Inflammation ( # 2014) DOI: 10.1007/s10753-014-9935-9
Interleukin-21 Polymorphism Affects Gene Expression and is Associated with Risk of Ischemic Stroke Guanggang Li,1 Ruxiang Xu,1 Yinghua Cao,1 Xiaodong Xie,2 and Zhendong Zheng2,3
Abstract—There has been more and more evidence to confirm the essential role of inflammatory processes in the development of ischemic stroke. Interleukin-21 (IL-21), the most recently discovered CD132-dependent cytokine, plays a key role in regulating inflammation. The aim of the study was to understand the association between IL-21 polymorphisms and ischemic stroke, and the effects of these polymorphisms on gene expression. Two polymorphisms in IL-21, rs907715G/A and rs4833837A/G, were identified in 278 ischemic stroke patients and 282 healthy controls. Results showed that frequencies of rs907715GA and AA genotypes were significantly increased in cases than in controls (odd ratio (OR)=1.49, 95 % confidence interval (CI): 1.01–2.14, P=0.042; OR=2.21, 95 % CI: 1.38–3.53, P=0.001). Similarly, rs907715A allele revealed a positive association with the disease (OR=1.52, P=0.001). The rs4833837A/G polymorphism did not show any correlation with ischemic stroke. We further evaluated IL-21 messenger RNA (mRNA) and protein levels in peripheral blood mononuclear cells (PBMCs) from subjects carrying different polymorphism genotypes. Results revealed that subjects carrying polymorphic rs907715GA and AA genotypes had significantly higher IL-21 mRNA levels, whereas protein level was increased only in subjects with rs907715AA genotype. Serum level of IL-21 was also significantly elevated in subjects with rs907715AA genotype. These data suggest that IL-21 polymorphism is associated with increased susceptibility to ischemic stroke possibly by upregulating gene expression. KEY WORDS: interleukin-21; polymorphism; ischemic stroke.
INTRODUCTION Stroke is a common cerebrovascular disorder, in which ischemic stroke is the major type [6, 7, 18]. There are an estimated 15 million new cases of ischemic stroke each year worldwide, and among these patients, 5 million die and 5 million become permanently disabled. Ischemic stroke accounts for 50 % of strokes, and risk for it is greatly influenced by an individual’s genetic background and various environmental factors. Mounting evidence indicates that inflammatory processes play an important role in the development of ischemic stroke, but the exact mechanism 1
Affiliated Bayi Brain Hospital, General Hospital of Beijing Military Command, Beijing, 100700, China 2 Department of Oncology, General Hospital of Shenyang Military Area Command, No. 83 Wenhua Road, Shenhe District, Shenyang, 110016, China 3 To whom correspondence should be addressed at Department of Oncology, General Hospital of Shenyang Military Area Command, No. 83 Wenhua Road, Shenhe District, Shenyang, 110016, China. E-mail: [email protected]
is still unclear. Proinflammatory cytokines are associated with ischemic stroke, and variations in cytokine genes play an important role in altering the transcription profile to induce predisposition and penetrance and change the pattern of proinflammatory cytokine production [6, 7, 18]. Functional polymorphisms of inflammatory genes may thereby influence the incidence and outcome of ischemic stroke [6, 7, 18]. Interleukin-21 (IL-21), the most recently discovered CD132-dependent cytokine, is mainly produced by activated T lymphocytes, particularly the inflammatory Th17 subset [5, 8, 12]. IL-21 can regulate the proliferation, maturation, and function of many hematopoietic cells including the B cell, T cell, NK cell, and dendritic cell [20, 23, 22]. IL-21 plays a major role in immunoglobulin production and terminal B cell differentiation but in certain settings can cause B cell apoptosis [16, 18, 19]. Final outcome depends on a delicate balance maintained by the interaction of many co-stimulatory factors. IL-21 also acts on the T cell as a co-mitogen and causes T cell proliferation [12, 13, 15]. IL-21 works in synergy with IL-7 and IL-15 to
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Li, Xu, Cao, Xie, and Zheng achieve this affect. When overexpressed, IL-21 can induce autoimmunity and exhibits action as an antitumor agent [4, 5, 8]. NK and NKT cells play a crucial role in providing innate immunity against viral infections and tumor growth. IL-21 is both produced by and acts on NKT cells. IL-21 induces maturation and increases the cytotoxic potential of NK cell [2]. IL-21 induces the genes of cytokines such as IFN-γ, IL-10, and GM-CSF, which are involved in the activation of innate immunity as well as many other genes involved in adaptive immunity [23, 22]. IFN-γ promotes innate and TH1 immune response by activating macrophages, NK cell, and T cells [23, 22]. The role of IL-21 and IL-15 is also to modulate the differentiation of dendritic cells. The dendritic cell has a role in pathogen recognition, capturing, processing, and presenting [23, 22]. Studies have shown that all the immune cells and cytokines mentioned above are greatly involved in the ischemic cascade after cerebral ischemia. It has been reported that single-nucleotide polymorphisms (SNPs) rs907715G/A and rs4833837A/G in IL-21 gene may be associated with various diseases such as breast cancer and Graves’ disease [1, 3, 10, 11, 14, 21]. However, functions of these polymorphisms remain unknown. In the current study, we investigated associations between these two IL-21 polymorphisms and the risk of ischemic stroke and examined the effects of these SNP on gene expression in PBMCs and serum IL21 level.
MATERIALS AND METHODS Patients and Controls The study group included 278 ischemic stroke patients and 282 healthy controls. Patients with the first onset of ischemic stroke were recruited. All patients were confirmed by neuroimaging evidence with CT or MRI according to the World Health Organization’s diagnostic criteria for ischemic stroke. The subtype classification for the etiology of ischemic stroke was based on the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Patients with transient ischemic attacks, intracranial hemorrhage, postseizure palsy, brain tumors, or brain trauma were excluded from the study. In the same period, 282 subjects who underwent regular physical examinations at the same hospital were recruited as controls. They were diagnosed free of ischemic stroke by their medical history or angiography, free of clear ischemic changes by
electrocardiography and without stroke symptoms. Individuals with congestive heart failure, peripheral vascular disease, rheumatic heart disease, pulmonary heart disease, tumor, chronic kidney disease, or hepatic disease were excluded from the study. All individuals enrolled were from the Han population in China. All patients and controls signed written informed consents. The study was approved by the ethics committee of the hospital. A structured questionnaire was used to collect general information on the patients and control subjects, including age, sex, smoking and drinking habits, diabetes, and obesity. DNA Extraction and Genotyping DNA extraction and genotyping were conducted using previously published methods. In brief, the IL-21 rs907715G/A and rs4833837A/G polymorphisms were detected by the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. PCR was performed in a total reaction volume of 20 ul containing 2 ul of 10× PCR buffer (Qiagen Inc., Hilden, Germany), 1.5 mM MgCl2, 0.5 uM of each primer, 0.2 mM dNTP, 1.2 U Taq polymerase (Qiagen Inc., Hilden, Germany), and 200 ng of genomic DNA. After an initial denaturation at 95 °C for 5 min, the DNA was amplified for 35 cycles at 94 °C for 30 s, 55–60 °C for 40 s, and 72 ° C for 45 s, with a final elongation at 72 °C for 10 min on the GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA, USA). Primers for PCR are shown in Table 1. PCR products containing the two polymorphic sites were then amplified using primers designed for Taq I and Pst I (New England Biolabs, Beverly, MA, USA) and digested with these restriction enzymes, respectively. The digested PCR products were fractionated on 2 % agarose Tris–borate– EDTA gel (Agarose 1000; Gibco BRL, Rockville, MD, USA) and stained with ethidium bromide. To confirm the genotyping results, more than 10 % of PCR-amplified Table 1. Primers Used for Detecting the IL-21 SNPs Primers rs907715 For PCR For sequence rs4833837 For PCR For sequence
F: 5′-ATAGATGAGGAAAGTGAGATC-3′ R: 5′- CTTTGCTTATTTGATATATTCC-3′ R: 5′- GTAGAATTACTAAATTAAAGGG-3′ F: 5′-GAAGACACCAGCAGCCCTGG-3′ R: 5′-GGTGAAATCATACCTCACTC-3′ F: 5′-CTACAGGTGTGTGTGTATGT-3′
IL-21 Polymorphism and Ischemic Stroke
Fig. 1. Typical sequence maps of IL-21 rs907715G/A and rs4833837A/G polymorphisms, in which the rs907715G/A polymorphism was detected by a reverse primer and the rs4833837A/G polymorphism was detected by a forward primer.
DNA samples were examined by DNA sequencing. Results between PCR and DNA sequencing analysis were
100 % concordant. Typical sequence maps of these two polymorphisms are shown (Fig. 1).
Table 2. Distributions of Demographic Characteristics Characteristics
Cases (n=278)
Age Sex Male Female BMI Diabetes No Yes Hypertension No Yes TOAST criteria Large-artery atherosclerosis Cardioembolism Small-vessel occlusion Undetermined etiology
46.4±9.7
Percent
Controls (n=282)
Percent
47.2±10.1
172 106 25.5±6.1
61.9 30.1
243 35
P value >0.05 >0.05
166 116 23.4±4.6
58.9 41.1
87.4 12.6
267 15
94.7 5.3
175 103
62.9 37.1
242 40
85.8 14.2
181 23 52 22
65.1 8.3 18.7 7.9
Interleukin-21 Polymorphism Affects Gene Expression and is Associated with Risk of Ischemic Stroke Guanggang Li,1 Ruxiang Xu,1 Yinghua Cao,1 Xiaodong Xie,2 and Zhendong Zheng2,3
Abstract—There has been more and more evidence to confirm the essential role of inflammatory processes in the development of ischemic stroke. Interleukin-21 (IL-21), the most recently discovered CD132-dependent cytokine, plays a key role in regulating inflammation. The aim of the study was to understand the association between IL-21 polymorphisms and ischemic stroke, and the effects of these polymorphisms on gene expression. Two polymorphisms in IL-21, rs907715G/A and rs4833837A/G, were identified in 278 ischemic stroke patients and 282 healthy controls. Results showed that frequencies of rs907715GA and AA genotypes were significantly increased in cases than in controls (odd ratio (OR)=1.49, 95 % confidence interval (CI): 1.01–2.14, P=0.042; OR=2.21, 95 % CI: 1.38–3.53, P=0.001). Similarly, rs907715A allele revealed a positive association with the disease (OR=1.52, P=0.001). The rs4833837A/G polymorphism did not show any correlation with ischemic stroke. We further evaluated IL-21 messenger RNA (mRNA) and protein levels in peripheral blood mononuclear cells (PBMCs) from subjects carrying different polymorphism genotypes. Results revealed that subjects carrying polymorphic rs907715GA and AA genotypes had significantly higher IL-21 mRNA levels, whereas protein level was increased only in subjects with rs907715AA genotype. Serum level of IL-21 was also significantly elevated in subjects with rs907715AA genotype. These data suggest that IL-21 polymorphism is associated with increased susceptibility to ischemic stroke possibly by upregulating gene expression. KEY WORDS: interleukin-21; polymorphism; ischemic stroke.
INTRODUCTION Stroke is a common cerebrovascular disorder, in which ischemic stroke is the major type [6, 7, 18]. There are an estimated 15 million new cases of ischemic stroke each year worldwide, and among these patients, 5 million die and 5 million become permanently disabled. Ischemic stroke accounts for 50 % of strokes, and risk for it is greatly influenced by an individual’s genetic background and various environmental factors. Mounting evidence indicates that inflammatory processes play an important role in the development of ischemic stroke, but the exact mechanism 1
Affiliated Bayi Brain Hospital, General Hospital of Beijing Military Command, Beijing, 100700, China 2 Department of Oncology, General Hospital of Shenyang Military Area Command, No. 83 Wenhua Road, Shenhe District, Shenyang, 110016, China 3 To whom correspondence should be addressed at Department of Oncology, General Hospital of Shenyang Military Area Command, No. 83 Wenhua Road, Shenhe District, Shenyang, 110016, China. E-mail: [email protected]
is still unclear. Proinflammatory cytokines are associated with ischemic stroke, and variations in cytokine genes play an important role in altering the transcription profile to induce predisposition and penetrance and change the pattern of proinflammatory cytokine production [6, 7, 18]. Functional polymorphisms of inflammatory genes may thereby influence the incidence and outcome of ischemic stroke [6, 7, 18]. Interleukin-21 (IL-21), the most recently discovered CD132-dependent cytokine, is mainly produced by activated T lymphocytes, particularly the inflammatory Th17 subset [5, 8, 12]. IL-21 can regulate the proliferation, maturation, and function of many hematopoietic cells including the B cell, T cell, NK cell, and dendritic cell [20, 23, 22]. IL-21 plays a major role in immunoglobulin production and terminal B cell differentiation but in certain settings can cause B cell apoptosis [16, 18, 19]. Final outcome depends on a delicate balance maintained by the interaction of many co-stimulatory factors. IL-21 also acts on the T cell as a co-mitogen and causes T cell proliferation [12, 13, 15]. IL-21 works in synergy with IL-7 and IL-15 to
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Li, Xu, Cao, Xie, and Zheng achieve this affect. When overexpressed, IL-21 can induce autoimmunity and exhibits action as an antitumor agent [4, 5, 8]. NK and NKT cells play a crucial role in providing innate immunity against viral infections and tumor growth. IL-21 is both produced by and acts on NKT cells. IL-21 induces maturation and increases the cytotoxic potential of NK cell [2]. IL-21 induces the genes of cytokines such as IFN-γ, IL-10, and GM-CSF, which are involved in the activation of innate immunity as well as many other genes involved in adaptive immunity [23, 22]. IFN-γ promotes innate and TH1 immune response by activating macrophages, NK cell, and T cells [23, 22]. The role of IL-21 and IL-15 is also to modulate the differentiation of dendritic cells. The dendritic cell has a role in pathogen recognition, capturing, processing, and presenting [23, 22]. Studies have shown that all the immune cells and cytokines mentioned above are greatly involved in the ischemic cascade after cerebral ischemia. It has been reported that single-nucleotide polymorphisms (SNPs) rs907715G/A and rs4833837A/G in IL-21 gene may be associated with various diseases such as breast cancer and Graves’ disease [1, 3, 10, 11, 14, 21]. However, functions of these polymorphisms remain unknown. In the current study, we investigated associations between these two IL-21 polymorphisms and the risk of ischemic stroke and examined the effects of these SNP on gene expression in PBMCs and serum IL21 level.
MATERIALS AND METHODS Patients and Controls The study group included 278 ischemic stroke patients and 282 healthy controls. Patients with the first onset of ischemic stroke were recruited. All patients were confirmed by neuroimaging evidence with CT or MRI according to the World Health Organization’s diagnostic criteria for ischemic stroke. The subtype classification for the etiology of ischemic stroke was based on the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Patients with transient ischemic attacks, intracranial hemorrhage, postseizure palsy, brain tumors, or brain trauma were excluded from the study. In the same period, 282 subjects who underwent regular physical examinations at the same hospital were recruited as controls. They were diagnosed free of ischemic stroke by their medical history or angiography, free of clear ischemic changes by
electrocardiography and without stroke symptoms. Individuals with congestive heart failure, peripheral vascular disease, rheumatic heart disease, pulmonary heart disease, tumor, chronic kidney disease, or hepatic disease were excluded from the study. All individuals enrolled were from the Han population in China. All patients and controls signed written informed consents. The study was approved by the ethics committee of the hospital. A structured questionnaire was used to collect general information on the patients and control subjects, including age, sex, smoking and drinking habits, diabetes, and obesity. DNA Extraction and Genotyping DNA extraction and genotyping were conducted using previously published methods. In brief, the IL-21 rs907715G/A and rs4833837A/G polymorphisms were detected by the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. PCR was performed in a total reaction volume of 20 ul containing 2 ul of 10× PCR buffer (Qiagen Inc., Hilden, Germany), 1.5 mM MgCl2, 0.5 uM of each primer, 0.2 mM dNTP, 1.2 U Taq polymerase (Qiagen Inc., Hilden, Germany), and 200 ng of genomic DNA. After an initial denaturation at 95 °C for 5 min, the DNA was amplified for 35 cycles at 94 °C for 30 s, 55–60 °C for 40 s, and 72 ° C for 45 s, with a final elongation at 72 °C for 10 min on the GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA, USA). Primers for PCR are shown in Table 1. PCR products containing the two polymorphic sites were then amplified using primers designed for Taq I and Pst I (New England Biolabs, Beverly, MA, USA) and digested with these restriction enzymes, respectively. The digested PCR products were fractionated on 2 % agarose Tris–borate– EDTA gel (Agarose 1000; Gibco BRL, Rockville, MD, USA) and stained with ethidium bromide. To confirm the genotyping results, more than 10 % of PCR-amplified Table 1. Primers Used for Detecting the IL-21 SNPs Primers rs907715 For PCR For sequence rs4833837 For PCR For sequence
F: 5′-ATAGATGAGGAAAGTGAGATC-3′ R: 5′- CTTTGCTTATTTGATATATTCC-3′ R: 5′- GTAGAATTACTAAATTAAAGGG-3′ F: 5′-GAAGACACCAGCAGCCCTGG-3′ R: 5′-GGTGAAATCATACCTCACTC-3′ F: 5′-CTACAGGTGTGTGTGTATGT-3′
IL-21 Polymorphism and Ischemic Stroke
Fig. 1. Typical sequence maps of IL-21 rs907715G/A and rs4833837A/G polymorphisms, in which the rs907715G/A polymorphism was detected by a reverse primer and the rs4833837A/G polymorphism was detected by a forward primer.
DNA samples were examined by DNA sequencing. Results between PCR and DNA sequencing analysis were
100 % concordant. Typical sequence maps of these two polymorphisms are shown (Fig. 1).
Table 2. Distributions of Demographic Characteristics Characteristics
Cases (n=278)
Age Sex Male Female BMI Diabetes No Yes Hypertension No Yes TOAST criteria Large-artery atherosclerosis Cardioembolism Small-vessel occlusion Undetermined etiology
46.4±9.7
Percent
Controls (n=282)
Percent
47.2±10.1
172 106 25.5±6.1
61.9 30.1
243 35
P value >0.05 >0.05
166 116 23.4±4.6
58.9 41.1
87.4 12.6
267 15
94.7 5.3
175 103
62.9 37.1
242 40
85.8 14.2
181 23 52 22
65.1 8.3 18.7 7.9
