Association Between Inflammatory Markers and the Interleukin-6 -174 G/C Polymorphism is Abolished in Peripheral Arterial Occlusive Disease
By Potaczek, D P Undas, A; Nowakowski, T; Szczeklik, A
Aim. The interleukin-6 (IL-6) -174 G/C polymorphism has been reported to determine IL-6 levels and contribute to the development of cardiovascular disorders. The aim of our study was to evaluate the effect of the IL-6 -174 G/C polymorphism on hemostatic or inflammatory markers in patients with peripheral arterial occlusive disease (PAOD), a common manifestation of obliterative atherosclerosis. Methods. Plasma IL-6, fibrinogen, C-reactive protein (CRP), tissue plasminogen activator, plasminogen activator inhibitor-1 (PAI-1), fibrinopeptide A and intercellular adhesion molecule-1 levels were determined in PAOD patients (n=50) and healthy controls (n=30) genotyped for the IL-6 -174 G/C polymorphism.
Results. In the control group, IL-6, CRP and fibrinogen levels were significantly associated with the IL-6 -174 G/C polymorphism with a gene-dosage effect being the highest in the CC subjects and the lowest in those with the GG genotype (P<0.0001, P=0.0002 and P=0.0001, respectively). Interestingly, the CC homozygotes had lower PAI-1 levels than carriers of the G allele (P=0.04). In PAOD patients, the IL-6 -174 G/C polymorphism had no effect on all the variables measured.
Conclusion. In contrast to apparently healthy subjects, the IL-6 – 174 G/C polymorphism showed no association with plasma IL-6, CRP, fibrinogen and PAI-1 levels in PAOD patients.
[Int Angiol 2007;26:318-23]
Key words: Atherosclerosis – Inflammation – Interleukin-6 – Arterial occlusive disease – Polymorphism, genetic.
Interleukin-6 (IL-6) is a pleiotropic cytokine produced by a variety of cells, such as monocytes, endothelial cells, fibroblasts, adipocytes, mast cells and T cells. It exerts many immune, hematologic, endocrine and metabolic effects. In particular, IL-6 plays a central role in acute inflammatory response and liver acute- phase reactant synthesis.1,2
A genetic regulation of serum IL-6 levels has been extensively investigated, especially in atherosclerotic vascular disease. The IL- 6 gene -174 G/C polymorphism has been postulated to be the most important genetic determinant of serum IL-6 concentrations. However, data on the relationship between the IL-6 -174 G/C polymorphism and IL-6 levels obtained in patients with different manifestations of atherosclerosis and in control groups are conflicting.3-10 Some authors reported that the C allele (or CC genotype) of the IL-6 – 174 G/C polymorphism is associated with higher serum IL6 levels,3, 5, 8 while others showed its lower levels,9, 10 or no effect.4, 6, 7 Moreover, C-reactive protein (CRP) 4, 6, 11 and fibrinogen4 concentrations have been found to be significantly higher in carriers of the C allele. However, some investigators reported no relationship between the IL-6 -174 G/C polymorphism and CRP3, 7, 8 and/or fibrinogen 3, 7 levels. In one report lower levels of both proteins have been shown in carriers of the C allele.10
Studies performed in a broad spectrum of cardiovascular disorders provided evidence that cardiovascular risk might be linked to the IL- 6 -174 G/C polymorphism; however, data are conflicting.3-6
Peripheral arterial occlusive disease (PAOD) is a common manifestation of obliterative atherosclerosis. It has been shown that various inflammatory and hemostatic factors are increased in PAOD patients, suggesting involvement of both inflammatory and prothrombotic state in the etiology and pathogenesis of PAOD.10, 12- 16
To our knowledge, so far three studies addressed the role of the IL-6 -174 G/C polymorphism in PAOD.10, 17, 18 In one study, Flex et al.,17 indicated a strong association between the IL-6 -174 G/C polymorphism and the presence of PAOD. They found the GG genotype to be more frequent in patients suffering from lower limb atherosclerotic ischemia.17 This study did not provide, however, data on interactions between this polymorphism and inflammatory markers. Another study, performed in patients with diabetes type 2 by Libra et al.,10 corroborated these findings by showing that the GG genotype is associated with PAOD development. Analysis of IL-6, CRP, fibrinogen and vascular endothelial growth factor (VEGF) showed that the first three variables are higher in diabetics not carrying the C allele, regardless of the presence or absence of PAOD.10 VEGF was, however, not related to the IL-6 -174 G/C polymorphism regardless of PAOD.10 In the study by Danielsson et al.,18 comparison of genotypic frequencies did not show any significant association between the IL-6 -174 G/C polymorphism and PAOD; an interaction between the polymorphism and inflammatory markers was not analyzed.18
Thus, a relationship between the IL-6 -174 G/C polymorphism and inflammation and/or blood coagulation in PAOD remains unclear. Therefore, we decided to perform a study on a potential association between inflammatory and hemostatic parameters with the IL-6 -174 G/ C polymorphism in the groups of PAOD patients and healthy controls.
Materials and methods
Study population
We enrolled 50 ambulatory patients diagnosed with PAOD in Fontaine stage II (evidenced by a history of intermittent claudication), physical examination, an ankle-brachial index (ABI) of <0.8 and arterial angiography (50% to 99% stenosis of the major arteries). Exclusion criteria were: age >70 years, acute infection, cancer, acute cardiovascular event within the previous 3 months, hepatic (alanine aminotransferase > 1.5-fold of the upper limit of the reference range) or renal (creatinine > 177 [mu]mol/L) dysfunction, and autoimmune diseases. The control group included 30 apparently healthy subjects with ABI of >0.9, without any signs or symptoms of acute infection or cancer, no history of coronary heart disease, arterial hypertension, intermittent claudication, stroke or other cardiovascular disorders, diabetes and any other metabolic or biochemical abnormalities, and no smokers. None of controls was taking any medication. The study was approved by the Jagiellonian University Ethics Committee and the subjects provided written informed consent.
Clinical tests
ABI was determined by Doppler ultrasonography (Multi Dopplex II [MD2], Huntleigh Healthcare, NJ, USA). A treadmill walking test was performed at a constant speed of 3.2 km/h and slope of 10[degrees] (Treadmill Series 2000, Marquette Electronics, WI, USA). Angiography was carried out by intravenous administration of contrast agent into the antecubital vein (Integris V3000, Philips Medical Systems, The Netherlands). Angiographic staging of vascular changes in low extremity arteries was performed using the adjusted runoff score (ROS).19 In brief, low extremity arteries were given points according to their importance for the extremity blood supply: common iliac, common femoral and popliteal arteries were given 3 points, superficial femoral artery 2 points, and the others were given 1 point. Artery occlusion rate was staged using 4-point scale: 0, artery not occluded; 1, occlusion <50%; 2, occlusion >/=50%; 3, complete artery occlusion (lower leg arteries occlusion was staged using 3-point scale: 0, unchanged vessel; 1, arterial wall irregularity without occlusion; 2, occlusion). ROS value was formed by a product of points for artery ‘importance’ and for the occlusion rate.
Laboratory investigations
Blood samples were drawn from the antecubital vein. Lipid profile was measured using standard laboratory methods. Fibrinogen (Dade Behring, Germany) and CRP (Dade Behring, Germany) were measured by latex nephelometry. Tissue plasminogen activator (t-PA) (American Diagnostica, CT, USA), plasminogen activator inhibitor-1 (PAI-I) (American Diagnostica, CT, USA), fibrinopeptide A (FPA) (American Diagnostica, CT, USA); IL-6 (R&D Systems, Great Britain) and intercellular adhesion molecule-1 (ICAM-I) (R&D Systems, Great Britain) were determined by commercial enzyme-linked immunosorbent assays.
Genotyping
Genotyping for the IL-6 -174 G/C polymorphism was performed as described previously.7,8
Statistical analysis
Comparisons of quantitative data (expressed as median [interquartile range, IQR]) were made by Mann-Whitney U test or by Kruskal-Wallis ANOVA, as appropriate. Comparisons of qualitative data (given as proportion [%]) were performed by chi^sup 2^ test or Fishers exact test, as appropriate. Correlations were assessed by Spearman’s rank correlation coefficient. A P value<0.05 was considered significant.
Results
Characteristics of both study groups are given in Table I. The genotype distribution of the IL-6 -174 G/C polymorphism was 17, 23 and 10 in the PAOD subjects, and 15, 7, 8 in controls (GG, GC and CC, respectively). These gave the C allele frequency of 0.43 in the former and 0.38 in the latter group. The genotype distribution was in Hardy-Weinberg equilibrium in PAOD patients (P=0.91), but not in controls (P=0.008), and did not differ between those groups (P=0.13).
In the control group, the highest IL-6 levels were found in subjects with the CC genotype and the lowest in those carrying two G alleles (P<0.0001 ) (Table II). Also contrasting CC homozygotes against G allele carriers and subjects with GG genotype against those carrying the C allele gave statistically significant results (P=000002 and P<0.000001, respectively). Moreover, the same stepwise patterns were seen for CRP and fibrinogen levels (Table II). For these proteins, the differences between all three genotypes, between carriers of the C allele and those with GG genotype, and between carriers of the G allele and subjects with CC genotype were all significant (P=0.0002, 0.00009 and 0.00001 for CRP, and P=0.0001, 0.00003 and 0.00001 for fibrinogen; respectively) (Table II). There were significant correlations between IL-6 levels and CRP (R=0.83, P<0.000001 ) or fibrinogen (R=0.87, P<0.000001) levels (Table III). Also PAI-1 levels differed significantly being the lowest in those with CC genotype (P=0.04) (Table II). However, we did not notice any statistically significant differences between any of the studied parameters in PAOD patients (all P not significant) (Table II). In this group, we found a negative correlation between ICAM-I and IL-6 levels (R=-0.56, P=0.002) (Table III).
ABI, claudication distance or ROS (median values: 0.6 [0.27], 100 [90] m and 24 [20.0], respectively) determined in PAOD patients were not related to the IL-6 -174 G/C polymorphism (all P not significant, data not shown). There were no differences in laboratory variables and clinical characteristics in relation to the IL-6 -174 G/C polymorphism in PAOD patients who self-reported taking statins (n=35) (all P >0.05, data not shown).
Discussion
Despite a number of reports regarding the role of inflammatory and hemostatic factors in PAOD and their genetic determinants,10, 12- 16 the interaction between the IL-6 -174 G/C polymorphism and PAOD remains unclear.
Our study compared for the first time the association of IL-6 – 174 G/C polymorphism with inflammatory and hemostatic parameters (IL- 6, CRP, fibrinogen, t-PA, PAI-I, FPA and ICAM-I) in patients with documented PAOD and in apparently healthy controls. In contrast to the study by Libra et al.,10 in which all patients were diagnosed with diabetes type 2, the majority of the participants in the present study were free of diabetes, which by itself affects largely the inflammatory response and enhances prothrombotic mechanisms in vivo.14, 16, 20-22
We showed here an interesting pattern of genetic association in healthy individuals, in whom the IL-6 -174 G/C polymorphism C allele (or CC genotype) was related to higher levels of IL-6, CRP and fibrinogen (the last two proteins correlated with IL-6 levels) in a dose-dependent manner. A novel finding is that the CC genotype is related to significantly lower PAI-I levels in healthy individuals. This corroborates the occurrence of tight links between fibrinolysis and inflammation under physiologic conditions in vivo. All these associations were, however, abolished in patients with PAOD, indicating that pathological states associated with enhanced inflammatory response and hypercoagulability are likely to disturb a subtle physiologic balance, largely genetically determined. Interestingly, no significant associations in relation to the IL-6 polymorphism were also observed in 35 PAOD subjects taking inhibitors of 3hydroxy-3-methylglutaryl coenzyme reductase (statins) that can affect inflammatory and hemostatic markers.7, 8 This might indicate that such a cholesterol-lowering medication of multiple additional actions is not able to restore a physiologic balance in PAOD patients usually with marked atherosclerotic burden and several cardiovascular risk factors.
Previous reports on the IL-6 -174 G/C polymorphism and IL-6 levels are conflicting.3-10 We observed the characteristic pattern in healthy controls (in whom the C allele or CC genotype determine higher IL-6 levels). This finding is in line with other reports.3, 5, 8 However, some studies reported opposite results, for example the study by Libra et al.,10 in which the GG genotype was associated with higher IL-6 levels in type 2 diabetics regardless to the presence or absence of PAOD. These discrepancies might be explained by different effects of the IL-6 -174 G/C polymorphism in various clinical settings and in populations which differ with respect to classical risk factors e.g., age, diabetes, hyperlipidemia. We suggest that the dose-dependent manner genetic association reported in the current study in apparently healthy subjects might be, therefore, a good frame of reference for the effect of the IL-6 – 174 G/C polymorphism on IL-6, CRP, fibrinogen and PAI-1 levels. In this group, the exclusion of pathological states known for their proinflammatory effects such as acute and chronic infections, cancer, metabolic or endocrine disorders, any organ dysfunction, etc. enabled us to observe the impact of genetic regulation on IL-6 synthesis and related humeral changes. Given the small size of our study population, the results presented here need, however, to be interpreted with caution.
Contrary to Flex et al.17 and Libra et al.,10 and similarly to Danielsson et al.,18 the allelic frequencies in the present study did not differ between both study subgroups. However, the limited number of the participants makes it impossible to speculate about the associations of allele frequencies or genotype distributions and the risk of developing PAOD.
Conclusions
In summary, we confirmed that the IL-6 -174 G/C polymorphism is involved in the regulation of inflammatory response in healthy controls. However, this effect is abolished in PAOD patients, which might be associated with the extent of atherosclerosis. Since the IL- 6 -174 G/C polymorphism-mediated effects appear to vary depending on the population studied, further investigation in well-defined patient groups are necessary to assess the actual role of this polymorphism in vivo.
Funding.-D. P. P. was awarded the stipend of Foundation for Polish Science START 2007.
Received on February 1, 2007; acknowledged on March 1, 2007; sent for revision on May 22, 2007; resubmitted on May 30, 2007; accepted for publication on June 20, 2007.
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D. P. POTACZEK1, A. UNDAS 2, T. NOWAKOWSKI1,A. SZCZEKLIK1
1 Department of Medicine, Jagiellonian University School of Medicine, Cracow, Poland
2 Institute of Cardiology, Jagiellonian University School of Medicine, Cracow, Poland
Address reprint requests to: A. Szczeklik, MD, PhD, Department of Medicine, Jagiellonian University School of Medicine, 8 Skawinska Str., 31-066 Cracow, Poland.
E-mail: mmszczek@cyf-kr.edu.pl
Copyright Edizioni Minerva Medica Dec 2007
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