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Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease
Cellular Immunology xxx (2016) xxx–xxx
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Research paper
Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease A.L. Shim a,b, A.A. Aksyonov b, V.M. Mitrokhin b,⇑, I.B. Lovchikova a, M.A. Konoplyannikov a, A.V. Konev a, A.S. Zotov a, R.S. Ovchinnikov a,b, E. Antova c, M.I. Mladenov b,d,⇑, A. Kamkin b a Federal Scientific Clinical Center for Specialized Types of Medical Assistance and Medical Technologies for the Federal Medical and Biological Agency, Orekhoviy Boulevard 28, Moscow 115682, Russia b Department of Fundamental and Applied Physiology, Russian National Research Medical University, Ostrovitjanova 1, Moscow 117997, Russia c Medical Faculty, University Clinic of Cardiology, ‘‘Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia d Faculty of Natural Sciences and Mathematics, Institute of Biology, ‘‘Ss. Cyril and Methodius” University, P.O. Box 162, 1000 Skopje, Macedonia
a r t i c l e
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Article history: Received 10 April 2016 Revised 28 July 2016 Accepted 8 September 2016 Available online xxxx Keywords: Cytokines Heart Sinus arrhythmia Interleukin-6
a b s t r a c t In this study, we were focused on the differences between certain circulating cytokine levels in patients with or without sinus arrhythmia, according to the median IL-6 level. All patients were stable with regards to symptoms and therapy for at least one month prior to the measurements conducted within this study. Exclusion criteria were: patients with sleep apnea, asthma, respiratory insufficiency of any genesis, active infection, allergy, inflammatory diseases, cancer, diabetes of any type and treatment with anti-inflammatory drugs. The study was approved by the Institutional Review Board. All recruited patients gave their verbal and written consent for participation in the study. The study group consisted of 74 patients divided into two groups: with (38) and without sinus arrhythmia but with diagnosed coronary artery disease (36). Sinus arrhythmia was confirmed by 24 h Holter monitoring. From all test parameters only cytokines IL-2, IL-8, IL-10, IL-17 and IL-18, showed statistically significant increasing in patients with statistically higher IL-6 levels. It is possible that IL-6 may not be a marker for the selection of patients with sinus arrhythmia or coronary artery disease. The findings indicate that IL-6 represents a reliable indicator for increased expression of IL-2, IL-8, IL-10, IL-17 and IL-18 in patients with sinus arrhythmia or coronary artery disease. Further studies in a large number of patients would be necessary to confirm our observations. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction The mechanisms underlying increased immunogenicity of body proteins in the cardiovascular system, the roles of immunogenic auto-antigens in eliciting inflammatory autoimmune responses, and the immunosuppressive mechanisms involved in controlling inflammatory and autoimmune cardiovascular diseases remain to Abbreviations: LVEF, left ventricular ejection fraction; LAV, left atrial volume; PASP, pulmonary artery systolic pressure; LVSV, left ventricular stroke volume; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; BPM, beats per minute; RAV, right atrial volume; SA, sinus arrhythmia; CAD, coronary artery disease. ⇑ Corresponding authors at: Department of Fundamental and Applied Physiology, Russian National Research Medical University, Ostrovitjanova 1, Moscow 117997, Russia (M.I. Mladenov). E-mail addresses: [email protected] (V.M. Mitrokhin), [email protected] (M.I. Mladenov).
be well-understood [1]. A significant association of inflammation indicating increased concentration of tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6), along with heavy meals, decrease in magnesium, potassium, coenzyme Q10 and nitrite in relation to circadian rhythms with risk of acute coronary syndromes have been commonly observed [2]. These findings pose a possibility that rhythm disturbances of any kind may be associated with increase in cytokines. An increased concentration of cytokines has also been observed among patients of chronic heart failure which may be associated with arrhythmogenesis and class III and IV heart failure compared to class I and II heart failure [3]. Chronic heart failure may be associated with neurohumoral dysfunction characterized with increased concentrations of catecholamines, cortisol and angiotensin II which may cause oxidative stress and inflammation and promote the loss of myocytes by apoptosis leading to rhythm disturbances [4].
http://dx.doi.org/10.1016/j.cellimm.2016.09.007 0008-8749/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
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IL-6 as a pleiotropic cytokine, functions as a mediator of inflammatory response and has both, pro- and anti-inflammatory properties. IL-6 has been identified as a differentiation factor of B and T-lymphocytes, hepatocytes, neuronal cells, and hemopoetic precursors [5]. Humphries et al. [6], reported that IL-6 was involved in the pathogenesis of cardiovascular disorders. Additional studies demonstrated that IL-6-174 G/C polymorphisms are associated with higher serum levels of IL-6, risk of coronary heart disease, acute coronary syndrome and atrial fibrillation [7]. Serum levels of IL-6 higher than 20 pg/ml in the first 24 h after ST-segment elevation at acute myocardial infarction are significantly associated with the higher frequency of in-hospital onset such is death [8]. Significant relationships between preoperative IL-6 and atrial fibrillation in patients who underwent an onpump coronary artery bypass grafting was also reported [9]. According Ucar et al. [10], elevated IL-6 and highly sensitive C-reactive protein (hsCRP) in patients with postoperative atrial fibrillation, suggests that inflammatory components play very important roles in the pathogenesis of atrial fibrillation. Also, reduction of long-term heart rate variability is associated with increased levels of IL-6 in patients with de-compensated heart failure [11]. The IL-6 level provides prognostic information that are complementary to clinical risk scores for the prediction of long-term cardiovascular events and death, suggesting that this bio-marker may potentially be used to refine clinical risk stratification in atrial fibrillation [12]. Elevated IL-6 serum concentrations were prospectively associated with an increased risk of spontaneous ventricular tachyarrhythmia in patients with an implantable cardioverter-defibrillator. These preliminary findings support a possible association of pro-inflammatory activity and an increased susceptibility to spontaneous ventricular tachyarrhythmia in the same patients [13]. All above studies reported a positive association between increased circulatory IL-6 concentration and different heart pathologies. Despite growing body of data in the field, it is unclear up to date whether elevated circulatory IL-6 plays an important role in patients with sinus arrhythmia (SA). Consequently, the objectives of our study were: to find out the role of IL-6 in patients with SA and in those without arrhythmia but with coronary artery disease (CAD); and to correlate the association of IL-6 levels with other subclasses of cytokines in the studied groups of patients. 2. Materials and methods 2.1. Patient data This was a single-center prospective study carried out between 2014 and 2015 in 74 consecutive patients with SA (n = 38) and without SA but with diagnosed CAD (n = 36). Patients were treated according to the guidelines of the American Heart Association/ American College of Cardiology. The study conformed to the principles outlined in the Helsinki Declaration and was approved by the Institutional Review Board (Ethics Committee of the Russian National Research Medical University N.I. Pirogov). After receiving appropriate information all recruited patients gave their verbal and written consent for participation in the study. SA was confirmed by 24 h Holter monitoring, instituted the same day when an echo-cardiogram had been evaluated and blood samples taken for biochemical analysis. SA included sinus node dysfunction (SND) (n = 22), bradycardia (n = 7) and tachycardia (n = 9). The study group consisted of 46 male and 28 female patients. As the study was designed to avoid mixing entities that are known to have a very different prognosis only patients with heart rate dysfunction and CAD were included, while patients with
Table 1 Study patient characteristics. Medications b-blockers (n, %) Hypolipidemic agents (n, %) Diuretics (n, %) Proton-pump inhibitor (n, %) ACE inhibitor (n, %) Antiplatelet drug (n, %) Antiarrhythmic agents (n, %) Anticoagulants (n, %) Calcium channel blockers (n, %)
43(57.3%) 51(68.0%) 13(17.3%) 19(25.3%) 26(34.7%) 32(42.7%) 13(17.3%) 29(38.7%) 26(34.7%)
Diseases Systemic hypertension (n, %) Chronic renal failure (n, %) Systolic blood pressure (mmHg ± SD)
48(64%) 0 1(1.33%) 0 135.96 ± 19.07
sleep apnea, asthma, respiratory insufficiency of any genesis, active infection, allergy, inflammatory diseases, cancer, diabetes of any type and treatment with anti-inflammatory drugs were excluded. All patients were stable with regards to symptoms and therapy for at least one month prior to the measurements conducted within this study. The patients were on standard medication consisting of b-blockers (57.3%), hypolipidemic agents (68%), diuretics (17.3%), proton-pump inhibitor (25.3%), ACE inhibitor (34.66%), antiplatelet drug (42.66%), antiarrhythmic agents (17.33%), anticoagulants (38.66%) and calcium channel blockers (34.66%). History of systemic hypertension (64%), chronic renal failure (1.33%) and systolic blood pressure were also assessed (Table 1). 2.2. Biochemical analysis Venous blood samples were taken from the patient’s ulnar vein, between 8.00 and 9.00 a.m. for biochemical tests after overnight fasting, using vials without any anticoagulant. Serum was prepared by centrifugation (2000g for 20 min.), separated into aliquots and stored at 80 °C until analyses were performed. Serum samples were analysed for: sodium (Na), glucose (Glu), creatinine (CRT), triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), and urea (U) by an Automatic biochemical Analyzer (Abbott Architect c8000 Chemistry Analyzer). 2.3. IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18 and VEGFimmunoassays IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18 and VEGF in the serum were analysed by the newly developed ELISA for quantitative analysis of IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18, and VEGF levels from Bender Med-Systems. The limits of detection of the assays were about 1.0 pg ml 1 for IL-1b, 2.0 pg ml 1 for IL-2, 0.4 pg ml 1 for IL-4, 0.92 pg ml 1 for IL-6, 2.0 pg ml 1 for IL-8, 1.0 pg ml 1 for IL-10, 2.0 pg ml 1 for IL-17, 2.0 pg ml 1 for IL-18 and 10.0 pg ml 1 for VEGF. Inter- and intra-assay CVs were 6.4% and 3.1% for IL-1b, 7.7% and 5.8% for IL-2, 8.3% and 8.6% for IL-4, 5.2% and 3.4% for IL-6, 8.9% and 6.1% for IL-8, 9.8% and 9.4% for IL-10, 4.8% and 8.5% for IL-17, 5.8% and 7.6% for IL-18, and 8.1% and 9.7% for VEGF. 2.4. Instrumental diagnostic methods Electrocardiograms (ECG) from the patients were recorded before blood sampling. The following time domain variables were computed for each subject: heart rate (HR), PQ interval, QRS complex, QT interval. Echo-cardiograms (Echo-CG) were also recorded before blood sampling measuring the following parameters: left
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
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ventricular ejection fraction (LVEF), left atrial volume (LAV), pulmonary artery systolic pressure (PASP), left ventricular stroke volume (LVSV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), beats per minute (BPM) and right atrial volume (RAV). 2.5. Statistical analysis Data was summarized and displayed as means ± SD for continuous variables, inclusive of the number of patients plus percentages in each group for categorical variables. The distribution was tested by the one-sample Kolmogorov-Smirnov test, while the Mann Whitney U analysis was used for non-normally distributed continuous variables. For categorical variables, chi-square statistics were used for assessing overall significance between the two groups of patients comparing the high and low plasma levels of IL-6. Correlations between the markers were calculated using Pearson correlation analysis. The level of significance for analysis was two-tailed and p < 0.05 was considered statistically significant. Based on the median serum levels of IL-6, we compared patients’ clinical parameters, including ECG and Echo-CG measurements of the atrial and ventricular size and function. We also examined the relationships to other laboratory markers such as Na, GLU, CRT, TG, TC, HDL, LDL, U and serum cytokine level. All analyses were performed with Graph Pad Prism 5.0 (San Diego, CA, USA). 3. Results 3.1. Statistical differences between groups with and without SA The first goal of this study was to check the differences between IL-6 serum levels, ECG, and Echo-CG parameters in patients with SA versus patients without SA but with CAD. The IL-6 levels of these two groups were compared and there were no significant differences between patients with or without SA (P = 0.826). ECG and Echo-CG parameters were also insignificant in both tested groups (P = 0.349, 0.805, 0.255, 0.841, 0.993, 0.706, 0.359, 0.766, 0.518, 0.726 and 0.624; for HR, PQ, QRS, QT, LVEF, LAV, PASP, LVSV, LVEDV, LVESV and RAV, respectively, Table 2). Further, in order to check the influence of gender on the circulatory IL-6 levels within both groups (with and without) SA, we have been compared: male with (n = 20) versus female with Table 2 Comparison between patients with and without Sinus arrhythmia. Number of patients (%)
Patients without Sinus arrhythmia (36)
Patients with Sinus arrhythmia (38)
P
Age (yrs. mean ± SD) Man (n, %) Women (n, %) LAV (ml) RAV (ml) LVSV (ml) LVEDV (ml) LVESV (ml) BPM PQ (ms) QRS (ms) QT (ms) LVEF (%. mean ± SD) PASP (mmHg) IL-6 (pg/ml)
68.91 ± 9.52 27 (57.5%) 10 (27.0%) 65.73 ± 28.62 56.03 ± 19.18 77.81 ± 10.74 131.08 ± 7.64 53.27 ± 6.99 70.57 ± 17.84 172 ± 43 110 ± 35 405 ± 44 59.20 ± 6.01 46.46 ± 19.28 3.37 ± 6.55
63.35 ± 16.06 20 (42.5%) 18 (47.4%) 65.05 ± 27.76 55.03 ± 18.45 78.53 ± 10.03 132.32 ± 8.76 53.79 ± 5.76 66.82 ± 16.68 170 ± 34 101 ± 34 403 ± 47 59.19 ± 4.90 50.50 ± 18.58 4.16 ± 6.62
0.156 / / 0.706 0.624 0.766 0.518 0.726 0.349 0.805 0.255 0.841 0.993 0.359 0.826
PQ interval (PQ), QRS complex (QRS), QT interval (QT), left ventricular ejection fraction (LVEF), left atrial volume (LAV), pulmonary artery systolic pressure (PASP), left ventricular stroke volume (LVSV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), beats per minute (BPM), right ventricular systolic pressure (RVSP), left atrial diameter (LAD), right atrial diameter (RAD), pulmonary artery diameter (PAD) and interleukin-6 (IL-6).
Table 3 Influence of gender on the circulatory IL-6 levels, within both groups (with and without) Sinus arrhythmia. Gender
With Sinus arrhythmia
Without Sinus arrhythmia
P
Female, (pg/ml) Male, (pg/ml) P
2.49 ± 2.79 5.96 ± 7.73 0.284
3.57 ± 3,56 3.39 ± 7,25 0.811
0.563 0.464
(n = 18) SA (P = 0.284) and male without (n = 26) versus female without (n = 10) SA (P = 0.811). The changes in circulatory IL-6 levels within the same gender were followed by comparing male with (n = 20) versus male without (n = 26) SA (P = 0.464) and female with (n = 18) versus female without (n = 10) SA (P = 0.563). Obtained results indicated that there were no statistical differences within all tested group pairs, which excludes gender as a factor involved in the changes of IL-6 in the patients with SA in comparison to patients without SA but with CAD (Table 3). 3.2. Statistical differences between ECG parameters in dependence of serum IL-6 level Taking into account, insignificant differences between all tested parameters in our experimental setting and to ascertain the one and predictive role of the IL-6 in different onsets of cardiovascular events [14], from the other side, we decided to check if there was any relationship among ECG parameters in dependence of median IL-6 values. Since, median IL-6 values in samples were 1.48 mg/ml, we classified patients into a high IL-6 group (P1.48 pg/ml) and low IL-6 group (<1.48 pg/ml) and compared ECG variances between these two groups. There were no significant differences based on Table 4 Differences between patients with low and high IL-6 levels (below and above median IL-6). Number of patients (n)
Low IL-6(38)
High IL-6(36)
P
Age (yrs. mean ± SD) Man (n. %) Women (n. %) LAV (ml) RAV (ml) LVSV (ml) LVEDV (ml) LVESV (ml) BPM PQ (ms) QRS (ms) QT (ms) LVEF (%. mean ± SD) PASP (mmHg) Na (mmol/L) TC (mmol/L) LDL (mmol/L) HDL (mmol/L) TG (mmol/L) CRT (lmol/L) U (mmol/L) GLU (mmol/L) IL-1b (pg/ml) IL-2 (pg/ml) IL-4 (pg/ml) IL-8 (pg/ml) IL-10 (pg/ml) IL-17 (pg/ml) IL-18 (pg/ml) VEGF (pg/ml)
66.00 ± 11.27 23 (60.5%) 15 (39.5%) 65.40 ± 26.70 55.92 ± 18.91 78.95 ± 10.80 131.89 ± 8.55 52.95 ± 5.75 65.89 ± 15.99 168 ± 49 102 ± 34 403 ± 47 59.66 ± 5.33 49.69 ± 19.33 139.81 ± 7.79 5.32 ± 1.42 3.11 ± 0.76 1.57 ± 0.35 1.44 ± 0.43 94.05 ± 31.87 7.26 ± 3.39 5.98 ± 1,27 0.87 ± 0,92 0.47 ± 0.64 0.83 ± 0.52 0.58 ± 1.03 1.57 ± 1.65 0.28 ± 0.41 107.28 ± 59.64 217.76 ± 231.01
64.26 ± 15.34 24 (64.9%) 13 (35.1%) 65.41 ± 27.62 55.11 ± 18.72 77.38 ± 9.89 131.51 ± 7.91 54.14 ± 6.95 71.51 ± 18.23 174 ± 29 109 ± 37 404 ± 45 58.73 ± 5.59 47.30 ± 18.65 139.8 ± 8.41 5.17 ± 1.32 2.94 ± 0.69 1.52 ± 0.34 1.37 ± 0.43 92.39 ± 18.70 6.71 ± 2.06 6.01 ± 1,08 2,42 ± 6,67 1.00 ± 0.85 0.74 ± 0.45 1.40 ± 1.56 4.71 ± 9.53 0.87 ± 0.70 137.85 ± 65.91 235.63 ± 251.99
0.591 / / 0.236 0.691 0.514 0.842 0.422 0.159 0.574 0.354 0.926 0.464 0.587 0.993 0.641 0.321 0.571 0.464 0.785 0.396 0.907 0.162 0.010⁄ 0.401 0.010⁄ 0.050⁄ 0.010⁄ 0.041⁄ 0.752
All abbreviations for Echo-CG and ECG parameters are the same as in Table 2. Sodium (Na), total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), triglycerides (TG), creatinine (CRT), urea (U), glucose (GLU), interleukin (IL) vascular endothelial growth factor (VEGF). ⁄p < 0.05.
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
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age (P = 0.591) or other tested variables like heart rate, PQ interval, QRS complex and QT interval (P = 0.159, 0.574, 0.354 and 0.924, respectively, Table 4). 3.3. Statistical differences between Echo-CG parameters in dependence of median serum IL-6 level Further, the Echo-CG parameters were compared between the groups with high and low IL-6 levels, and there were no significant statistical differences in LVEF, LAV, PASP, LVSV, LVEDV, LVESV and RAV; (P = 0.464, 0.236, 0.587, 0.514, 0.842, 0.422 and 0.691; respectively, Table 4). 3.4. Statistical differences between biochemical markers in dependence of median serum IL-6 level The biochemical analyses of taken blood samples were also compared between groups with low and high IL-6 serum levels. There were no significant statistical differences in sodium (Na), total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), triglycerides (TG), creatinine (CRT), urea (U) and glucose (GLU); (P = 0.993, 0.641, 0.321, 0.571, 0.464, 0.785, 0.396 and 0.907; respectively, Table 4). 3.5. Statistical differences between cytokines in dependence of median serum IL-6 level The values of IL-1b, IL-2, IL-4, IL-8, IL-10, IL-17, IL-18 and VEGF between groups with low and high IL-6 serum levels were compared as well. There were no significant differences in IL-1b, IL-4 and VEGF levels (P = 0.162, 0.401, 0.752; respectively, Table 4). It is interesting to note that significant differences were registered for IL-2, IL-8, IL-17 (P = 0.01, for all; respectively, Table 4) and IL-10 and IL-18 (P = 0.050, 0.041; respectively, Table 4). It is important to notice that, higher levels of these cytokines were observed in the group with a higher IL-6 level and a lower was detected in the group with a lower IL-6 level. 3.6. Correlation between IL-6 and other cytokines Another important goal of this study was to find a correlation between IL-6 level and other cytokines. The Pearson correlation coefficients with significance levels for the correlations between various cytokines and IL-6 levels are shown in (Table 5). There were significant correlations between IL-6 and IL-2, IL-8, IL-10, IL-17 and IL-18 (P = 0.010, 0.011, 0.050, 0.008 and 0.044; respectively) in the group with low IL-6 level. Similarly, in the group of patients with high IL-6 level, the coefficients of correlation were with the level of significance for the same group of cytokines
Table 5 Correlation between IL-6 and other ILs within groups with high and low IL-6 levels (below and above median IL-6).
⁄
Cytokine
Patients with Low IL-6 (38) P
r
P
r
IL-1b IL-2 (pg/ml) IL-4 (pg/ml) IL-8 (pg/ml) IL-10 (pg/ml) IL-17 (pg/ml) IL-18 (pg/ml) VEGF (pg/ml)
0.162 0.010⁄ 0.401 0.011⁄ 0.050⁄ 0.008⁄ 0.044⁄ 0.752
0.245 0.428 0.192 0.404 0.361 0.472 0.378 0.102
0.223 0.020⁄ 0.326 0.046⁄ 0.348 0.009⁄ 0.049⁄ 0.407
0.161 0.395 0.104 0.368 0.077 0.447 0.354 0.098
p < 0.05.
Patients with High IL-6 (36)
Table 6 Differences in Medications and Syndromes between patients with low and high IL-6 (below and above median IL-6). Number of patients (n, %)
Low IL-6 (38)
High IL-6 (36)
Medications b-blockers (n,%) AT1-receptor antagonists (n, %) Hypolipidemic agents (n, %) Diuretics (n, %) Proton-pump inhibitor (n, %) ACE inhibitor (n, %) Antiplatelet drug (n, %) Antiarrhythmic agents (n, %) Anticoagulants (n, %) Calcium channel blockers (n, %)
22 (57.9%) 6 (15.8%) 23 (60.5%) 6 (15.8%) 9 (23.7%) 11 (28.9%) 19 (50%) 6 (15.8%) 9 (23.7%) 15 (39.5%)
21 (56.8%) 4 (10.8%) 18 (48.6%) 7 (18.9%) 10 (27%) 14 (37.8%) 13 (35.1%) 7 (18.9%) 19 (51.4%) 11 (29.7%)
Diseases Systemic hypertension (n, %) Chronic renal failure (n, %) Systolic blood pressure (mmHg ± SD)
25 (65.8%) 1 (2.6%) 137.03 ± 18.79
23 (62.2%) 0 (0%) 135.48 ± 20.43
(IL-2, IL-8, IL-17 and IL-18; P = 0.020; 0.046; 0.009 and 0.049, respectively), with exception of IL-10 (P = 0.348). 3.7. Statistical differences in medications and diseases between patients with low and high IL-6 serum levels Two subgroups of patients, classified according to the serum IL-6 level, did not differ significantly in the use of b-blockers, AT1-receptor antagonists, hypolipidemic agents, diuretics, proton pump inhibitors, ACE inhibitors, antiplatelet drugs, antiarrhythmic agents, anticoagulants, and calcium channel blockers (Table 6). As shown in the same table, higher IL-6 level is not associated with systemic hypertension and systolic blood pressure (Table 6). Chronic renal failure was registered only in one patient in the group with low IL-6 serum level (Table 6). 4. Discussion Inflammation in heart disease patients is produced by a complex of humoral and cellular interactions with numerous pathways, including activation or expression of cytokines and multiple inflammatory mediators [3,4,15]. In this direction, Kubota et al. (2000), finding correlation between serum IL-6 levels and its myocardial mRNA expression, lies down the basis for its classification as an appropriate activator of myocardial cytokine expression [16]. A growing body of data for its involvement in the signaling pathways of a wide variety of other cytokines, fostered us to analyze its relationships with other cytokines among patients with or without SA. By checking the relationship between IL-6 and IL-8, we have shown that circulatory IL-8 levels were higher in patients with circulatory higher IL-6 levels. Assuming that one of the key mechanisms by which circulating IL-6 contributes to the development of coronary heart disease is the autocrine and paracrine activation of monocytes, such a close association between IL-6 and IL-8 could be related to the activation of the monocyte-macrophage branch of cell-mediated immunity [17]. In addition, Maggiore et al. [18], asserted that IL-6 and IL-8 levels are elevated in children with untreated autoimmune hepatitis. This association indicates that serum IL-6 is the main stimulator of the hepatic acute-phase response, which is further associated with increased IL-8 level, increased blood viscosity and increased platelet numbers and activity [18]. It seems that such an inter-adjustment among both cytokines (IL-6 and IL-8) together with the other induced proinflammatory mechanisms refers to heart damage. Another important mechanism by which circulating IL-6 contributes to the development of CAD is to stimulate the
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
A.L. Shim et al. / Cellular Immunology xxx (2016) xxx–xxx
hypothalamic–pituitary adrenal (HPA) axis [4]. This activation is associated with central obesity, hypertension and insulin resistance [19], from one aspect, and circulatory IL-2 alteration from the other [20]. Our results for significant increasing in IL-2 in patients with higher IL-6 levels could be related to a biphasic pattern of action of the IL-2 [20]. Such a pattern of action, is related to dose-dependent modulation of the release of endogenous dopamine (increasing the release at lower concentrations and inhibiting release at a higher concentrations of the IL-2) [21]. Actually, the positive association between IL-6 and IL-2 manifested through a bi-phasic modality of action, demonstrates the determining role of IL-6 upon the circulatory IL-2 levels in the pathophysiology of different heart diseases. In our study, there was a significant association between IL-6 and IL-10. IL-10 is considered an ‘‘anti-inflammatory” cytokine that decreases the production of IL-6 in the peripheral mononuclear cells and is traditionally assumed to blunt its deleterious effects in heart failure patients [22,23]. The mechanism of combined elevation of IL-6 and IL-10 in patients with SA is unclear and beyond the scope of the current study. We are on the opinion that partial explanation, lay in the increased circulatory levels of IL-18 in patients with a higher IL-6 concentration. Actually, an increased pro-inflammatory profile undoubtedly leads to the elevation of the serum concentration of IL-18 as an important cytokine for initiating and perpetuating the catabolic and inflammatory responses in the patients with SA [24]. IL-18 can also act as a co-inducer of the T-helper 2 (Th2) cytokine IL-10 from IL-6 fostered T cells and basophils [25,26]. Therefore, the over-expression of the IL-18 could be one of the reasons for a significant increase in serum levels of IL-10 in the patients with SA. Taken together, the elevated production of the pro-inflammatory IL-6 and IL-18 and antiinflammatory IL-10 cytokine, exert a complexity of inflammatory reactions that cause heart damage in the patient study group. The biggest issue that arises here, is why we did not find significant correlation between IL-6 and IL-10 in the group of patients with a higher IL-6 level. The response probably lies in the relatively huge IL-10 variations, which may mask the correlation between IL-6 and IL-10 within the same group of patients. IL-6 can interact with both membrane-bound and soluble forms of its ligand-binding receptor (IL-6R alpha), triggering signaling via dimerization of the glikoprotein (gp130), the signaling subunit of the IL-6 receptor complex [27]. This leads to activation of the Janus kinase/signal transducer and activator of the transcription (JAK/STAT) pathway and mainly culminates in the activation of the STAT3 transcription factor [28,29]. Both IL-6 and STAT3 have recently emerged as main regulators of the differentiation and function of Th17 cells, via a positive feedback loop enhancing expression and activation of IL-6 itself, IL-17 and STAT3 [27]. Based on that, it seems that significant increasing in the IL-17 levels in patients with higher circulatory IL-6 levels in our study, is probably associated with the IL-6 fostered differentiation of the Th17 cells and their involvement in the heart failure. Since we didn’t find any correlation between categorical parameters and IL-6, we suggest that IL-6 induces systemic inflammation in both groups of patients (with or without SA), and probably can not be taken as a marker for noninvasive selection of patients with SA or CAD. The IL-6 only represent a reliable indicator for increased expression of IL-2, IL-8, IL-10, IL-17 and IL-18 in patients with SA or CAD.
5. Limitations The patient sample was rather small for statistical evaluation of the interaction forces of investigated parameters. In all probability, this could be the reason why elevated IL-6 wasn’t associated with
5
other clinical prognostic parameters. In addition, some studies have provided evidence that cardiac production is not the main source of raising peripheral cytokine concentrations, especially in patients with CAD and that the bioactivity of IL-6 in the serum in some cardial onsets is modulated by the serum IL-6-R receptor [30]. The same study, actually revealed that cytokine induction in myocardium is a relatively late event in the pathogenesis of CAD, which partly explains why obtained results from human studies are contradictory. Taking that, we measured pro/anti-inflammatory markers in the ulnar vein, it would be interesting to know the local degree of inflammation at the atrial level or in the pulmonary veins, which could not be correctly assessed from a systemic point. The study is limited by the absence of additional parameters which would help to interpret the sources of IL-6 elevation. The study is also hampered by its cross-sectional design and we can only explore the associations, no causality is implied. In order to elucidate the complexity of the inflammatory model in patients with or without SA, additional studies with the profound immunological milieu should be done. Conflict of interest None declared Acknowledgments We thank subjects and staff at Federal Scientific Clinical Center for Specialized Types of Medical Assistance and Medical Technologies for participating in the study. References [1] M. Jan, A.T. Virtue, M. Pansuria, J. Liu, X. Xiong, P. Fang, S. Meng, H. Wang, X.F. Yang, The role of immunogenicity in cardiovascular disease, World Heart J. 3 (2011) 1–29. [2] R.B. Singh, J. Fedacko, J.P. Sharma, V. Vargova, M. Sharma, F.D. Moshiri, K. Otsuka. Meester, Association of inflammation, heavy meals, magnesium, nitrite, and coenzyme Q10 deficiency and circadian rhythms with risk of acute coronary syndromes, World Heart J. 2 (2010) 219–228. [3] A. Kumar, R.B. Singh, M. Saxena, M.A. Niaz, S.R. Josh, P. Chattopadhyay, V. Mechirova, D. Pella, R. Chopra, Effect of carni Q-gel (ubiquinol and carnitine) on cytokines in patients with heart failure in the Tishcon study, Acta Cardiol. 62 (2007) 349–354. [4] R.B. Singh, G. Cornelissen, T. Takahashi, S. Shastun, K. Hristova, S. Chibisov, M. Keim, M. Abramova, K. Otsuka, B. Saboo, R.K. Singh, N.S. Verma, A. Gvozdjáková, J. Fedacko, D. Pella, R. Singh, A. Maheshwari, A.K. Pandey, D.W. Wilson, Brain-heart interactions and circadian rhythms in chronic heart failure, World Heart J. 7 (2015) 129–142. [5] T. Kishimoto, S. Akira, M. Narazaki, T. Taga, Interleukin-6 family of cytokines and gp130, Blood 86 (1995) 1243–1254. [6] S.E. Humphries, L.A. Luong, M.S. Ogg, E. Hawe, G.J. Miller, The interleukin-6174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men, Eur. Heart J. 22 (2001) 2243–2252. [7] D.P. Potaczek, A. Undas, M. Celinska-Lowenhoff, A. Szczeklik, Interleukin-6-174 G/C promoter polymorphism and effects of fenofibrate and simvastatin on inflammatory markers in hypercholesterolemic patients, Blood Coagul. Fibrinolysis 17 (2006) 35–38. [8] G. Borrayo-Sanchez, A. Pacheco-Bouthillier, L. Mendoza-Valdez, I. Isordia-Salas, R. Arguero-Sanchez, G. Careaga Reyna, Prognostic value of serum levels of interleukin-6 in patients with ST-segment elevation acute myocardial infarction, Cir. Cir. 78 (2010) 25–30. [9] S. Ziabakhsh-Tabari, Can perioperative C-reactive protein and interleukin-6 levels predict atrial fibrillation after coronary artery bypass surgery, Saudi Med. J. 29 (2008) 1429–1431. [10] H.I. Ucar, M. Tok, E. Atalar, O.F. Dogan, M. Oc, B. Farsak, M. Guvener, M. Yilmaz, R. Dogan, M. Demircin, I. Pasaoglu, Predictive significance of plasma levels of interleukin-6 and high-sensitivity C-reactive protein in atrial fibrillation after coronary artery bypass surgery, Heart Surg. Forum 10 (2010) 131–135. [11] D. Aronson, M.A. Mittleman, A.J. Burger, Interleukin-6 levels are inversely correlated with heart rate variability in patients with decompensated heart failure, J. Cardiovasc. Electrophysiol. 12 (2001) 294–300. [12] V. Roldán, F. Marín, J. Díaz, P. Gallego, E. Jover, M. Romera, S. ManzanoFernández, T. Casas, M. Valdés, V. Vicente, G.Y. Lip, High sensitivity cardiac troponin T and interleukin-6 predict adverse cardiovascular events and
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Research paper
Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease A.L. Shim a,b, A.A. Aksyonov b, V.M. Mitrokhin b,⇑, I.B. Lovchikova a, M.A. Konoplyannikov a, A.V. Konev a, A.S. Zotov a, R.S. Ovchinnikov a,b, E. Antova c, M.I. Mladenov b,d,⇑, A. Kamkin b a Federal Scientific Clinical Center for Specialized Types of Medical Assistance and Medical Technologies for the Federal Medical and Biological Agency, Orekhoviy Boulevard 28, Moscow 115682, Russia b Department of Fundamental and Applied Physiology, Russian National Research Medical University, Ostrovitjanova 1, Moscow 117997, Russia c Medical Faculty, University Clinic of Cardiology, ‘‘Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia d Faculty of Natural Sciences and Mathematics, Institute of Biology, ‘‘Ss. Cyril and Methodius” University, P.O. Box 162, 1000 Skopje, Macedonia
a r t i c l e
i n f o
Article history: Received 10 April 2016 Revised 28 July 2016 Accepted 8 September 2016 Available online xxxx Keywords: Cytokines Heart Sinus arrhythmia Interleukin-6
a b s t r a c t In this study, we were focused on the differences between certain circulating cytokine levels in patients with or without sinus arrhythmia, according to the median IL-6 level. All patients were stable with regards to symptoms and therapy for at least one month prior to the measurements conducted within this study. Exclusion criteria were: patients with sleep apnea, asthma, respiratory insufficiency of any genesis, active infection, allergy, inflammatory diseases, cancer, diabetes of any type and treatment with anti-inflammatory drugs. The study was approved by the Institutional Review Board. All recruited patients gave their verbal and written consent for participation in the study. The study group consisted of 74 patients divided into two groups: with (38) and without sinus arrhythmia but with diagnosed coronary artery disease (36). Sinus arrhythmia was confirmed by 24 h Holter monitoring. From all test parameters only cytokines IL-2, IL-8, IL-10, IL-17 and IL-18, showed statistically significant increasing in patients with statistically higher IL-6 levels. It is possible that IL-6 may not be a marker for the selection of patients with sinus arrhythmia or coronary artery disease. The findings indicate that IL-6 represents a reliable indicator for increased expression of IL-2, IL-8, IL-10, IL-17 and IL-18 in patients with sinus arrhythmia or coronary artery disease. Further studies in a large number of patients would be necessary to confirm our observations. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction The mechanisms underlying increased immunogenicity of body proteins in the cardiovascular system, the roles of immunogenic auto-antigens in eliciting inflammatory autoimmune responses, and the immunosuppressive mechanisms involved in controlling inflammatory and autoimmune cardiovascular diseases remain to Abbreviations: LVEF, left ventricular ejection fraction; LAV, left atrial volume; PASP, pulmonary artery systolic pressure; LVSV, left ventricular stroke volume; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; BPM, beats per minute; RAV, right atrial volume; SA, sinus arrhythmia; CAD, coronary artery disease. ⇑ Corresponding authors at: Department of Fundamental and Applied Physiology, Russian National Research Medical University, Ostrovitjanova 1, Moscow 117997, Russia (M.I. Mladenov). E-mail addresses: [email protected] (V.M. Mitrokhin), [email protected] (M.I. Mladenov).
be well-understood [1]. A significant association of inflammation indicating increased concentration of tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6), along with heavy meals, decrease in magnesium, potassium, coenzyme Q10 and nitrite in relation to circadian rhythms with risk of acute coronary syndromes have been commonly observed [2]. These findings pose a possibility that rhythm disturbances of any kind may be associated with increase in cytokines. An increased concentration of cytokines has also been observed among patients of chronic heart failure which may be associated with arrhythmogenesis and class III and IV heart failure compared to class I and II heart failure [3]. Chronic heart failure may be associated with neurohumoral dysfunction characterized with increased concentrations of catecholamines, cortisol and angiotensin II which may cause oxidative stress and inflammation and promote the loss of myocytes by apoptosis leading to rhythm disturbances [4].
http://dx.doi.org/10.1016/j.cellimm.2016.09.007 0008-8749/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
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IL-6 as a pleiotropic cytokine, functions as a mediator of inflammatory response and has both, pro- and anti-inflammatory properties. IL-6 has been identified as a differentiation factor of B and T-lymphocytes, hepatocytes, neuronal cells, and hemopoetic precursors [5]. Humphries et al. [6], reported that IL-6 was involved in the pathogenesis of cardiovascular disorders. Additional studies demonstrated that IL-6-174 G/C polymorphisms are associated with higher serum levels of IL-6, risk of coronary heart disease, acute coronary syndrome and atrial fibrillation [7]. Serum levels of IL-6 higher than 20 pg/ml in the first 24 h after ST-segment elevation at acute myocardial infarction are significantly associated with the higher frequency of in-hospital onset such is death [8]. Significant relationships between preoperative IL-6 and atrial fibrillation in patients who underwent an onpump coronary artery bypass grafting was also reported [9]. According Ucar et al. [10], elevated IL-6 and highly sensitive C-reactive protein (hsCRP) in patients with postoperative atrial fibrillation, suggests that inflammatory components play very important roles in the pathogenesis of atrial fibrillation. Also, reduction of long-term heart rate variability is associated with increased levels of IL-6 in patients with de-compensated heart failure [11]. The IL-6 level provides prognostic information that are complementary to clinical risk scores for the prediction of long-term cardiovascular events and death, suggesting that this bio-marker may potentially be used to refine clinical risk stratification in atrial fibrillation [12]. Elevated IL-6 serum concentrations were prospectively associated with an increased risk of spontaneous ventricular tachyarrhythmia in patients with an implantable cardioverter-defibrillator. These preliminary findings support a possible association of pro-inflammatory activity and an increased susceptibility to spontaneous ventricular tachyarrhythmia in the same patients [13]. All above studies reported a positive association between increased circulatory IL-6 concentration and different heart pathologies. Despite growing body of data in the field, it is unclear up to date whether elevated circulatory IL-6 plays an important role in patients with sinus arrhythmia (SA). Consequently, the objectives of our study were: to find out the role of IL-6 in patients with SA and in those without arrhythmia but with coronary artery disease (CAD); and to correlate the association of IL-6 levels with other subclasses of cytokines in the studied groups of patients. 2. Materials and methods 2.1. Patient data This was a single-center prospective study carried out between 2014 and 2015 in 74 consecutive patients with SA (n = 38) and without SA but with diagnosed CAD (n = 36). Patients were treated according to the guidelines of the American Heart Association/ American College of Cardiology. The study conformed to the principles outlined in the Helsinki Declaration and was approved by the Institutional Review Board (Ethics Committee of the Russian National Research Medical University N.I. Pirogov). After receiving appropriate information all recruited patients gave their verbal and written consent for participation in the study. SA was confirmed by 24 h Holter monitoring, instituted the same day when an echo-cardiogram had been evaluated and blood samples taken for biochemical analysis. SA included sinus node dysfunction (SND) (n = 22), bradycardia (n = 7) and tachycardia (n = 9). The study group consisted of 46 male and 28 female patients. As the study was designed to avoid mixing entities that are known to have a very different prognosis only patients with heart rate dysfunction and CAD were included, while patients with
Table 1 Study patient characteristics. Medications b-blockers (n, %) Hypolipidemic agents (n, %) Diuretics (n, %) Proton-pump inhibitor (n, %) ACE inhibitor (n, %) Antiplatelet drug (n, %) Antiarrhythmic agents (n, %) Anticoagulants (n, %) Calcium channel blockers (n, %)
43(57.3%) 51(68.0%) 13(17.3%) 19(25.3%) 26(34.7%) 32(42.7%) 13(17.3%) 29(38.7%) 26(34.7%)
Diseases Systemic hypertension (n, %) Chronic renal failure (n, %) Systolic blood pressure (mmHg ± SD)
48(64%) 0 1(1.33%) 0 135.96 ± 19.07
sleep apnea, asthma, respiratory insufficiency of any genesis, active infection, allergy, inflammatory diseases, cancer, diabetes of any type and treatment with anti-inflammatory drugs were excluded. All patients were stable with regards to symptoms and therapy for at least one month prior to the measurements conducted within this study. The patients were on standard medication consisting of b-blockers (57.3%), hypolipidemic agents (68%), diuretics (17.3%), proton-pump inhibitor (25.3%), ACE inhibitor (34.66%), antiplatelet drug (42.66%), antiarrhythmic agents (17.33%), anticoagulants (38.66%) and calcium channel blockers (34.66%). History of systemic hypertension (64%), chronic renal failure (1.33%) and systolic blood pressure were also assessed (Table 1). 2.2. Biochemical analysis Venous blood samples were taken from the patient’s ulnar vein, between 8.00 and 9.00 a.m. for biochemical tests after overnight fasting, using vials without any anticoagulant. Serum was prepared by centrifugation (2000g for 20 min.), separated into aliquots and stored at 80 °C until analyses were performed. Serum samples were analysed for: sodium (Na), glucose (Glu), creatinine (CRT), triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), and urea (U) by an Automatic biochemical Analyzer (Abbott Architect c8000 Chemistry Analyzer). 2.3. IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18 and VEGFimmunoassays IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18 and VEGF in the serum were analysed by the newly developed ELISA for quantitative analysis of IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18, and VEGF levels from Bender Med-Systems. The limits of detection of the assays were about 1.0 pg ml 1 for IL-1b, 2.0 pg ml 1 for IL-2, 0.4 pg ml 1 for IL-4, 0.92 pg ml 1 for IL-6, 2.0 pg ml 1 for IL-8, 1.0 pg ml 1 for IL-10, 2.0 pg ml 1 for IL-17, 2.0 pg ml 1 for IL-18 and 10.0 pg ml 1 for VEGF. Inter- and intra-assay CVs were 6.4% and 3.1% for IL-1b, 7.7% and 5.8% for IL-2, 8.3% and 8.6% for IL-4, 5.2% and 3.4% for IL-6, 8.9% and 6.1% for IL-8, 9.8% and 9.4% for IL-10, 4.8% and 8.5% for IL-17, 5.8% and 7.6% for IL-18, and 8.1% and 9.7% for VEGF. 2.4. Instrumental diagnostic methods Electrocardiograms (ECG) from the patients were recorded before blood sampling. The following time domain variables were computed for each subject: heart rate (HR), PQ interval, QRS complex, QT interval. Echo-cardiograms (Echo-CG) were also recorded before blood sampling measuring the following parameters: left
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
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ventricular ejection fraction (LVEF), left atrial volume (LAV), pulmonary artery systolic pressure (PASP), left ventricular stroke volume (LVSV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), beats per minute (BPM) and right atrial volume (RAV). 2.5. Statistical analysis Data was summarized and displayed as means ± SD for continuous variables, inclusive of the number of patients plus percentages in each group for categorical variables. The distribution was tested by the one-sample Kolmogorov-Smirnov test, while the Mann Whitney U analysis was used for non-normally distributed continuous variables. For categorical variables, chi-square statistics were used for assessing overall significance between the two groups of patients comparing the high and low plasma levels of IL-6. Correlations between the markers were calculated using Pearson correlation analysis. The level of significance for analysis was two-tailed and p < 0.05 was considered statistically significant. Based on the median serum levels of IL-6, we compared patients’ clinical parameters, including ECG and Echo-CG measurements of the atrial and ventricular size and function. We also examined the relationships to other laboratory markers such as Na, GLU, CRT, TG, TC, HDL, LDL, U and serum cytokine level. All analyses were performed with Graph Pad Prism 5.0 (San Diego, CA, USA). 3. Results 3.1. Statistical differences between groups with and without SA The first goal of this study was to check the differences between IL-6 serum levels, ECG, and Echo-CG parameters in patients with SA versus patients without SA but with CAD. The IL-6 levels of these two groups were compared and there were no significant differences between patients with or without SA (P = 0.826). ECG and Echo-CG parameters were also insignificant in both tested groups (P = 0.349, 0.805, 0.255, 0.841, 0.993, 0.706, 0.359, 0.766, 0.518, 0.726 and 0.624; for HR, PQ, QRS, QT, LVEF, LAV, PASP, LVSV, LVEDV, LVESV and RAV, respectively, Table 2). Further, in order to check the influence of gender on the circulatory IL-6 levels within both groups (with and without) SA, we have been compared: male with (n = 20) versus female with Table 2 Comparison between patients with and without Sinus arrhythmia. Number of patients (%)
Patients without Sinus arrhythmia (36)
Patients with Sinus arrhythmia (38)
P
Age (yrs. mean ± SD) Man (n, %) Women (n, %) LAV (ml) RAV (ml) LVSV (ml) LVEDV (ml) LVESV (ml) BPM PQ (ms) QRS (ms) QT (ms) LVEF (%. mean ± SD) PASP (mmHg) IL-6 (pg/ml)
68.91 ± 9.52 27 (57.5%) 10 (27.0%) 65.73 ± 28.62 56.03 ± 19.18 77.81 ± 10.74 131.08 ± 7.64 53.27 ± 6.99 70.57 ± 17.84 172 ± 43 110 ± 35 405 ± 44 59.20 ± 6.01 46.46 ± 19.28 3.37 ± 6.55
63.35 ± 16.06 20 (42.5%) 18 (47.4%) 65.05 ± 27.76 55.03 ± 18.45 78.53 ± 10.03 132.32 ± 8.76 53.79 ± 5.76 66.82 ± 16.68 170 ± 34 101 ± 34 403 ± 47 59.19 ± 4.90 50.50 ± 18.58 4.16 ± 6.62
0.156 / / 0.706 0.624 0.766 0.518 0.726 0.349 0.805 0.255 0.841 0.993 0.359 0.826
PQ interval (PQ), QRS complex (QRS), QT interval (QT), left ventricular ejection fraction (LVEF), left atrial volume (LAV), pulmonary artery systolic pressure (PASP), left ventricular stroke volume (LVSV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), beats per minute (BPM), right ventricular systolic pressure (RVSP), left atrial diameter (LAD), right atrial diameter (RAD), pulmonary artery diameter (PAD) and interleukin-6 (IL-6).
Table 3 Influence of gender on the circulatory IL-6 levels, within both groups (with and without) Sinus arrhythmia. Gender
With Sinus arrhythmia
Without Sinus arrhythmia
P
Female, (pg/ml) Male, (pg/ml) P
2.49 ± 2.79 5.96 ± 7.73 0.284
3.57 ± 3,56 3.39 ± 7,25 0.811
0.563 0.464
(n = 18) SA (P = 0.284) and male without (n = 26) versus female without (n = 10) SA (P = 0.811). The changes in circulatory IL-6 levels within the same gender were followed by comparing male with (n = 20) versus male without (n = 26) SA (P = 0.464) and female with (n = 18) versus female without (n = 10) SA (P = 0.563). Obtained results indicated that there were no statistical differences within all tested group pairs, which excludes gender as a factor involved in the changes of IL-6 in the patients with SA in comparison to patients without SA but with CAD (Table 3). 3.2. Statistical differences between ECG parameters in dependence of serum IL-6 level Taking into account, insignificant differences between all tested parameters in our experimental setting and to ascertain the one and predictive role of the IL-6 in different onsets of cardiovascular events [14], from the other side, we decided to check if there was any relationship among ECG parameters in dependence of median IL-6 values. Since, median IL-6 values in samples were 1.48 mg/ml, we classified patients into a high IL-6 group (P1.48 pg/ml) and low IL-6 group (<1.48 pg/ml) and compared ECG variances between these two groups. There were no significant differences based on Table 4 Differences between patients with low and high IL-6 levels (below and above median IL-6). Number of patients (n)
Low IL-6(38)
High IL-6(36)
P
Age (yrs. mean ± SD) Man (n. %) Women (n. %) LAV (ml) RAV (ml) LVSV (ml) LVEDV (ml) LVESV (ml) BPM PQ (ms) QRS (ms) QT (ms) LVEF (%. mean ± SD) PASP (mmHg) Na (mmol/L) TC (mmol/L) LDL (mmol/L) HDL (mmol/L) TG (mmol/L) CRT (lmol/L) U (mmol/L) GLU (mmol/L) IL-1b (pg/ml) IL-2 (pg/ml) IL-4 (pg/ml) IL-8 (pg/ml) IL-10 (pg/ml) IL-17 (pg/ml) IL-18 (pg/ml) VEGF (pg/ml)
66.00 ± 11.27 23 (60.5%) 15 (39.5%) 65.40 ± 26.70 55.92 ± 18.91 78.95 ± 10.80 131.89 ± 8.55 52.95 ± 5.75 65.89 ± 15.99 168 ± 49 102 ± 34 403 ± 47 59.66 ± 5.33 49.69 ± 19.33 139.81 ± 7.79 5.32 ± 1.42 3.11 ± 0.76 1.57 ± 0.35 1.44 ± 0.43 94.05 ± 31.87 7.26 ± 3.39 5.98 ± 1,27 0.87 ± 0,92 0.47 ± 0.64 0.83 ± 0.52 0.58 ± 1.03 1.57 ± 1.65 0.28 ± 0.41 107.28 ± 59.64 217.76 ± 231.01
64.26 ± 15.34 24 (64.9%) 13 (35.1%) 65.41 ± 27.62 55.11 ± 18.72 77.38 ± 9.89 131.51 ± 7.91 54.14 ± 6.95 71.51 ± 18.23 174 ± 29 109 ± 37 404 ± 45 58.73 ± 5.59 47.30 ± 18.65 139.8 ± 8.41 5.17 ± 1.32 2.94 ± 0.69 1.52 ± 0.34 1.37 ± 0.43 92.39 ± 18.70 6.71 ± 2.06 6.01 ± 1,08 2,42 ± 6,67 1.00 ± 0.85 0.74 ± 0.45 1.40 ± 1.56 4.71 ± 9.53 0.87 ± 0.70 137.85 ± 65.91 235.63 ± 251.99
0.591 / / 0.236 0.691 0.514 0.842 0.422 0.159 0.574 0.354 0.926 0.464 0.587 0.993 0.641 0.321 0.571 0.464 0.785 0.396 0.907 0.162 0.010⁄ 0.401 0.010⁄ 0.050⁄ 0.010⁄ 0.041⁄ 0.752
All abbreviations for Echo-CG and ECG parameters are the same as in Table 2. Sodium (Na), total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), triglycerides (TG), creatinine (CRT), urea (U), glucose (GLU), interleukin (IL) vascular endothelial growth factor (VEGF). ⁄p < 0.05.
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
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A.L. Shim et al. / Cellular Immunology xxx (2016) xxx–xxx
age (P = 0.591) or other tested variables like heart rate, PQ interval, QRS complex and QT interval (P = 0.159, 0.574, 0.354 and 0.924, respectively, Table 4). 3.3. Statistical differences between Echo-CG parameters in dependence of median serum IL-6 level Further, the Echo-CG parameters were compared between the groups with high and low IL-6 levels, and there were no significant statistical differences in LVEF, LAV, PASP, LVSV, LVEDV, LVESV and RAV; (P = 0.464, 0.236, 0.587, 0.514, 0.842, 0.422 and 0.691; respectively, Table 4). 3.4. Statistical differences between biochemical markers in dependence of median serum IL-6 level The biochemical analyses of taken blood samples were also compared between groups with low and high IL-6 serum levels. There were no significant statistical differences in sodium (Na), total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), triglycerides (TG), creatinine (CRT), urea (U) and glucose (GLU); (P = 0.993, 0.641, 0.321, 0.571, 0.464, 0.785, 0.396 and 0.907; respectively, Table 4). 3.5. Statistical differences between cytokines in dependence of median serum IL-6 level The values of IL-1b, IL-2, IL-4, IL-8, IL-10, IL-17, IL-18 and VEGF between groups with low and high IL-6 serum levels were compared as well. There were no significant differences in IL-1b, IL-4 and VEGF levels (P = 0.162, 0.401, 0.752; respectively, Table 4). It is interesting to note that significant differences were registered for IL-2, IL-8, IL-17 (P = 0.01, for all; respectively, Table 4) and IL-10 and IL-18 (P = 0.050, 0.041; respectively, Table 4). It is important to notice that, higher levels of these cytokines were observed in the group with a higher IL-6 level and a lower was detected in the group with a lower IL-6 level. 3.6. Correlation between IL-6 and other cytokines Another important goal of this study was to find a correlation between IL-6 level and other cytokines. The Pearson correlation coefficients with significance levels for the correlations between various cytokines and IL-6 levels are shown in (Table 5). There were significant correlations between IL-6 and IL-2, IL-8, IL-10, IL-17 and IL-18 (P = 0.010, 0.011, 0.050, 0.008 and 0.044; respectively) in the group with low IL-6 level. Similarly, in the group of patients with high IL-6 level, the coefficients of correlation were with the level of significance for the same group of cytokines
Table 5 Correlation between IL-6 and other ILs within groups with high and low IL-6 levels (below and above median IL-6).
⁄
Cytokine
Patients with Low IL-6 (38) P
r
P
r
IL-1b IL-2 (pg/ml) IL-4 (pg/ml) IL-8 (pg/ml) IL-10 (pg/ml) IL-17 (pg/ml) IL-18 (pg/ml) VEGF (pg/ml)
0.162 0.010⁄ 0.401 0.011⁄ 0.050⁄ 0.008⁄ 0.044⁄ 0.752
0.245 0.428 0.192 0.404 0.361 0.472 0.378 0.102
0.223 0.020⁄ 0.326 0.046⁄ 0.348 0.009⁄ 0.049⁄ 0.407
0.161 0.395 0.104 0.368 0.077 0.447 0.354 0.098
p < 0.05.
Patients with High IL-6 (36)
Table 6 Differences in Medications and Syndromes between patients with low and high IL-6 (below and above median IL-6). Number of patients (n, %)
Low IL-6 (38)
High IL-6 (36)
Medications b-blockers (n,%) AT1-receptor antagonists (n, %) Hypolipidemic agents (n, %) Diuretics (n, %) Proton-pump inhibitor (n, %) ACE inhibitor (n, %) Antiplatelet drug (n, %) Antiarrhythmic agents (n, %) Anticoagulants (n, %) Calcium channel blockers (n, %)
22 (57.9%) 6 (15.8%) 23 (60.5%) 6 (15.8%) 9 (23.7%) 11 (28.9%) 19 (50%) 6 (15.8%) 9 (23.7%) 15 (39.5%)
21 (56.8%) 4 (10.8%) 18 (48.6%) 7 (18.9%) 10 (27%) 14 (37.8%) 13 (35.1%) 7 (18.9%) 19 (51.4%) 11 (29.7%)
Diseases Systemic hypertension (n, %) Chronic renal failure (n, %) Systolic blood pressure (mmHg ± SD)
25 (65.8%) 1 (2.6%) 137.03 ± 18.79
23 (62.2%) 0 (0%) 135.48 ± 20.43
(IL-2, IL-8, IL-17 and IL-18; P = 0.020; 0.046; 0.009 and 0.049, respectively), with exception of IL-10 (P = 0.348). 3.7. Statistical differences in medications and diseases between patients with low and high IL-6 serum levels Two subgroups of patients, classified according to the serum IL-6 level, did not differ significantly in the use of b-blockers, AT1-receptor antagonists, hypolipidemic agents, diuretics, proton pump inhibitors, ACE inhibitors, antiplatelet drugs, antiarrhythmic agents, anticoagulants, and calcium channel blockers (Table 6). As shown in the same table, higher IL-6 level is not associated with systemic hypertension and systolic blood pressure (Table 6). Chronic renal failure was registered only in one patient in the group with low IL-6 serum level (Table 6). 4. Discussion Inflammation in heart disease patients is produced by a complex of humoral and cellular interactions with numerous pathways, including activation or expression of cytokines and multiple inflammatory mediators [3,4,15]. In this direction, Kubota et al. (2000), finding correlation between serum IL-6 levels and its myocardial mRNA expression, lies down the basis for its classification as an appropriate activator of myocardial cytokine expression [16]. A growing body of data for its involvement in the signaling pathways of a wide variety of other cytokines, fostered us to analyze its relationships with other cytokines among patients with or without SA. By checking the relationship between IL-6 and IL-8, we have shown that circulatory IL-8 levels were higher in patients with circulatory higher IL-6 levels. Assuming that one of the key mechanisms by which circulating IL-6 contributes to the development of coronary heart disease is the autocrine and paracrine activation of monocytes, such a close association between IL-6 and IL-8 could be related to the activation of the monocyte-macrophage branch of cell-mediated immunity [17]. In addition, Maggiore et al. [18], asserted that IL-6 and IL-8 levels are elevated in children with untreated autoimmune hepatitis. This association indicates that serum IL-6 is the main stimulator of the hepatic acute-phase response, which is further associated with increased IL-8 level, increased blood viscosity and increased platelet numbers and activity [18]. It seems that such an inter-adjustment among both cytokines (IL-6 and IL-8) together with the other induced proinflammatory mechanisms refers to heart damage. Another important mechanism by which circulating IL-6 contributes to the development of CAD is to stimulate the
Please cite this article in press as: A.L. Shim et al., Serum interleukin-6: Association with circulating cytokine serum levels in patients with sinus arrhythmia and patients with coronary artery disease, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.09.007
A.L. Shim et al. / Cellular Immunology xxx (2016) xxx–xxx
hypothalamic–pituitary adrenal (HPA) axis [4]. This activation is associated with central obesity, hypertension and insulin resistance [19], from one aspect, and circulatory IL-2 alteration from the other [20]. Our results for significant increasing in IL-2 in patients with higher IL-6 levels could be related to a biphasic pattern of action of the IL-2 [20]. Such a pattern of action, is related to dose-dependent modulation of the release of endogenous dopamine (increasing the release at lower concentrations and inhibiting release at a higher concentrations of the IL-2) [21]. Actually, the positive association between IL-6 and IL-2 manifested through a bi-phasic modality of action, demonstrates the determining role of IL-6 upon the circulatory IL-2 levels in the pathophysiology of different heart diseases. In our study, there was a significant association between IL-6 and IL-10. IL-10 is considered an ‘‘anti-inflammatory” cytokine that decreases the production of IL-6 in the peripheral mononuclear cells and is traditionally assumed to blunt its deleterious effects in heart failure patients [22,23]. The mechanism of combined elevation of IL-6 and IL-10 in patients with SA is unclear and beyond the scope of the current study. We are on the opinion that partial explanation, lay in the increased circulatory levels of IL-18 in patients with a higher IL-6 concentration. Actually, an increased pro-inflammatory profile undoubtedly leads to the elevation of the serum concentration of IL-18 as an important cytokine for initiating and perpetuating the catabolic and inflammatory responses in the patients with SA [24]. IL-18 can also act as a co-inducer of the T-helper 2 (Th2) cytokine IL-10 from IL-6 fostered T cells and basophils [25,26]. Therefore, the over-expression of the IL-18 could be one of the reasons for a significant increase in serum levels of IL-10 in the patients with SA. Taken together, the elevated production of the pro-inflammatory IL-6 and IL-18 and antiinflammatory IL-10 cytokine, exert a complexity of inflammatory reactions that cause heart damage in the patient study group. The biggest issue that arises here, is why we did not find significant correlation between IL-6 and IL-10 in the group of patients with a higher IL-6 level. The response probably lies in the relatively huge IL-10 variations, which may mask the correlation between IL-6 and IL-10 within the same group of patients. IL-6 can interact with both membrane-bound and soluble forms of its ligand-binding receptor (IL-6R alpha), triggering signaling via dimerization of the glikoprotein (gp130), the signaling subunit of the IL-6 receptor complex [27]. This leads to activation of the Janus kinase/signal transducer and activator of the transcription (JAK/STAT) pathway and mainly culminates in the activation of the STAT3 transcription factor [28,29]. Both IL-6 and STAT3 have recently emerged as main regulators of the differentiation and function of Th17 cells, via a positive feedback loop enhancing expression and activation of IL-6 itself, IL-17 and STAT3 [27]. Based on that, it seems that significant increasing in the IL-17 levels in patients with higher circulatory IL-6 levels in our study, is probably associated with the IL-6 fostered differentiation of the Th17 cells and their involvement in the heart failure. Since we didn’t find any correlation between categorical parameters and IL-6, we suggest that IL-6 induces systemic inflammation in both groups of patients (with or without SA), and probably can not be taken as a marker for noninvasive selection of patients with SA or CAD. The IL-6 only represent a reliable indicator for increased expression of IL-2, IL-8, IL-10, IL-17 and IL-18 in patients with SA or CAD.
5. Limitations The patient sample was rather small for statistical evaluation of the interaction forces of investigated parameters. In all probability, this could be the reason why elevated IL-6 wasn’t associated with
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other clinical prognostic parameters. In addition, some studies have provided evidence that cardiac production is not the main source of raising peripheral cytokine concentrations, especially in patients with CAD and that the bioactivity of IL-6 in the serum in some cardial onsets is modulated by the serum IL-6-R receptor [30]. The same study, actually revealed that cytokine induction in myocardium is a relatively late event in the pathogenesis of CAD, which partly explains why obtained results from human studies are contradictory. Taking that, we measured pro/anti-inflammatory markers in the ulnar vein, it would be interesting to know the local degree of inflammation at the atrial level or in the pulmonary veins, which could not be correctly assessed from a systemic point. The study is limited by the absence of additional parameters which would help to interpret the sources of IL-6 elevation. The study is also hampered by its cross-sectional design and we can only explore the associations, no causality is implied. In order to elucidate the complexity of the inflammatory model in patients with or without SA, additional studies with the profound immunological milieu should be done. Conflict of interest None declared Acknowledgments We thank subjects and staff at Federal Scientific Clinical Center for Specialized Types of Medical Assistance and Medical Technologies for participating in the study. References [1] M. Jan, A.T. Virtue, M. Pansuria, J. Liu, X. Xiong, P. Fang, S. Meng, H. Wang, X.F. Yang, The role of immunogenicity in cardiovascular disease, World Heart J. 3 (2011) 1–29. [2] R.B. Singh, J. Fedacko, J.P. Sharma, V. Vargova, M. Sharma, F.D. Moshiri, K. Otsuka. Meester, Association of inflammation, heavy meals, magnesium, nitrite, and coenzyme Q10 deficiency and circadian rhythms with risk of acute coronary syndromes, World Heart J. 2 (2010) 219–228. [3] A. Kumar, R.B. Singh, M. Saxena, M.A. Niaz, S.R. Josh, P. Chattopadhyay, V. Mechirova, D. Pella, R. Chopra, Effect of carni Q-gel (ubiquinol and carnitine) on cytokines in patients with heart failure in the Tishcon study, Acta Cardiol. 62 (2007) 349–354. [4] R.B. Singh, G. Cornelissen, T. Takahashi, S. Shastun, K. Hristova, S. Chibisov, M. Keim, M. Abramova, K. Otsuka, B. Saboo, R.K. Singh, N.S. Verma, A. Gvozdjáková, J. Fedacko, D. Pella, R. Singh, A. Maheshwari, A.K. Pandey, D.W. Wilson, Brain-heart interactions and circadian rhythms in chronic heart failure, World Heart J. 7 (2015) 129–142. [5] T. Kishimoto, S. Akira, M. Narazaki, T. Taga, Interleukin-6 family of cytokines and gp130, Blood 86 (1995) 1243–1254. [6] S.E. Humphries, L.A. Luong, M.S. Ogg, E. Hawe, G.J. Miller, The interleukin-6174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men, Eur. Heart J. 22 (2001) 2243–2252. [7] D.P. Potaczek, A. Undas, M. Celinska-Lowenhoff, A. Szczeklik, Interleukin-6-174 G/C promoter polymorphism and effects of fenofibrate and simvastatin on inflammatory markers in hypercholesterolemic patients, Blood Coagul. Fibrinolysis 17 (2006) 35–38. [8] G. Borrayo-Sanchez, A. Pacheco-Bouthillier, L. Mendoza-Valdez, I. Isordia-Salas, R. Arguero-Sanchez, G. Careaga Reyna, Prognostic value of serum levels of interleukin-6 in patients with ST-segment elevation acute myocardial infarction, Cir. Cir. 78 (2010) 25–30. [9] S. Ziabakhsh-Tabari, Can perioperative C-reactive protein and interleukin-6 levels predict atrial fibrillation after coronary artery bypass surgery, Saudi Med. J. 29 (2008) 1429–1431. [10] H.I. Ucar, M. Tok, E. Atalar, O.F. Dogan, M. Oc, B. Farsak, M. Guvener, M. Yilmaz, R. Dogan, M. Demircin, I. Pasaoglu, Predictive significance of plasma levels of interleukin-6 and high-sensitivity C-reactive protein in atrial fibrillation after coronary artery bypass surgery, Heart Surg. Forum 10 (2010) 131–135. [11] D. Aronson, M.A. Mittleman, A.J. Burger, Interleukin-6 levels are inversely correlated with heart rate variability in patients with decompensated heart failure, J. Cardiovasc. Electrophysiol. 12 (2001) 294–300. [12] V. Roldán, F. Marín, J. Díaz, P. Gallego, E. Jover, M. Romera, S. ManzanoFernández, T. Casas, M. Valdés, V. Vicente, G.Y. Lip, High sensitivity cardiac troponin T and interleukin-6 predict adverse cardiovascular events and
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