Associations of Serum IL-18 Levels With Carotid Intima-Media Thickness
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动脉硬化血栓血管生物学 2005年第7期
From the Department of Internal Medicine and Therapeutics (A8) (H.Y., K.K., T.H., S.F., H.H., M.H.), Osaka University Graduate School of Medicine, Japan; and the Department of Clinical Study Management (Y.N.), Translational Research Informatics Center, Kobe, Japan.
Correspondence to Hiroshi Yamagami, MD, PhD, Department of Internal Medicine and Therapeutics (A8), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail yamagami@medone.med.osaka-u.ac.jp
Abstract
Objective— Elevated circulating levels of IL-18 can predict future coronary heart disease. Although IL-18 is thought to play a crucial role in atherosclerosis, whether circulating IL-18 levels are associated with the severity of atherosclerosis remains to be determined. With the use of B-mode ultrasound, this study examines the relationships of serum IL-18 levels with carotid intima-media thickness (IMT) as a reflector for systemic atherosclerosis.
Methods and Results— The study comprised 366 patients without histories of cardiovascular accidents. Severity of carotid atherosclerosis was evaluated by the mean max IMT, ie, mean of the maximal wall thickness at 12 carotid segments. Serum IL-18, IL-6, and high-sensitive C-reactive protein (hs-CRP) levels were determined in all patients. Log-transformed IL-18 concentrations were positively correlated with IMT (r=0.36, P<0.001), and the association remained significant (?=0.20, P<0.001) when controlling for traditional atherosclerotic risk factors, IL-6 and hs-CRP levels. Also, IMT was greater in the highest and the middle tertile of IL-18 levels than in the lowest tertile.
Conclusion— Higher serum IL-18 levels appear to be associated with greater carotid IMT, suggesting the link between IL-18 and atherosclerosis.
With the use of B-mode ultrasound, this study examined the relationships of serum IL-18 levels with carotid intima-media thickness (IMT). We have found that higher IL-18 levels are associated with greater IMT, suggesting the link between IL-18 and atherosclerosis.
Key Words: atherosclerosis ? carotid arteries ? cytokines ? inflammation ? ultrasonic diagnosis
Introduction
With the recognition that inflammation plays a significant role in atherosclerosis and its complications, studies have shown that circulating inflammatory markers are predictive of cardiovascular diseases (CVD).1 Particularly, elevated circulating levels of C-reactive protein (CRP) and IL-6 (IL-6) have been associated with the risk for CVD.2–4 Additionally, circulating levels of IL-18 are found to be a strong predictor of CVD deaths in patients with coronary artery disease,5 as well as an independent predictor for coronary events in healthy men.6
IL-18 was originally identified as an interferon-–inducing factor,7 which may play a central role in the inflammatory cascades.8 To date, experimental studies have shown that expression of IL-18 is related with atherosclerotic plaque progression and its instability.9,10 However, clinical studies relating IL-18 to atherosclerotic severity are limited.11 Moreover, we are unaware of studies that examined the relationships with IL-6 and high-sensitive CRP (hs-CRP) taken into account.
Carotid intima-media thickness (IMT), as assessed by B-mode ultrasound, is a commonly used clinical marker that reflects systemic burden of atherosclerosis.12 Moreover, increased IMT has been a predictor of future coronary events and stroke.13–15 On the basis of such findings, to clarify the relationships between such inflammatory markers and IMT would be of value to extend the current knowledge regarding the link between inflammation and atherosclerotic diseases.
With the use of B-mode ultrasound, this study examines the relationships of serum IL-18 levels with carotid IMT as a reflector for systemic atherosclerosis.
Methods
Subjects
The subjects for this investigation were enrolled from patients of the Department of Internal Medicine and Therapeutics at Osaka University Hospital, who had undergone carotid ultrasound examination between October 2000 and November 2004.
In the current study, patients with the histories of ischemic heart disease, stroke or arteriosclerosis obliterans were excluded, because circulating IL-18 levels can be substantially modified in such patients.16–18 Also, patients with acute inflammatory diseases, collagen diseases, or malignant neoplasm were excluded, because levels of inflammation can be greatly enhanced by such diseases. Additionally, patients with occluded carotid arteries and those with the history of carotid endarterectomy were excluded, because IMT could not be correctly determined in such patients.
Consequently, this study comprised 366 patients (mean±SD, age 64.8±9.1 years) equally including men and women. Patients’ characteristics are shown in Table 1, demonstrating higher prevalence of atherosclerotic risk factors in the study sample. Institutional ethical committee approved this study, and written informed consent was obtained from all patients. Also, the investigation conforms to the principles outlined in the Declaration of Helsinki.
TABLE 1. Patient Characteristics (n=366)
Carotid Ultrasonography
All ultrasound examinations were performed with the use of Phillips SONOS 5500 equipped with a 3- to 11-MHz linear-array transducer. Three different longitudinal (anterior oblique, lateral, and posterior oblique) and transverse images of the bilateral carotid arteries were obtained, and IMT was measured as the distance between the luminal–intimal interface and the medial–adventitial interface. It was measured with the use of an electronic caliper on the frozen frame of a suitable longitudinal B-mode image in which putative maximal IMT was visualized. Thereby, severity of carotid atherosclerosis was evaluated by the mean max-IMT, which is the mean of maximal wall thickness at 12 carotid segments (near and far wall of the left and right common carotid artery, carotid bifurcation, and internal carotid artery).
All examinations were performed by one sonographer (H.Y.) who was blinded from the patients’ clinical details. Before this study, reproducibility of the mean max-IMT was examined for randomly selected 70 patients without carotid occlusion, in which IMT measurements were performed twice by the same examiner. Intraclass correlation coefficient for the mean max-IMT measurements was 0.96, with a similar average between the two measurements.
Measurement of Serum Inflammatory Markers
After carotid ultrasound examinations, blood was drawn with minimally traumatic venipuncture for the measurement of serum inflammatory markers. Thereafter, the blood was centrifuged at 3000 rpm at 4°C for 15 minutes, and aliquots were stored at –70°C. Serum concentration of IL-18 was measured by single determination with enzyme-linked immunosorbent assay method (MBL Co, Ltd. Nagoya, Japan). In this assay system, mean interclass coefficient of variation (CV) of IL-18 measurements was 5.9%. Also, in 52 randomly selected patients, within-person correlation coefficient by 1-year interval was 0.84 (P<0.001).
Additionally, level of IL-6 was measured by enzyme-linked immunosorbent assay method (R & D system, Minneapolis, Minn), and hs-CRP was measured by latex turbidimetric immunoassay (Shionogi Biomedical Laboratory Inc, Osaka, Japan).
Evaluation of Atherosclerotic Risk Factors
Levels of fasting blood glucose, serum total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TG) were determined from the blood sample taken for inflammatory marker evaluations. Information on the patients’ medical histories and medication usages was obtained from the clinical records. Hypertension was defined by casual blood pressure 140/90 mm Hg or the current use of antihypertensive agents. Diabetes mellitus was defined by fasting blood glucose 7.0 mmol/L or by the use of glucose-lowering agents. Dyslipidemia was defined by fasting serum total cholesterol >5.7 mmol/L, TG >1.7 mmol/L, HDL cholesterol <1.04 mmol/L, or by the use of cholesterol-lowering agents. Smoking status was categorically evaluated based on self-reports, with a smoker defined by the history of smoking 10 cigarettes per day >1 year.
Statistical Analyses
All analyses were performed with SPSS 11.5J (SPSS Japan Inc). Because distributions of inflammatory markers levels appeared to be left-skewed, they were normalized by log-transformation. Thereafter, associations between IL-18 levels and atherosclerotic risk factors were examined by Pearson correlation analysis for continuous variables, and by unpaired t test for categorical variables. Also, relationships between inflammatory marker levels and mean max-IMT were examined by Pearson correlation analysis. Subsequently, multiple linear regression analyses were used to examine associations between IL-18 levels and mean max-IMT: (1) by controlling for age and sex; (2) by additionally controlling for traditional atherosclerotic risk factors (body mass index, hypertension, diabetes, smoking status, total cholesterol, TGs, and HDL cholesterol); and (3) by further controlling for IL-6 and hs-CRP levels. Finally, mean max-IMT was compared across the IL-18 tertiles by the general linear model, followed by Bonferroni post-hoc test. Probability values were 2-tailed and were considered significant when <0.05.
Results
Associations between IL-18 levels and atherosclerotic risk factors are shown in Table 2. Levels of IL-18 were higher in men than in women, in patients with hypertension than in those without, and in patients with smoking history than in those without. Also, IL-18 was positively correlated with age, body mass index, and TGs, and negatively with HDL cholesterol. Additionally, IL-18 levels showed modest correlations with IL-6 and hs-CRP (r=0.23 and 0.29; both P<0.001). Of note, IL-18 levels were similar between patients on aspirin, statins, angiotensin-converting enzyme inhibitors, or angiotensin II type 1 receptor blocker, and those not using them (data not shown).
TABLE 2. Associations of Serum IL-18 Levels With Atherosclerotic Risk Factors (n=366)
To clarify the link between IL-18 and severity of atherosclerosis, associations of serum IL-18 levels with the mean max-IMT were examined. By univariate analysis, log-transformed concentration of IL-18 was positively correlated with IMT (r=0.36, P<0.001). By multiple regression analyses (Table 3), the association between IL-18 and IMT remained significant when controlling for age and sex (model 1), and additionally controlling for traditional atherosclerotic risk factors (model 2). Moreover, the association was little attenuated when further controlling for IL-6 and hs-CRP (model 3). Of note, although both IL-6 and hs-CRP had significant correlations with carotid IMT (r=0.25 and r=0.23, both P<0.001), neither of such associations was significant when IL-18 and traditional atherosclerotic risk factors were simultaneously included in the model (model 3).
TABLE 3. Multivariate Analyses of Mean Max-IMT
Additionally, given different IL-18 levels between men and women, we have performed separate analyses. Similar to the results obtained for all 366 patients, IL-18 levels were correlated with IMT both in men (r=0.36, P<0.001) and in women (r=0.31, P<0.001), and the associations remained significant when controlling for age, body mass index, and traditional atherosclerotic risk factors (in men, ?=0.18, P=0.01; and in women, ?=0.25, P<0.001).
Given the association between IL-18 and carotid IMT, the mean max-IMT was compared across the tertiles of IL-18 levels (Figure). IMT was greater in the highest and the middle tertile of IL-18 than in the lowest tertile (Table 4). Moreover, the differences persisted when adjusting traditional atherosclerotic risk factors, log-transformed IL-6 and hs-CRP, and medication usages.
Mean max-IMT according to tertiles of IL-18. Error bars are 95% CI. *P<0.001
TABLE 4. Mean Max-IMT Stratified by IL-18 Tertiles
Discussion
In the present study, we have found that elevated serum IL-18 levels are associated with increased carotid IMT as evaluated by B-mode ultrasound. Also, the association was independent of traditional atherosclerotic risk factors, IL-6, and hs-CRP levels. To our knowledge, this is the first study that demonstrates the associations between IL-18 levels and carotid atherosclerosis, with IL-6 and hs-CRP taken into account.
In the current study, IL-18 levels were higher in hypertensive patients and in smokers than in those who were not, and had significant correlations with traditional atherosclerotic risk factors (Table 2). These findings are approximately in line with those of Ferrucci et al,19 who showed associations of higher IL-18 levels with such risk factors. Also, in accordance with previous studies,5,6,20 IL-18 levels had modest correlations with other inflammatory markers. Nevertheless, studies that examined the associations of IL-18 levels with atherosclerotic risk factors and other inflammatory markers are limited, requiring further studies to clarify their linkages.
Although elevated IL-18 levels can predict the development of CVD,5,6 their association with carotid IMT remains to be examined. In the present study, we have found that higher IL-18 levels are associated with greater IMT, suggesting their link with carotid atherosclerosis. However, because of the impact of atherosclerotic risk factors,21–23 the association between IL-18 and IMT may need to be examined with such factors taken into accounts. When controlling for age and sex, IL-18 was significantly associated with IMT (Table 3, model 1), and the association was independent of traditional atherosclerotic risk factors (Table 3, model 2). Furthermore, similar results were obtained when separate analyses were performed for men and women. These findings support the association between IL-18 and carotid atherosclerosis. Additionally, such association was little modified when levels of IL-6 and hs-CRP were further controlled for (Table 3, model 3), suggesting that the association is independent of such inflammatory markers. Of note, despite the associations of IL-6 or hs-CRP with IMT,24–26 neither had significant association with IMT in the multiple regression model, which could be because of the lack of our statistical power.
To further demonstrate the associations between carotid atherosclerosis and IL-18, mean max-IMT was compared across the tertiles of IL-18 levels. IMT was greater in patients belonging to the highest and the middle tertiles than in those belonging to the lowest tertile (Figure, Table 4), and the differences persisted when adjusting traditional atherosclerotic risk factors. The greater IMT in patients with higher IL-18 appears to be congruent with Aso et al11 Moreover, although inflammatory markers levels can be modified by aspirin, statins, angiotensin-converting enzyme inhibitors, or angiotensin II type 1 receptor blocker,27–30 the differences between IL-18 and IMT were not virtually modified when such medication usages were considered (Table 4), further supporting the link between IL-18 and carotid IMT.
IL-18 is highly expressed in human carotid atherosclerotic plaques, predominantly colocalized with macrophages.9 Thus, increased IL-18 production from severe atherosclerotic lesions could contribute to the higher IL-18 found in this study. Also, experimental studies have shown that IL-18 enhances atherosclerosis through release of interferon-10 and induces expression of IL-6 in vascular endothelial and smooth muscle cells.31 Inversely, IL-18 deficiency reduces the extent of atherosclerosis in apolipoprotein E–knockout mice.32 These findings are in accordance with the hypothesis that IL-18 plays a key role in atherogenesis, supporting the link between IL-18 and carotid atherosclerosis.
There are some limitations for the current study. First, because this study is cross-sectionally designed, we cannot determine the causal relationships between higher IL-18 levels and greater IMT. Second, we used single blood sampling for the measurements of IL-18 levels, which does not guarantee the average levels in our patients. However, our IL-18 measurements were relatively stable over 1 year, supporting the link between IL-18 and chronic atherosclerosis. Third, this study included substantial number of patients on medications, requiring larger studies to separate the effects of such medications.
In conclusion, we have demonstrated an association between higher serum IL-18 level and greater carotid IMT, suggesting the link between IL-18 and atherosclerosis. This finding can offer a clue to understand the role of IL-18 in the development of atherosclerotic diseases.
Acknowledgments
The present study was supported in part by the Smoking Research Foundation of Japan and a research grant for cardiovascular diseases from the Japanese Ministry of Health, Labor, and Welfare. We appreciate A. Kanzawa and S. Higa for their secretarial assistance.
References
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Bots ML, Hoes AW, Koudstaal PJ, Hofman A, Grobbee DE. Common carotid intima-media thickness and risk of stroke and myocardial infarction: the Rotterdam Study. Circulation. 1997; 96: 1432–1437.
O’Leary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Wolfson SK Jr. Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group . N Engl J Med. 1999; 340: 14–22.
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Gerdes N, Sukhova GK, Libby P, Reynolds RS, Young JL, Schonbeck U. Expression of interleukin (IL)-18 and functional IL-18 receptor on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for atherogenesis. J Exp Med. 2002; 195: 245–257.
Elhage R, Jawien J, Rudling M, Ljunggren HG, Takeda K, Akira S, Bayard F, Hansson GK. Reduced atherosclerosis in interleukin-18 deficient apolipoprotein E-knockout mice. Cardiovasc Res. 2003; 59: 234–240.(Hiroshi Yamagami; Kazuo K)
Correspondence to Hiroshi Yamagami, MD, PhD, Department of Internal Medicine and Therapeutics (A8), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail yamagami@medone.med.osaka-u.ac.jp
Abstract
Objective— Elevated circulating levels of IL-18 can predict future coronary heart disease. Although IL-18 is thought to play a crucial role in atherosclerosis, whether circulating IL-18 levels are associated with the severity of atherosclerosis remains to be determined. With the use of B-mode ultrasound, this study examines the relationships of serum IL-18 levels with carotid intima-media thickness (IMT) as a reflector for systemic atherosclerosis.
Methods and Results— The study comprised 366 patients without histories of cardiovascular accidents. Severity of carotid atherosclerosis was evaluated by the mean max IMT, ie, mean of the maximal wall thickness at 12 carotid segments. Serum IL-18, IL-6, and high-sensitive C-reactive protein (hs-CRP) levels were determined in all patients. Log-transformed IL-18 concentrations were positively correlated with IMT (r=0.36, P<0.001), and the association remained significant (?=0.20, P<0.001) when controlling for traditional atherosclerotic risk factors, IL-6 and hs-CRP levels. Also, IMT was greater in the highest and the middle tertile of IL-18 levels than in the lowest tertile.
Conclusion— Higher serum IL-18 levels appear to be associated with greater carotid IMT, suggesting the link between IL-18 and atherosclerosis.
With the use of B-mode ultrasound, this study examined the relationships of serum IL-18 levels with carotid intima-media thickness (IMT). We have found that higher IL-18 levels are associated with greater IMT, suggesting the link between IL-18 and atherosclerosis.
Key Words: atherosclerosis ? carotid arteries ? cytokines ? inflammation ? ultrasonic diagnosis
Introduction
With the recognition that inflammation plays a significant role in atherosclerosis and its complications, studies have shown that circulating inflammatory markers are predictive of cardiovascular diseases (CVD).1 Particularly, elevated circulating levels of C-reactive protein (CRP) and IL-6 (IL-6) have been associated with the risk for CVD.2–4 Additionally, circulating levels of IL-18 are found to be a strong predictor of CVD deaths in patients with coronary artery disease,5 as well as an independent predictor for coronary events in healthy men.6
IL-18 was originally identified as an interferon-–inducing factor,7 which may play a central role in the inflammatory cascades.8 To date, experimental studies have shown that expression of IL-18 is related with atherosclerotic plaque progression and its instability.9,10 However, clinical studies relating IL-18 to atherosclerotic severity are limited.11 Moreover, we are unaware of studies that examined the relationships with IL-6 and high-sensitive CRP (hs-CRP) taken into account.
Carotid intima-media thickness (IMT), as assessed by B-mode ultrasound, is a commonly used clinical marker that reflects systemic burden of atherosclerosis.12 Moreover, increased IMT has been a predictor of future coronary events and stroke.13–15 On the basis of such findings, to clarify the relationships between such inflammatory markers and IMT would be of value to extend the current knowledge regarding the link between inflammation and atherosclerotic diseases.
With the use of B-mode ultrasound, this study examines the relationships of serum IL-18 levels with carotid IMT as a reflector for systemic atherosclerosis.
Methods
Subjects
The subjects for this investigation were enrolled from patients of the Department of Internal Medicine and Therapeutics at Osaka University Hospital, who had undergone carotid ultrasound examination between October 2000 and November 2004.
In the current study, patients with the histories of ischemic heart disease, stroke or arteriosclerosis obliterans were excluded, because circulating IL-18 levels can be substantially modified in such patients.16–18 Also, patients with acute inflammatory diseases, collagen diseases, or malignant neoplasm were excluded, because levels of inflammation can be greatly enhanced by such diseases. Additionally, patients with occluded carotid arteries and those with the history of carotid endarterectomy were excluded, because IMT could not be correctly determined in such patients.
Consequently, this study comprised 366 patients (mean±SD, age 64.8±9.1 years) equally including men and women. Patients’ characteristics are shown in Table 1, demonstrating higher prevalence of atherosclerotic risk factors in the study sample. Institutional ethical committee approved this study, and written informed consent was obtained from all patients. Also, the investigation conforms to the principles outlined in the Declaration of Helsinki.
TABLE 1. Patient Characteristics (n=366)
Carotid Ultrasonography
All ultrasound examinations were performed with the use of Phillips SONOS 5500 equipped with a 3- to 11-MHz linear-array transducer. Three different longitudinal (anterior oblique, lateral, and posterior oblique) and transverse images of the bilateral carotid arteries were obtained, and IMT was measured as the distance between the luminal–intimal interface and the medial–adventitial interface. It was measured with the use of an electronic caliper on the frozen frame of a suitable longitudinal B-mode image in which putative maximal IMT was visualized. Thereby, severity of carotid atherosclerosis was evaluated by the mean max-IMT, which is the mean of maximal wall thickness at 12 carotid segments (near and far wall of the left and right common carotid artery, carotid bifurcation, and internal carotid artery).
All examinations were performed by one sonographer (H.Y.) who was blinded from the patients’ clinical details. Before this study, reproducibility of the mean max-IMT was examined for randomly selected 70 patients without carotid occlusion, in which IMT measurements were performed twice by the same examiner. Intraclass correlation coefficient for the mean max-IMT measurements was 0.96, with a similar average between the two measurements.
Measurement of Serum Inflammatory Markers
After carotid ultrasound examinations, blood was drawn with minimally traumatic venipuncture for the measurement of serum inflammatory markers. Thereafter, the blood was centrifuged at 3000 rpm at 4°C for 15 minutes, and aliquots were stored at –70°C. Serum concentration of IL-18 was measured by single determination with enzyme-linked immunosorbent assay method (MBL Co, Ltd. Nagoya, Japan). In this assay system, mean interclass coefficient of variation (CV) of IL-18 measurements was 5.9%. Also, in 52 randomly selected patients, within-person correlation coefficient by 1-year interval was 0.84 (P<0.001).
Additionally, level of IL-6 was measured by enzyme-linked immunosorbent assay method (R & D system, Minneapolis, Minn), and hs-CRP was measured by latex turbidimetric immunoassay (Shionogi Biomedical Laboratory Inc, Osaka, Japan).
Evaluation of Atherosclerotic Risk Factors
Levels of fasting blood glucose, serum total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TG) were determined from the blood sample taken for inflammatory marker evaluations. Information on the patients’ medical histories and medication usages was obtained from the clinical records. Hypertension was defined by casual blood pressure 140/90 mm Hg or the current use of antihypertensive agents. Diabetes mellitus was defined by fasting blood glucose 7.0 mmol/L or by the use of glucose-lowering agents. Dyslipidemia was defined by fasting serum total cholesterol >5.7 mmol/L, TG >1.7 mmol/L, HDL cholesterol <1.04 mmol/L, or by the use of cholesterol-lowering agents. Smoking status was categorically evaluated based on self-reports, with a smoker defined by the history of smoking 10 cigarettes per day >1 year.
Statistical Analyses
All analyses were performed with SPSS 11.5J (SPSS Japan Inc). Because distributions of inflammatory markers levels appeared to be left-skewed, they were normalized by log-transformation. Thereafter, associations between IL-18 levels and atherosclerotic risk factors were examined by Pearson correlation analysis for continuous variables, and by unpaired t test for categorical variables. Also, relationships between inflammatory marker levels and mean max-IMT were examined by Pearson correlation analysis. Subsequently, multiple linear regression analyses were used to examine associations between IL-18 levels and mean max-IMT: (1) by controlling for age and sex; (2) by additionally controlling for traditional atherosclerotic risk factors (body mass index, hypertension, diabetes, smoking status, total cholesterol, TGs, and HDL cholesterol); and (3) by further controlling for IL-6 and hs-CRP levels. Finally, mean max-IMT was compared across the IL-18 tertiles by the general linear model, followed by Bonferroni post-hoc test. Probability values were 2-tailed and were considered significant when <0.05.
Results
Associations between IL-18 levels and atherosclerotic risk factors are shown in Table 2. Levels of IL-18 were higher in men than in women, in patients with hypertension than in those without, and in patients with smoking history than in those without. Also, IL-18 was positively correlated with age, body mass index, and TGs, and negatively with HDL cholesterol. Additionally, IL-18 levels showed modest correlations with IL-6 and hs-CRP (r=0.23 and 0.29; both P<0.001). Of note, IL-18 levels were similar between patients on aspirin, statins, angiotensin-converting enzyme inhibitors, or angiotensin II type 1 receptor blocker, and those not using them (data not shown).
TABLE 2. Associations of Serum IL-18 Levels With Atherosclerotic Risk Factors (n=366)
To clarify the link between IL-18 and severity of atherosclerosis, associations of serum IL-18 levels with the mean max-IMT were examined. By univariate analysis, log-transformed concentration of IL-18 was positively correlated with IMT (r=0.36, P<0.001). By multiple regression analyses (Table 3), the association between IL-18 and IMT remained significant when controlling for age and sex (model 1), and additionally controlling for traditional atherosclerotic risk factors (model 2). Moreover, the association was little attenuated when further controlling for IL-6 and hs-CRP (model 3). Of note, although both IL-6 and hs-CRP had significant correlations with carotid IMT (r=0.25 and r=0.23, both P<0.001), neither of such associations was significant when IL-18 and traditional atherosclerotic risk factors were simultaneously included in the model (model 3).
TABLE 3. Multivariate Analyses of Mean Max-IMT
Additionally, given different IL-18 levels between men and women, we have performed separate analyses. Similar to the results obtained for all 366 patients, IL-18 levels were correlated with IMT both in men (r=0.36, P<0.001) and in women (r=0.31, P<0.001), and the associations remained significant when controlling for age, body mass index, and traditional atherosclerotic risk factors (in men, ?=0.18, P=0.01; and in women, ?=0.25, P<0.001).
Given the association between IL-18 and carotid IMT, the mean max-IMT was compared across the tertiles of IL-18 levels (Figure). IMT was greater in the highest and the middle tertile of IL-18 than in the lowest tertile (Table 4). Moreover, the differences persisted when adjusting traditional atherosclerotic risk factors, log-transformed IL-6 and hs-CRP, and medication usages.
Mean max-IMT according to tertiles of IL-18. Error bars are 95% CI. *P<0.001
TABLE 4. Mean Max-IMT Stratified by IL-18 Tertiles
Discussion
In the present study, we have found that elevated serum IL-18 levels are associated with increased carotid IMT as evaluated by B-mode ultrasound. Also, the association was independent of traditional atherosclerotic risk factors, IL-6, and hs-CRP levels. To our knowledge, this is the first study that demonstrates the associations between IL-18 levels and carotid atherosclerosis, with IL-6 and hs-CRP taken into account.
In the current study, IL-18 levels were higher in hypertensive patients and in smokers than in those who were not, and had significant correlations with traditional atherosclerotic risk factors (Table 2). These findings are approximately in line with those of Ferrucci et al,19 who showed associations of higher IL-18 levels with such risk factors. Also, in accordance with previous studies,5,6,20 IL-18 levels had modest correlations with other inflammatory markers. Nevertheless, studies that examined the associations of IL-18 levels with atherosclerotic risk factors and other inflammatory markers are limited, requiring further studies to clarify their linkages.
Although elevated IL-18 levels can predict the development of CVD,5,6 their association with carotid IMT remains to be examined. In the present study, we have found that higher IL-18 levels are associated with greater IMT, suggesting their link with carotid atherosclerosis. However, because of the impact of atherosclerotic risk factors,21–23 the association between IL-18 and IMT may need to be examined with such factors taken into accounts. When controlling for age and sex, IL-18 was significantly associated with IMT (Table 3, model 1), and the association was independent of traditional atherosclerotic risk factors (Table 3, model 2). Furthermore, similar results were obtained when separate analyses were performed for men and women. These findings support the association between IL-18 and carotid atherosclerosis. Additionally, such association was little modified when levels of IL-6 and hs-CRP were further controlled for (Table 3, model 3), suggesting that the association is independent of such inflammatory markers. Of note, despite the associations of IL-6 or hs-CRP with IMT,24–26 neither had significant association with IMT in the multiple regression model, which could be because of the lack of our statistical power.
To further demonstrate the associations between carotid atherosclerosis and IL-18, mean max-IMT was compared across the tertiles of IL-18 levels. IMT was greater in patients belonging to the highest and the middle tertiles than in those belonging to the lowest tertile (Figure, Table 4), and the differences persisted when adjusting traditional atherosclerotic risk factors. The greater IMT in patients with higher IL-18 appears to be congruent with Aso et al11 Moreover, although inflammatory markers levels can be modified by aspirin, statins, angiotensin-converting enzyme inhibitors, or angiotensin II type 1 receptor blocker,27–30 the differences between IL-18 and IMT were not virtually modified when such medication usages were considered (Table 4), further supporting the link between IL-18 and carotid IMT.
IL-18 is highly expressed in human carotid atherosclerotic plaques, predominantly colocalized with macrophages.9 Thus, increased IL-18 production from severe atherosclerotic lesions could contribute to the higher IL-18 found in this study. Also, experimental studies have shown that IL-18 enhances atherosclerosis through release of interferon-10 and induces expression of IL-6 in vascular endothelial and smooth muscle cells.31 Inversely, IL-18 deficiency reduces the extent of atherosclerosis in apolipoprotein E–knockout mice.32 These findings are in accordance with the hypothesis that IL-18 plays a key role in atherogenesis, supporting the link between IL-18 and carotid atherosclerosis.
There are some limitations for the current study. First, because this study is cross-sectionally designed, we cannot determine the causal relationships between higher IL-18 levels and greater IMT. Second, we used single blood sampling for the measurements of IL-18 levels, which does not guarantee the average levels in our patients. However, our IL-18 measurements were relatively stable over 1 year, supporting the link between IL-18 and chronic atherosclerosis. Third, this study included substantial number of patients on medications, requiring larger studies to separate the effects of such medications.
In conclusion, we have demonstrated an association between higher serum IL-18 level and greater carotid IMT, suggesting the link between IL-18 and atherosclerosis. This finding can offer a clue to understand the role of IL-18 in the development of atherosclerotic diseases.
Acknowledgments
The present study was supported in part by the Smoking Research Foundation of Japan and a research grant for cardiovascular diseases from the Japanese Ministry of Health, Labor, and Welfare. We appreciate A. Kanzawa and S. Higa for their secretarial assistance.
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