1.25%)

83 (30%)

76 (27.3%)

65 (23.4%)

51 (18.3%)

2 (0.7%)

1 (0.3%)

0 (0%)

0 (0%)

0.032

0.027

< 0.0001

0.039

0.024

0.397

0.011

0.223

0.5 (0.27–0.94)

0.47 (0.24–0.91)

2.97 (1.76–4.98)

0.43 (0.19–0.98)

7.26 (1.31–40.43)

3.51 (0.22–56.73)

25.15 (1.29–492.6)

10.51 (0.42–260.7)

CI — confidence interval; OR — odds ratio

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).

References

  1. 1. Lloyd-Jones DM, Larson MG, Leip EP et al. Lifetime risk for developing congestive heart failure: The Framingham Heart Study. Circulation, 2002; 106: 3068–3072.
  2. 2. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med, 1990; 323: 236–241.
  3. 3. Aukrust P, Ueland T, Lien E et al. Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol, 1999; 83: 376–382.
  4. 4. Hirota H, Izumi M, Hamaguchi T et al. Circulating interleukin-6 family cytokines and their receptors in patients with congestive heart failure. Heart Vessels, 2004; 19: 237–241.
  5. 5. Satoh S, Oyama J, Suematsu N et al. Increased productivity of tumor necrosis factor-alpha in helper T cells in patients with systolic heart failure. Int J Cardiol, 2006; 111: 405–412.
  6. 6. El-Menyar AA. Cytokines and myocardial dysfunction: State of the art. J Card Fail, 2008; 14: 61–74.
  7. 7. Hedayat M, Mahmoudi MJ, Rose NR, Rezaei N. Proinflammatory cytokines in heart failure: Double-edged swords. Heart Fail Rev, 2010; 15: 543–562.
  8. 8. Essner R, Rhoades K, McBride WH, Morton DL, Economou JS. IL-4 down-regulates IL-1 and TNF gene expression in human monocytes. J Immunol, 1989; 142: 3857–3861.
  9. 9. Damle NK, Doyle LV. Distinct regulatory effects of IL-4 and TNF-alpha during CD3-dependent and CD3-independent initiation of human T-cell activation. Lymphokine Res, 1989; 8: 85–97.
  10. 10. Rezaei N, Aghamohammadi A, Mahmoudi M et al. Association of IL-4 and IL-10 gene promoter polymorphisms with common variable immunodeficiency. Immunobiology, 2010; 215: 81–87.
  11. 11. Shahram F, Nikoopour E, Rezaei N, Saeedfar K, Ziaei N, Davatchi F, Amirzargar A. Association of interleukin-2, interleukin-4 and transforming growth factor-beta gene polymorphisms with Behcet’s disease. Clin Exp Rheumatol, 2011; 29 (suppl. 67): S28–S31.
  12. 12. Amirzargar AA, Bagheri M, Ghavamzadeh A et al. Cytokine gene polymorphism in Iranian patients with chronic myelogenous leukaemia. Int J Immunogenet, 2005; 32: 167–171.
  13. 13. Amirzargar AA, Rezaei N, Jabbari H et al. Cytokine single nucleotide polymorphisms in Iranian patients with pulmonary tuberculosis. Eur Cytokine Netw, 2006; 17: 84–89.
  14. 14. Amirzargar AA, Movahedi M, Rezaei N et al. Polymorphisms in IL4 and iLARA confer susceptibility to asthma. J Investig Allergol Clin Immunol, 2009; 19: 433–438.
  15. 15. Amirzargar AA, Naroueynejad M, Khosravi F et al. Cytokine single nucleotide polymorphisms in Iranian populations. Eur Cytokine Netw, 2008; 19: 104–112.
  16. 16. Rosenwasser LJ, Klemm DJ, Dresback JK et al. Promoter polymorphisms in the chromosome 5 gene cluster in asthma and atopy. Clin Exp Allergy, 1995; 25 (suppl. 2): 74–78.
  17. 17. Bijlsma FJ, vanKuik J, Tilanus MG et al. Donor interleukin-4 promoter gene polymorphism influences allograft rejection after heart transplantation. J Heart Lung Transplant, 2002; 21: 340–346.
  18. 18. Kim BS, Park SM, Uhm TG et al. Effect of single nucleotide polymorphisms within the interleukin-4 promoter on aspirin intolerance in asthmatics and interleukin-4 promoter activity. Pharmacogenet Genomics, 2010; 20: 748–758.
  19. 19. Gervaziev YV, Kaznacheev VA, Gervazieva VB. Allelic polymorphisms in the interleukin-4 promoter regions and their association with bronchial asthma among the Russian population. Int Arch Allergy Immunol, 2006; 141: 257–264.
  20. 20. Abehsira-Amar O, Gibert M, Joliy M, Thèze J, Jankovic DL. IL-4 plays a dominant role in the differential development of Tho into Th1 and Th2 cells. J Immunol, 1992; 148: 3820–3829.
  21. 21. Rogge L. A genomic view of helper T cell subsets. Ann N Y Acad Sci, 2002; 975: 57–67.
  22. 22. Timonen P, Magga J, Risteli J et al. Cytokines, interstitial collagen and ventricular remodelling in dilated cardiomyopathy. Int J Cardiol, 2008; 124: 293–300.
  23. 23. Soylemezoglu O, Wild G, Dalley AJ et al. Urinary and serum type III collagen: markers of renal fibrosis. Nephrol Dial Transplant, 1997; 12: 1883–1889.
  24. 24. Teppo AM, Törnroth T, Honkanen E, Grönhagen-Riska C. Urinary amino-terminal propeptide of type III procollagen (PIIINP) as a marker of interstitial fibrosis in renal transplant recipients. Transplantation, 2003; 75: 2113–2119.
  25. 25. Rivera M, Taléns-Visconti R, Jordán A et al. Myocardial remodeling and immunologic activation in patients with heart failure. Rev Esp Cardiol, 2006; 59: 911–918.
  26. 26. Kanellakis P, Ditiatkovski M, Kostolias G, Bobik A. A pro-fibrotic role for interleukin-4 in cardiac pressure overload. Cardiovasc Res, 2012; 95: 77–85.
  27. 27. Roselló-Lletí E, Rivera M, Bertomeu V, Cortés R, Jordán A, González-Molina A. Interleukin-4 and cardiac fibrosis in patients with heart failure. Rev Esp Cardiol, 2007; 60: 777–780.
  28. 28. Fukunaga T, Soejima H, Irie A et al. Expression of inter- feron-gamma and interleukin-4 production in CD4+ T cells in patients with chronic heart failure. Heart Vessels, 2007; 22: 178–183.
  29. 29. Cheng X Liao YH, Li B et al. The significance of Th1/Th2 function imbalance in patients with post-infarction cardiac insufficiency. Zhonghua Xin Xue Guan Bing Za Zhi, 2005; 33: 526–528.
  30. 30. Fukunaga T, Soejima H, Irie A et al. Relation between CD4+ T-cell activation and severity of chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol, 2007; 100: 483–488.

Important: This website uses cookies. More >>

The cookies allow us to identify your computer and find out details about your last visit. They remembering whether you've visited the site before, so that you remain logged in - or to help us work out how many new website visitors we get each month. Most internet browsers accept cookies automatically, but you can change the settings of your browser to erase cookies or prevent automatic acceptance if you prefer.

By "Via Medica sp. z o.o." sp.k., ul. Świętokrzyska 73, 80–180 Gdańsk, Poland
tel.:+48 58 320 94 94, fax:+48 58 320 94 60, e-mail: viamedica@viamedica.pl