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Serum Interleukin-15 Concentration in Patients With Essential Hypertension
AJH
Epidemiology
2005; 18:1019 –1025
ORIGINAL CONTRIBUTIONS
Serum Interleukin-15 Concentration in Patients With Essential Hypertension Masaharu Kaibe, Mitsuru Ohishi, Norihisa Ito, Ming Yuan, Takashi Takagi, Minako Terai, Yuji Tatara, Norio Komai, Hiromi Rakugi, and Toshio Ogihara Background: Interleukin (IL)-15 is one of the cytokines produced by neutrophils and monocytes/macrophages, and its expression is found immunohistochemically in inflammatory cells adjacent to vulnerable atherosclerotic plaques. However, the influence of systemic IL-15 on cardiovascular disease is still unclear. Therefore, we designed clinical investigations to clarify the relationship between cardiovascular complications and serum IL-15 levels. Methods and Results: Three hundred ninety-nine patients with essential hypertension were analyzed. We divided the study subjects into the following three groups according to the modified World Health Organization–International Society of Hypertension classification of 1999: patients with no organ damage (n ⫽ 213), patients with mild organ damage (n ⫽ 128), and patients with severe organ damage (n ⫽ 58). We measured serum IL-15, highly sensitive C reactive protein, IL-6, soluble intercellular adhesion molecule, and soluble vascular cell adhesion molecule levels. Serum IL-15 concentration in patients with severe organ
R
ecently local inflammatory response has been suggested to be one of the major mechanisms of the development of atherosclerosis, which is the principal change in arteries in cardiovascular disease. Many cytokines, chemokines, and cell adhesion molecules are involved in this phenomenon, and inflammatory cells, such as white blood cells, lymphocytes, and monocytes/macrophages, also play an important role in the development of atherosclerosis. Landmark articles first illustrated the potential association between C-reactive protein (CRP) level and cardiovascular outcome in patients with unstable coronary syndromes1 and numerous studies have found that the baseline level of highly sensitive CRP (hs-CRP) is associated with the risk of future
See related article on page 1017
Received December 17, 2004. First decision February 18, 2005. Accepted February 23, 2005. From the Department of Geriatric Medicine, Osaka University Graduate School of Medicine, Suita, Japan. This study was partially funded by a Japan Heart Foundation/ Pfizer Grant for Research on Hypertension and Vascular Metabolism and by the Research Foundation for Community Medicine, Research © 2005 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
damage was significantly higher than that in those with no organ damage (P ⬍ .01) and those with mild organ damage (P ⬍ .01). Serum IL-15 concentration in patients with coronary artery disease or peripheral artery disease was significantly higher than that in those without coronary artery disease or peripheral artery disease. Moreover, serum IL-15 concentration in patients with lacunar infarction was significantly higher than that in those without lacunar infarction (P ⬍ .005). By multiple linear logistic regression analysis, serum IL-15 concentration was independently correlated with cardiovascular disease. Conclusions: These data suggest that a systemic inflammatory response involving IL-15 might be involved in the occurrence of cardiovascular disease in patients with essential hypertension. Am J Hypertens 2005;18:1019 –1025 © 2005 American Journal of Hypertension, Ltd. Key Words: IL-15, cardiovascular disease, hs-CRP, IL-6.
myocardial infarction, stroke, peripheral vascular disease, and cardiovascular death.2 Several inflammatory biomarkers have been reported to be risk factors and predictors of cardiovascular disease. In the Physician’s Health Study, soluble intercellular adhesion molecule-1 (sICAM-1) was associated with blood pressure (BP)3 and was shown to be a predictor of myocardial infarction,4 and an increased baseline level of the proinflammatory cytokine interleukin-6 (IL-6) also predicted future cardiovascular events.5 In a study from the secondary prevention Cholesterol and Recurrent Events (CARE) trial, elevation of tumor necrosis factor-␣ in patients in the stable phase after myocardial infarction was associated with an increased risk of recurrent coronary events.6 The cytokine interleukin-15 (IL-15) is one of these
Meeting on Hypertension and Arteriosclerosis. MK was also supported by a Research Fellowship of the Japan Society for the Promotion of Science. Address correspondence and reprint requests to Dr. Hiromi Rakugi, Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita 565-0871, Japan; e-mail: rakugi@ geriat.med.osaka-u.ac.jp 0895-7061/05/$30.00 doi:10.1016/j.amjhyper.2005.02.014
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proinflammatory cytokines, and has been detected in different inflammatory conditions.7 Interleukin-15 is a 14-15kDa cytokine expressed at the mRNA level in numerous normal human tissues in a broad range of cell types, including activated monocytes, dendritic cells, osteoclasts, and fibroblasts.8 Pathologic study has demonstrated IL-15 expression in human atherosclerotic plaque. Expression of IL-15 mRNA and protein was found almost solely in fibrolipid and lipid-rich plaques, associated with oxidized LDLpositive macrophages.9 In gene expression arrays, a transcript corresponding to IL-15 mRNA was elevated in the atherosclerotic aorta of ApoE-deficient mice fed a Western diet for 10 or 20 weeks, corresponding to lesions of the fatty streak and fibrofatty plaque stage, respectively. Immunostaining for IL-15 was localized to aortic smooth muscle cells in nonatherosclerotic C57BL/6 mice, whereas both macrophages and smooth muscle cells in atherosclerotic lesions of ApoE-deficient mice stained positive for IL-15.10 These data show that IL-15 expression is upregulated in atherosclerosis and suggest that this cytokine may be important in its pathogenesis. Inflammatory cytokines, such as CRP, IL-6, and cell adhesion molecule, play local roles to be involved in atherogenic response adjacent to vulnerable plaque. Interleukin-15 could be also associated with cardiovascular disease; however, the relationship between systemic IL-15 concentration and cardiovascular events is still unclear. Therefore, we designed a clinical investigation to clarify the relationship between serum IL-15 concentration and cardiovascular complications in patients with essential hypertension.
Methods Subjects Four hundred seventy-eight patients with essential hypertension who attended Osaka University Hospital were enrolled in this study. Hypertension was defined as systolic BP of more than 140 mm Hg, diastolic BP of more than 90 mm Hg, or administration of antihypertensive drugs. We excluded patients with acute coronary syndrome and stroke within 3 months before participation. We excluded patients with acute or chronic infectious or inflammatory disease (n ⫽ 14) or malignant neoplasm (n ⫽ 8), which influence serum IL-15 concentration. We also excluded patients who were administered hormone replacement therapy (n ⫽ 0), steroid (n ⫽ 5) or aspirin (n ⫽ 47), to exclude their influence on serum IL-15 concentration. Five patients refused to undergo venipuncture. Finally, 399 patients with essential hypertension were analyzed in this study. The protocol of this study was approved by our hospital ethics committee, and written informed consent was obtained from all subjects before participation in this cross-sectional observational study. One hundred fortyeight patients were not administered any antihypertensive drugs, whereas 151 patients were treated with a calcium antagonist, 80 with an angiotensin-converting enzyme inhibitor, 122 with an angiotensin type I receptor antagonist, 43 with a -blocker, 16 with an ␣-blocker, and 35 with a diuretic.
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Cardiovascular Complications We evaluated organ damage due to hypertension according to the World Health Organization (WHO)–International Society of Hypertension (ISH) classification modified in 1999.11 We evaluated mild organ damage corresponding to target organ damage in WHO–ISH 1999, such as left ventricular hypertrophy (LVH), retinal artery narrowing, proteinuria (⬎20 mg/L), or increased plasma creatinine concentration (1.2 to 2.0 mg/dL) and carotid plaque. LVH was determined by electrocardiography using the Sokolow and Lyon criteria or by echocardiography—a septal or posterior wall thickness of more than 12 mm. Carotid plaque was determined by carotid ultrasonography as a score of five or more as previously reported.12 One special operator examined echocardiography and carotid ultrasonography (Power Vision 6000, Toshiba, Tokyo, Japan) and took the mean of three examinations as representative data for this study. The intraobserver coefficient of variation was 2.8% ⫾ 1.2%. We also evaluated severe organ damage corresponding to associated clinical conditions in WHO–ISH 1999, such as cerebrovascular disease by brain magnetic resource imaging (MRI) or computed tomographic scan and neurologic findings, coronary artery disease (CAD) by electrocardiography, echocardiography, or typical symptoms, congestive heart failure, or peripheral artery disease (PAD) without ulceration or gangrene. Moreover, we evaluated lacunar infarction by brain MRI. Serum Concentrations of Inflammatory Substances A venous blood sample was obtained from each patient in the morning after an overnight fast and stopping administration of drugs for at least 24 h. Serum was stored at ⫺80°C. Stored serum was thawed and assayed for serum IL-15, hs-CRP, IL-6, sICAM-1 and soluble vascular cell adhesion molecule (sVCAM-1) by commercially available ELISAs (serum IL-15 and IL-6: BIOSOURCE International, Camarillo, CA; hs-CRP: ALPHA DIAGNOSTIC International, San Antonio, TX; sICAM-1 and sVCAM-1: DIACLONE Research, Besançon Cedex, France). Serum levels of IL-15, hs-CRP, IL-6, sICAM-1, and sVCAM-1 were measured in duplicate for each patient. The intraobserver coefficient of variation for these inflammatory substances was 3.1% ⫾ 1.3%, 2.2% ⫾ 0.9%, 2.7% ⫾ 1.2%, 2.9% ⫾ 1.0%, and 2.8% ⫾ 1.2%, respectively. Statistical Analysis Data were analyzed with JMP ver. 4 (SAS Inc., Cary, NC) and presented as mean ⫾ SEM. We used Mann-Whitney U test to analyze nonparametrical data, such as hs-CRP, IL-6, sICAM-1, sVCAM-1, and IL-15 for significant differences among each hypertensive complication group. ANOVA and Student t test were also used to analyze other clinical data for significant differences among cardiovascular complications. We also used multiple linear logistic regression analysis to
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Table 1. Patients characteristics
Number Male/female Age (y) BMI (kg/m2) Current smokers (%) Smoking index SBP (mm Hg) DBP (mm Hg) HR (beats/min) TC (mg/dL) HDL-C (mg/dL) FBG (mg/dL) HbA1C (%) hs-CRP (ng/mL) IL-6 (pg/mL) sICAM-1 (ng/mL) sVCAM-1 (ng/mL)
No Organ Damage
Mild Organ Damage
Severe Organ Damage
213 103/110 58.5 ⫾ 0.9 24.5 ⫾ 0.3 19.2 238.3 ⫾ 27.5 148.3 ⫾ 1.4 87.3 ⫾ 1.0 73.8 ⫾ 1.0 210.8 ⫾ 2.6 59.3 ⫾ 1.2 100.5 ⫾ 1.4 6.1 ⫾ 0.8 1.2 ⫾ 0.1 19.1 ⫾ 2.9 837 ⫾ 33.4 2009 ⫾ 175
128 72/56 61.9 ⫾ 1.1* 24.3 ⫾ 0.3 31.1† 337.0 ⫾ 44.6* 153.4 ⫾ 2.0* 88.8 ⫾ 1.1 72.1 ⫾ 1.2 204.2 ⫾ 3.0 55.7 ⫾ 1.9 102.5 ⫾ 1.7 6.0 ⫾ 1.2 1.4 ⫾ 0.1* 37.8 ⫾ 4.5‡ 1009 ⫾ 48.9† 2438 ⫾ 243†
58 47/11* 68.1 ⫾ 1.1‡ 23.3 ⫾ 0.4* 46.6‡§ 672.1 ⫾ 89.3‡§ 146.6 ⫾ 2.9 81.5 ⫾ 1.9†§ 76.4 ⫾ 2.5 193.3 ⫾ 4.5†§ 54.2 ⫾ 2.1* 111.7 ⫾ 4.2†§ 5.8 ⫾ 0.2 2.5 ⫾ 0.5† 42.8 ⫾ 7.9‡ 1043 ⫾ 88.8† 2418 ⫾ 333
BMI ⫽ body mass index; DBP ⫽ diastolic blood pressure; FBG ⫽ fasting blood glucose; HDL-C ⫽ high-density lipoprotein cholesterol; HR ⫽ heart rate; SBP ⫽ systolic blood pressure; TC ⫽ total cholesterol. Analyzed by Student t test, except for inflammatory cytokines (Mann-Whitney U test) and male/female and current smokers (2 test). * P ⬍ .05, † P ⬍ .01, ‡ P ⬍ .001 v no organ damage; § P ⬍ .01 v mild organ damage.
determine the correlation of cardiovascular complications with risk factors, including serum IL-15 concentration. A value of P ⬍ .05 was taken as statistically significant. A power calculation demonstrates that the minimum number of study subjects to determine a relationship between serum IL-15 concentration and hypertensive complications was more than 129, and moreover statistical power of this study was more than 0.9.
Results Patients Characteristics The patients’ characteristics are shown in Table 1. Hypertensive patients were classified into three groups: no organ damage (NOD; n ⫽ 213), mild organ damage (MOD; n ⫽ 128), and severe organ damage (SOD; n ⫽ 58). Patients with SOD showed a higher frequency of the male gender, older age, lower body mass index (BMI), higher frequency of current smoking, lower diastolic BP, lower total cholesterol, lower HDL-cholesterol, and higher fasting blood glucose compared with those with NOD or MOD. Serum Concentrations of Inflammatory Substances and Cardiovascular Complications Serum concentrations of hs-CRP, IL-6, sICAM-1, and sVCAM-1 were measured as known inflammatory-related substances (Table 1 and Fig. 1). Serum hs-CRP concentration in patients with SOD (2.5 ⫾ 0.5 ng/mL; P ⬍ .01) and MOD (1.4 ⫾ 0.1 ng/mL; P ⬍ .05) was significantly higher than that in those with NOD (1.2 ⫾ 0.1 ng/mL). Serum IL-6 concen-
tration in patients with SOD (42.8 ⫾ 7.9 pg/mL; P ⬍ .001) and MOD (37.8 ⫾ 4.5 pg/mL; P ⬍ .001) was also significantly higher than that in those with NOD (19.1 ⫾ 2.9 pg/mL). The sICAM-1 concentration in patients with SOD (1043 ⫾ 88.8 ng/mL; P ⬍ .01) and MOD (1009 ⫾ 48.9 ng/mL; P ⬍ .01) was also significantly higher than that in those with NOD (837 ⫾ 33.4 ng/mL). The sVCAM-1 concentration in patients with NOD (2009 ⫾ 175 ng/mL; P ⬍ .05) was significantly lower than those in patients with MOD (2438 ⫾ 243 ng/mL), but there was no significant difference compared to those with SOD. We also analyzed the relationship between serum concentrations of hs-CRP, IL-6, sICAM-1, and sVCAM-1 and the subtype of cerebrocardiovascular disease with SOD (Table 2). Serum hs-CRP and sICAM-1 concentrations in patients with CAD or PAD was significantly higher than that in those without CAD or PAD (P ⬍ .05), and serum IL-6 concentration in patients with PAD was significantly higher than that in those without PAD (P ⬍ .01); however, there was no significant difference in serum concentration of sVCAM-1 in patients with or without each subtype of SOD. We also analyzed cumulative effect of target organ damage in MOD, but we could not find any cumulative effect on serum levels of hs-CRP, IL-6, IL-15, sICAM-1, or sVCAM-1 (data not shown). The relationship between serum IL-15 concentration and cardiovascular complications is shown in Fig. 2. Serum IL-15 concentration in patients with SOD (88.7 ⫾ 18.7 pg/mL) was significantly higher than that in those with NOD (55.2 ⫾ 6.9 pg/mL; P ⬍ .01) or MOD (51.0 ⫾ 8.3 pg/mL; P ⬍ .01). We also analyzed the relationship between serum IL-15 concentration and the subtype of cardiovascular disease with SOD (Table 2). Serum IL-15 concentration in patients with CAD,
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cerebrovascular disease, and lacunar infarction was significantly higher than that in those without CAD (P ⬍ .05).
(a)
(ng/mL)
** *
hs-CRP
16 14 12 10 8 6 4 2 0
IL-15 Concentration and Other Factors To clarify factors influencing serum IL-15 concentration, we analyzed the relationship between serum IL-15 concentration and other factors (Table 3). There was no relationship with these factors. Mild
No
Severe
organ damage
We performed logistic regression analysis for cardiovascular complications, using sex, age, BMI, current smoking, systolic BP, diastolic BP, total cholesterol, HDLcholesterol, fasting blood glucose, treatment with antihypertensive drugs, and IL-15, which were significantly related to organ damage (Table 4). Serum IL-15 concentration (P ⫽ .0089) was independently related to cardiovascular complications in addition to sex (P ⫽ .0037) and older age (P ⫽ .0017).
(b)
(pg/mL)
*** ***
300
IL-6
250
200 150
100 50
Discussion
0 No
Severe
Mild
organ damage (c)
**
(ng/mL)
**
sICAM-1
3000 2500
2000 1500
1000 500 0 No
Mild
Severe
organ damage (d)
**
(ng/mL)
sVCAM-1
Multiple Linear Logistic Regression Analysis
16000 14000 12000 10000 8000 6000 4000 2000 0 No
Mild
Severe
organ damage FIG. 1. Relationship between serum highly sensitive C-reactive protein (hs-CRP) (A), interleukin-6 (IL-6) (B), soluble intercellular adhesion molecule-1 (sICAM-1) (C), and sVCAM-1(D) concentrations and cardiovascular complications. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001 analyzed by Mann-Whitney U test.
In the present study, serum IL-15 concentration in patients with SOD was significantly increased, and was independently related to cardiovascular complications. Interleukin-15 is a member of the IL-2 family and stimulates T-lymphocyte and natural killer cell (NK cell) production. Although IL-15 mRNA is widely expressed constitutively in various tissues, IL-15 protein expression is regulated by inflammatory-related transcription factors, such as nuclear faction-B (NF-B)13 and interferon regulatory factor-1.14 Several previous reports have suggested that an elevated serum IL-15 concentration is associated with immunologic inflammatory disease, such as acute graft-versus-host disease,15 rheumatoid arthritis,7,16 Kawasaki disease,17 and renal allograft dysfunction18; however, to our knowledge, a relationship between serum IL-15 concentration and cardiovascular disease has not been reported. In the present study, systemic IL-15 concentration was related to cardiovascular disease, but the reason why serum IL-15 level was significantly elevated in patients with SOD is still unclear. Interleukin-15 can also promote T-cell adhesion and activation, and cytokine secretion as well as angiogenesis, and may thus play a role in atherosclerosis. Pathologically, IL-15 expression has been found in macrophages of atherosclerotic lesions in both humans and an animal model.10 The IL-15 mRNA expression was also found in oxidized LDL-positive macrophages and CD40 ligand-positive T cells.9 From this accumulated evidence, IL-15 might play an important role in the local inflammatory response in the development of atherosclerosis. Recently, local inflammatory reaction has been considered one of the main factors in the development of atherosclerosis and the occurrence of cardiovascular events. From this point of view, there have been several reports on
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Table 2. Association between inflammatory substances and types of severe organ damage With Each Type of Severe Organ Damage hs-CRP concentration (ng/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) IL-6 concentration (pg/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) sICAM-1 concentration (ng/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) sVCAM-1 concentration (ng/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) IL-15 concentration (pg/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73)
Without Each Type of Severe Organ Damage
P
2.11 2.71 3.04 1.40
⫾ ⫾ ⫾ ⫾
0.54 0.62 0.42 0.31
1.32 1.37 1.23 1.60
⫾ ⫾ ⫾ ⫾
0.11 0.22 0.11 0.30
.0413 .2364 .0278 .3382
48.4 45.7 51.6 30.4
⫾ ⫾ ⫾ ⫾
12.4 12.0 9.7 5.7
28.0 32.0 27.1 33.8
⫾ ⫾ ⫾ ⫾
2.6 4.1 2.6 5.4
.0642 .6244 .0093 .7757
1181.6 992.5 1043.5 973.5
⫾ ⫾ ⫾ ⫾
139.5 147.1 109.6 71.0
910.2 943.2 912.7 896.6
⫾ ⫾ ⫾ ⫾
28.6 50.6 29.3 65.3
.0219 .7517 .0323 .1048
2757.0 1954.2 2773.7 2751.0
⫾ ⫾ ⫾ ⫾
686.9 650.9 529.0 340.3
2246.9 2480.6 2210.3 2105.4
⫾ ⫾ ⫾ ⫾
139.4 234.9 141.2 317.0
.1669 .6357 .1783 .1572
131.4 93.3 92.0 94.2
⫾ ⫾ ⫾ ⫾
26.3 27.7 20.4 13.8
54.8 64.0 54.6 42.3
⫾ ⫾ ⫾ ⫾
5.3 10.0 5.4 12.9
.0453 .0332 .1506 .0329
CAD ⫽ coronary artery disease; PAD ⫽ peripheral artery disease analyzed by Mann-Whitney U test.
the relationship between interleukins and cardiovascular disease. An increased serum level of IL-6, which is wellknown as one of the major interleukins, was associated with myocardial infarction,19 and IL-1 and IL-6-stimulated CRP production.20 Moreover, elevation of serum IL-10 concentration was associated with decreased risk of coronary events in patients with unstable angina.21 More recently, serum IL-18 concentration has been watched with keen interest as a risk factor for stroke22 and coronary
heart disease.23 In the present study, we measured several already known inflammatory-related substances: hs-CRP and IL-6 as representative inflammatory markers, and sICAM-1 and sVCAM-1 as representative soluble cell adhesion molecules. The elevated serum levels of hs-CRP2 and IL-65 in patients with SOD were reasonable findings compatible with previous reports. A previous study sug-
Table 3. Relationship between IL-15 and other factors
**
IL-15
(pg/mL)
Correlation Coefficient
**
700 600 500 400 300 200 100 0 No
Mild
Severe
organ damage FIG. 2. Relationship between serum interleukin-15 (IL-15) concentration and cardiovascular complications. **P ⬍ .01 analyzed by Mann-Whitney U test.
Male/female Age BMI (kg/m2) Smoking index SBP (mm Hg) DBP (mm Hg) HR TC (mg/dL) HDL-C (mg/dL) FBG (mg/dL) HbA1c (%) Medication
0.029 0.017 0.073 0.037 0.030 0.006 0.039 0.088 0.055 0.023
P .3892 .5687 .7342 .1530 .4671 .5579 .9203 .4397 .0949 .3028 .7117 .0946
Abbreviations as in Table 1. Analyzed by single regression analysis, except for male/female and medication (Mann-Whitney U test).
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Table 4. Correlation with severe organ damage analyzed by multiple linear logistic regression analysis
Sex Age BMI (kg/m2) Current smoking SBP (mm Hg) DBP (mm Hg) TC (mg/dL) HDL-C (mg/dL) FBG (mg/dL) Medication IL-15 (pg/mL)
F
P
8.41 9.88 0.001 4.15 0.00 1.89 0.00 0.00 2.30 3.23 6.85
.0037 .0017 .9697 .1254 .9955 .1692 .9760 .9840 .1290 .0723 .0089
Abbreviations as in Table 1. Analyzed by multiple logistic regression analysis.
gested that serum level of sICAM-1 was associated with BP,3 which is one of the possible reasons why sICAM-1 level was higher in the MOD group. As a limitation of this study, we could not homogenize the baseline characteristics of the three groups and could not remove the influence of medication, especially antihypertensive drugs. In the present study, serum IL-15 concentration in patients taking antihypertensive drugs was increased compared to that in patients not taking antihypertensive drugs. This does not necessarily mean that administration of antihypertensive drugs increased the serum level of IL-15. Most of the patients with SOD were administered antihypertensive drugs, and the percentage of patients without antihypertensive drugs was relatively higher in the NOD group. This might be one of the reasons for the observation of increased IL-15 concentration in patients receiving antihypertensive drugs. To confirm the influence of antihypertensive drugs on serum level of IL-15, a cohort study comparing serum IL-15 concentration in patients before and after antihypertensive drug administration might be required. The present study showed a decreased level of IL-15 in patients with MOD compared with that in the NOD group, without statistical significance (P ⫽ .379), as shown in Fig. 2. This finding suggests that IL-15 might not be related to mild hypertensive complications, such as left ventricular hypertrophy and retinal artery stenosis, but only becomes elevated after the occurrence of cardiovascular disease. The present study investigated serum IL-15 concentration and cardiovascular disease in patients with essential hypertension, and showed that an elevated serum concentration of IL-15 in patients with essential hypertension was an independent correlative risk factor for SOD, including existent or preexistent cardiovascular disease. From this accumulated evidence, serum IL-15 concentration was only increased in patients with severe cardiovascular complications, whereas serum hs-CRP and IL-6 concentrations were associated with cardiovascular complications, serum
sICAM-1 concentration was associated with mild organ damage, and serum sVCAM-1 concentration was associated with only mild organ damage. Although there are many limitations in this study, these findings raise the possibility that IL-15 might not be related to hypertension and a substantial amount of this cytokine might only be detected when severe organ damage occurs and its detection would be the consequence of an inflammatory reaction. Study Limitations Although we examined several inflammatory markers in this study, the role of inflammatory reaction in the development of atherosclerosis might be quite complicated, with many other inflammatory substances involved in this phenomenon. In the present study, although serum IL-15 concentration was adopted as one of the independent factors by multiple linear logistic regression analysis, a larger case-controlled study might be required to confirm the independence of serum IL-15 concentration. As inflammatory cytokines and cardiovascular risk factors were modified by administration of renin-angiotensin inhibition, statins, or their combinations, we should more carefully conclude that additional study without administration would be required. This study was a cross-sectional, hospital-based study, which could not reveal whether IL-15 concentration might be a predictor of cardiovascular complications and it is unclear whether an elevated serum IL-15 concentration influences the occurrence of cardiovascular events. We should emphasize the importance of a larger cohort study to clarify and confirm the influence of serum IL-15 concentration on the occurrence of cardiovascular events and its mortality. However, the evidence that systemic IL-15 concentration may not be related to hypertension and could be raised when severe organ damage occurs, and that its detection would be the consequence of an inflammatory reaction, might be important in managing patients with essential hypertension.
Acknowledgments We are most grateful to Tomoko Ikeda, Seiko Kaji, and Kazuko Iwasa for their technical assistance.
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epidermal V gamma 3 cells in IL-15-deficient and IFN regulatory factor-1-deficient mice. J Immunol 2002;168:6486 – 6493. Chik K, Li K, Pong H, Shing M, Li C, Yuen P: Elevated serum interleukin-15 level in acute graft-versus-host disease after hematopoietic cell transplantation. J Pediatr Hematol Oncol 2003; 25:960 –964. McInnes I, al-Mughales M, Field M, Leung B, Huang F, Dixon R, Sturrock R, Wilkinson P, Liew F: The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis. Nat Med 1996;2:175–182. Jang G, Kim H, Ahn S, Kim D: Raised serum interleukin 15 levels in Kawasaki disease. Ann Rheum Dis 2003;62:264 –266. Hu R, Tsai M, Lee P: Serum profiles of IL-12, -15, and -18 in renal allograft dysfunction in man. Transplant Proc 2003;35:246 –248. Bennet A, Prince J, Fei G, Lyrenas L, Huang Y, Wiman B, Frostegard J, Faire U: Interleukin-6 serum levels and genotypes influence the risk for myocardial infarction. Atherosclerosis 2003;171:359 –367. Calabro P, Willerson J, Yeh E: Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells. Circulation 2003;108:1930 –1932. Anguera I, Miranda-Guardiola F, Bosch X, Filella X, Sitges M, Marin J, Betriu A, Sanz G: Elevation of serum levels of the antiinflammatory cytokine interleukin-10 and decreased risk of coronary events in patients with unstable angina. Am Heart J 2002;144: 811– 817. Zaremba J, Losy J: Interleukin-18 in acute ischaemic stroke patients. Neurol Sci 2003;24:117–124. Blankenberg S, Luc G, Ducimetiere P, Arveiler D, Ferrieres J, Amouyel P, Evans A, Cambien F, Tiret L, Group PS: Interleukin-18 and the risk of coronary heart disease in European men: the Prospective Epidemiological Study of Myocardial Infarction (PRIME). Circulation 2003;108:2453–2459.
Epidemiology
2005; 18:1019 –1025
ORIGINAL CONTRIBUTIONS
Serum Interleukin-15 Concentration in Patients With Essential Hypertension Masaharu Kaibe, Mitsuru Ohishi, Norihisa Ito, Ming Yuan, Takashi Takagi, Minako Terai, Yuji Tatara, Norio Komai, Hiromi Rakugi, and Toshio Ogihara Background: Interleukin (IL)-15 is one of the cytokines produced by neutrophils and monocytes/macrophages, and its expression is found immunohistochemically in inflammatory cells adjacent to vulnerable atherosclerotic plaques. However, the influence of systemic IL-15 on cardiovascular disease is still unclear. Therefore, we designed clinical investigations to clarify the relationship between cardiovascular complications and serum IL-15 levels. Methods and Results: Three hundred ninety-nine patients with essential hypertension were analyzed. We divided the study subjects into the following three groups according to the modified World Health Organization–International Society of Hypertension classification of 1999: patients with no organ damage (n ⫽ 213), patients with mild organ damage (n ⫽ 128), and patients with severe organ damage (n ⫽ 58). We measured serum IL-15, highly sensitive C reactive protein, IL-6, soluble intercellular adhesion molecule, and soluble vascular cell adhesion molecule levels. Serum IL-15 concentration in patients with severe organ
R
ecently local inflammatory response has been suggested to be one of the major mechanisms of the development of atherosclerosis, which is the principal change in arteries in cardiovascular disease. Many cytokines, chemokines, and cell adhesion molecules are involved in this phenomenon, and inflammatory cells, such as white blood cells, lymphocytes, and monocytes/macrophages, also play an important role in the development of atherosclerosis. Landmark articles first illustrated the potential association between C-reactive protein (CRP) level and cardiovascular outcome in patients with unstable coronary syndromes1 and numerous studies have found that the baseline level of highly sensitive CRP (hs-CRP) is associated with the risk of future
See related article on page 1017
Received December 17, 2004. First decision February 18, 2005. Accepted February 23, 2005. From the Department of Geriatric Medicine, Osaka University Graduate School of Medicine, Suita, Japan. This study was partially funded by a Japan Heart Foundation/ Pfizer Grant for Research on Hypertension and Vascular Metabolism and by the Research Foundation for Community Medicine, Research © 2005 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
damage was significantly higher than that in those with no organ damage (P ⬍ .01) and those with mild organ damage (P ⬍ .01). Serum IL-15 concentration in patients with coronary artery disease or peripheral artery disease was significantly higher than that in those without coronary artery disease or peripheral artery disease. Moreover, serum IL-15 concentration in patients with lacunar infarction was significantly higher than that in those without lacunar infarction (P ⬍ .005). By multiple linear logistic regression analysis, serum IL-15 concentration was independently correlated with cardiovascular disease. Conclusions: These data suggest that a systemic inflammatory response involving IL-15 might be involved in the occurrence of cardiovascular disease in patients with essential hypertension. Am J Hypertens 2005;18:1019 –1025 © 2005 American Journal of Hypertension, Ltd. Key Words: IL-15, cardiovascular disease, hs-CRP, IL-6.
myocardial infarction, stroke, peripheral vascular disease, and cardiovascular death.2 Several inflammatory biomarkers have been reported to be risk factors and predictors of cardiovascular disease. In the Physician’s Health Study, soluble intercellular adhesion molecule-1 (sICAM-1) was associated with blood pressure (BP)3 and was shown to be a predictor of myocardial infarction,4 and an increased baseline level of the proinflammatory cytokine interleukin-6 (IL-6) also predicted future cardiovascular events.5 In a study from the secondary prevention Cholesterol and Recurrent Events (CARE) trial, elevation of tumor necrosis factor-␣ in patients in the stable phase after myocardial infarction was associated with an increased risk of recurrent coronary events.6 The cytokine interleukin-15 (IL-15) is one of these
Meeting on Hypertension and Arteriosclerosis. MK was also supported by a Research Fellowship of the Japan Society for the Promotion of Science. Address correspondence and reprint requests to Dr. Hiromi Rakugi, Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita 565-0871, Japan; e-mail: rakugi@ geriat.med.osaka-u.ac.jp 0895-7061/05/$30.00 doi:10.1016/j.amjhyper.2005.02.014
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proinflammatory cytokines, and has been detected in different inflammatory conditions.7 Interleukin-15 is a 14-15kDa cytokine expressed at the mRNA level in numerous normal human tissues in a broad range of cell types, including activated monocytes, dendritic cells, osteoclasts, and fibroblasts.8 Pathologic study has demonstrated IL-15 expression in human atherosclerotic plaque. Expression of IL-15 mRNA and protein was found almost solely in fibrolipid and lipid-rich plaques, associated with oxidized LDLpositive macrophages.9 In gene expression arrays, a transcript corresponding to IL-15 mRNA was elevated in the atherosclerotic aorta of ApoE-deficient mice fed a Western diet for 10 or 20 weeks, corresponding to lesions of the fatty streak and fibrofatty plaque stage, respectively. Immunostaining for IL-15 was localized to aortic smooth muscle cells in nonatherosclerotic C57BL/6 mice, whereas both macrophages and smooth muscle cells in atherosclerotic lesions of ApoE-deficient mice stained positive for IL-15.10 These data show that IL-15 expression is upregulated in atherosclerosis and suggest that this cytokine may be important in its pathogenesis. Inflammatory cytokines, such as CRP, IL-6, and cell adhesion molecule, play local roles to be involved in atherogenic response adjacent to vulnerable plaque. Interleukin-15 could be also associated with cardiovascular disease; however, the relationship between systemic IL-15 concentration and cardiovascular events is still unclear. Therefore, we designed a clinical investigation to clarify the relationship between serum IL-15 concentration and cardiovascular complications in patients with essential hypertension.
Methods Subjects Four hundred seventy-eight patients with essential hypertension who attended Osaka University Hospital were enrolled in this study. Hypertension was defined as systolic BP of more than 140 mm Hg, diastolic BP of more than 90 mm Hg, or administration of antihypertensive drugs. We excluded patients with acute coronary syndrome and stroke within 3 months before participation. We excluded patients with acute or chronic infectious or inflammatory disease (n ⫽ 14) or malignant neoplasm (n ⫽ 8), which influence serum IL-15 concentration. We also excluded patients who were administered hormone replacement therapy (n ⫽ 0), steroid (n ⫽ 5) or aspirin (n ⫽ 47), to exclude their influence on serum IL-15 concentration. Five patients refused to undergo venipuncture. Finally, 399 patients with essential hypertension were analyzed in this study. The protocol of this study was approved by our hospital ethics committee, and written informed consent was obtained from all subjects before participation in this cross-sectional observational study. One hundred fortyeight patients were not administered any antihypertensive drugs, whereas 151 patients were treated with a calcium antagonist, 80 with an angiotensin-converting enzyme inhibitor, 122 with an angiotensin type I receptor antagonist, 43 with a -blocker, 16 with an ␣-blocker, and 35 with a diuretic.
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Cardiovascular Complications We evaluated organ damage due to hypertension according to the World Health Organization (WHO)–International Society of Hypertension (ISH) classification modified in 1999.11 We evaluated mild organ damage corresponding to target organ damage in WHO–ISH 1999, such as left ventricular hypertrophy (LVH), retinal artery narrowing, proteinuria (⬎20 mg/L), or increased plasma creatinine concentration (1.2 to 2.0 mg/dL) and carotid plaque. LVH was determined by electrocardiography using the Sokolow and Lyon criteria or by echocardiography—a septal or posterior wall thickness of more than 12 mm. Carotid plaque was determined by carotid ultrasonography as a score of five or more as previously reported.12 One special operator examined echocardiography and carotid ultrasonography (Power Vision 6000, Toshiba, Tokyo, Japan) and took the mean of three examinations as representative data for this study. The intraobserver coefficient of variation was 2.8% ⫾ 1.2%. We also evaluated severe organ damage corresponding to associated clinical conditions in WHO–ISH 1999, such as cerebrovascular disease by brain magnetic resource imaging (MRI) or computed tomographic scan and neurologic findings, coronary artery disease (CAD) by electrocardiography, echocardiography, or typical symptoms, congestive heart failure, or peripheral artery disease (PAD) without ulceration or gangrene. Moreover, we evaluated lacunar infarction by brain MRI. Serum Concentrations of Inflammatory Substances A venous blood sample was obtained from each patient in the morning after an overnight fast and stopping administration of drugs for at least 24 h. Serum was stored at ⫺80°C. Stored serum was thawed and assayed for serum IL-15, hs-CRP, IL-6, sICAM-1 and soluble vascular cell adhesion molecule (sVCAM-1) by commercially available ELISAs (serum IL-15 and IL-6: BIOSOURCE International, Camarillo, CA; hs-CRP: ALPHA DIAGNOSTIC International, San Antonio, TX; sICAM-1 and sVCAM-1: DIACLONE Research, Besançon Cedex, France). Serum levels of IL-15, hs-CRP, IL-6, sICAM-1, and sVCAM-1 were measured in duplicate for each patient. The intraobserver coefficient of variation for these inflammatory substances was 3.1% ⫾ 1.3%, 2.2% ⫾ 0.9%, 2.7% ⫾ 1.2%, 2.9% ⫾ 1.0%, and 2.8% ⫾ 1.2%, respectively. Statistical Analysis Data were analyzed with JMP ver. 4 (SAS Inc., Cary, NC) and presented as mean ⫾ SEM. We used Mann-Whitney U test to analyze nonparametrical data, such as hs-CRP, IL-6, sICAM-1, sVCAM-1, and IL-15 for significant differences among each hypertensive complication group. ANOVA and Student t test were also used to analyze other clinical data for significant differences among cardiovascular complications. We also used multiple linear logistic regression analysis to
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Table 1. Patients characteristics
Number Male/female Age (y) BMI (kg/m2) Current smokers (%) Smoking index SBP (mm Hg) DBP (mm Hg) HR (beats/min) TC (mg/dL) HDL-C (mg/dL) FBG (mg/dL) HbA1C (%) hs-CRP (ng/mL) IL-6 (pg/mL) sICAM-1 (ng/mL) sVCAM-1 (ng/mL)
No Organ Damage
Mild Organ Damage
Severe Organ Damage
213 103/110 58.5 ⫾ 0.9 24.5 ⫾ 0.3 19.2 238.3 ⫾ 27.5 148.3 ⫾ 1.4 87.3 ⫾ 1.0 73.8 ⫾ 1.0 210.8 ⫾ 2.6 59.3 ⫾ 1.2 100.5 ⫾ 1.4 6.1 ⫾ 0.8 1.2 ⫾ 0.1 19.1 ⫾ 2.9 837 ⫾ 33.4 2009 ⫾ 175
128 72/56 61.9 ⫾ 1.1* 24.3 ⫾ 0.3 31.1† 337.0 ⫾ 44.6* 153.4 ⫾ 2.0* 88.8 ⫾ 1.1 72.1 ⫾ 1.2 204.2 ⫾ 3.0 55.7 ⫾ 1.9 102.5 ⫾ 1.7 6.0 ⫾ 1.2 1.4 ⫾ 0.1* 37.8 ⫾ 4.5‡ 1009 ⫾ 48.9† 2438 ⫾ 243†
58 47/11* 68.1 ⫾ 1.1‡ 23.3 ⫾ 0.4* 46.6‡§ 672.1 ⫾ 89.3‡§ 146.6 ⫾ 2.9 81.5 ⫾ 1.9†§ 76.4 ⫾ 2.5 193.3 ⫾ 4.5†§ 54.2 ⫾ 2.1* 111.7 ⫾ 4.2†§ 5.8 ⫾ 0.2 2.5 ⫾ 0.5† 42.8 ⫾ 7.9‡ 1043 ⫾ 88.8† 2418 ⫾ 333
BMI ⫽ body mass index; DBP ⫽ diastolic blood pressure; FBG ⫽ fasting blood glucose; HDL-C ⫽ high-density lipoprotein cholesterol; HR ⫽ heart rate; SBP ⫽ systolic blood pressure; TC ⫽ total cholesterol. Analyzed by Student t test, except for inflammatory cytokines (Mann-Whitney U test) and male/female and current smokers (2 test). * P ⬍ .05, † P ⬍ .01, ‡ P ⬍ .001 v no organ damage; § P ⬍ .01 v mild organ damage.
determine the correlation of cardiovascular complications with risk factors, including serum IL-15 concentration. A value of P ⬍ .05 was taken as statistically significant. A power calculation demonstrates that the minimum number of study subjects to determine a relationship between serum IL-15 concentration and hypertensive complications was more than 129, and moreover statistical power of this study was more than 0.9.
Results Patients Characteristics The patients’ characteristics are shown in Table 1. Hypertensive patients were classified into three groups: no organ damage (NOD; n ⫽ 213), mild organ damage (MOD; n ⫽ 128), and severe organ damage (SOD; n ⫽ 58). Patients with SOD showed a higher frequency of the male gender, older age, lower body mass index (BMI), higher frequency of current smoking, lower diastolic BP, lower total cholesterol, lower HDL-cholesterol, and higher fasting blood glucose compared with those with NOD or MOD. Serum Concentrations of Inflammatory Substances and Cardiovascular Complications Serum concentrations of hs-CRP, IL-6, sICAM-1, and sVCAM-1 were measured as known inflammatory-related substances (Table 1 and Fig. 1). Serum hs-CRP concentration in patients with SOD (2.5 ⫾ 0.5 ng/mL; P ⬍ .01) and MOD (1.4 ⫾ 0.1 ng/mL; P ⬍ .05) was significantly higher than that in those with NOD (1.2 ⫾ 0.1 ng/mL). Serum IL-6 concen-
tration in patients with SOD (42.8 ⫾ 7.9 pg/mL; P ⬍ .001) and MOD (37.8 ⫾ 4.5 pg/mL; P ⬍ .001) was also significantly higher than that in those with NOD (19.1 ⫾ 2.9 pg/mL). The sICAM-1 concentration in patients with SOD (1043 ⫾ 88.8 ng/mL; P ⬍ .01) and MOD (1009 ⫾ 48.9 ng/mL; P ⬍ .01) was also significantly higher than that in those with NOD (837 ⫾ 33.4 ng/mL). The sVCAM-1 concentration in patients with NOD (2009 ⫾ 175 ng/mL; P ⬍ .05) was significantly lower than those in patients with MOD (2438 ⫾ 243 ng/mL), but there was no significant difference compared to those with SOD. We also analyzed the relationship between serum concentrations of hs-CRP, IL-6, sICAM-1, and sVCAM-1 and the subtype of cerebrocardiovascular disease with SOD (Table 2). Serum hs-CRP and sICAM-1 concentrations in patients with CAD or PAD was significantly higher than that in those without CAD or PAD (P ⬍ .05), and serum IL-6 concentration in patients with PAD was significantly higher than that in those without PAD (P ⬍ .01); however, there was no significant difference in serum concentration of sVCAM-1 in patients with or without each subtype of SOD. We also analyzed cumulative effect of target organ damage in MOD, but we could not find any cumulative effect on serum levels of hs-CRP, IL-6, IL-15, sICAM-1, or sVCAM-1 (data not shown). The relationship between serum IL-15 concentration and cardiovascular complications is shown in Fig. 2. Serum IL-15 concentration in patients with SOD (88.7 ⫾ 18.7 pg/mL) was significantly higher than that in those with NOD (55.2 ⫾ 6.9 pg/mL; P ⬍ .01) or MOD (51.0 ⫾ 8.3 pg/mL; P ⬍ .01). We also analyzed the relationship between serum IL-15 concentration and the subtype of cardiovascular disease with SOD (Table 2). Serum IL-15 concentration in patients with CAD,
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cerebrovascular disease, and lacunar infarction was significantly higher than that in those without CAD (P ⬍ .05).
(a)
(ng/mL)
** *
hs-CRP
16 14 12 10 8 6 4 2 0
IL-15 Concentration and Other Factors To clarify factors influencing serum IL-15 concentration, we analyzed the relationship between serum IL-15 concentration and other factors (Table 3). There was no relationship with these factors. Mild
No
Severe
organ damage
We performed logistic regression analysis for cardiovascular complications, using sex, age, BMI, current smoking, systolic BP, diastolic BP, total cholesterol, HDLcholesterol, fasting blood glucose, treatment with antihypertensive drugs, and IL-15, which were significantly related to organ damage (Table 4). Serum IL-15 concentration (P ⫽ .0089) was independently related to cardiovascular complications in addition to sex (P ⫽ .0037) and older age (P ⫽ .0017).
(b)
(pg/mL)
*** ***
300
IL-6
250
200 150
100 50
Discussion
0 No
Severe
Mild
organ damage (c)
**
(ng/mL)
**
sICAM-1
3000 2500
2000 1500
1000 500 0 No
Mild
Severe
organ damage (d)
**
(ng/mL)
sVCAM-1
Multiple Linear Logistic Regression Analysis
16000 14000 12000 10000 8000 6000 4000 2000 0 No
Mild
Severe
organ damage FIG. 1. Relationship between serum highly sensitive C-reactive protein (hs-CRP) (A), interleukin-6 (IL-6) (B), soluble intercellular adhesion molecule-1 (sICAM-1) (C), and sVCAM-1(D) concentrations and cardiovascular complications. *P ⬍ .05, **P ⬍ .01, ***P ⬍ .001 analyzed by Mann-Whitney U test.
In the present study, serum IL-15 concentration in patients with SOD was significantly increased, and was independently related to cardiovascular complications. Interleukin-15 is a member of the IL-2 family and stimulates T-lymphocyte and natural killer cell (NK cell) production. Although IL-15 mRNA is widely expressed constitutively in various tissues, IL-15 protein expression is regulated by inflammatory-related transcription factors, such as nuclear faction-B (NF-B)13 and interferon regulatory factor-1.14 Several previous reports have suggested that an elevated serum IL-15 concentration is associated with immunologic inflammatory disease, such as acute graft-versus-host disease,15 rheumatoid arthritis,7,16 Kawasaki disease,17 and renal allograft dysfunction18; however, to our knowledge, a relationship between serum IL-15 concentration and cardiovascular disease has not been reported. In the present study, systemic IL-15 concentration was related to cardiovascular disease, but the reason why serum IL-15 level was significantly elevated in patients with SOD is still unclear. Interleukin-15 can also promote T-cell adhesion and activation, and cytokine secretion as well as angiogenesis, and may thus play a role in atherosclerosis. Pathologically, IL-15 expression has been found in macrophages of atherosclerotic lesions in both humans and an animal model.10 The IL-15 mRNA expression was also found in oxidized LDL-positive macrophages and CD40 ligand-positive T cells.9 From this accumulated evidence, IL-15 might play an important role in the local inflammatory response in the development of atherosclerosis. Recently, local inflammatory reaction has been considered one of the main factors in the development of atherosclerosis and the occurrence of cardiovascular events. From this point of view, there have been several reports on
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Table 2. Association between inflammatory substances and types of severe organ damage With Each Type of Severe Organ Damage hs-CRP concentration (ng/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) IL-6 concentration (pg/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) sICAM-1 concentration (ng/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) sVCAM-1 concentration (ng/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73) IL-15 concentration (pg/mL) CAD (n ⫽ 17) Cerebrovascular disease (n ⫽ 19) PAD (n ⫽ 29) Lacunar infarction (n ⫽ 73)
Without Each Type of Severe Organ Damage
P
2.11 2.71 3.04 1.40
⫾ ⫾ ⫾ ⫾
0.54 0.62 0.42 0.31
1.32 1.37 1.23 1.60
⫾ ⫾ ⫾ ⫾
0.11 0.22 0.11 0.30
.0413 .2364 .0278 .3382
48.4 45.7 51.6 30.4
⫾ ⫾ ⫾ ⫾
12.4 12.0 9.7 5.7
28.0 32.0 27.1 33.8
⫾ ⫾ ⫾ ⫾
2.6 4.1 2.6 5.4
.0642 .6244 .0093 .7757
1181.6 992.5 1043.5 973.5
⫾ ⫾ ⫾ ⫾
139.5 147.1 109.6 71.0
910.2 943.2 912.7 896.6
⫾ ⫾ ⫾ ⫾
28.6 50.6 29.3 65.3
.0219 .7517 .0323 .1048
2757.0 1954.2 2773.7 2751.0
⫾ ⫾ ⫾ ⫾
686.9 650.9 529.0 340.3
2246.9 2480.6 2210.3 2105.4
⫾ ⫾ ⫾ ⫾
139.4 234.9 141.2 317.0
.1669 .6357 .1783 .1572
131.4 93.3 92.0 94.2
⫾ ⫾ ⫾ ⫾
26.3 27.7 20.4 13.8
54.8 64.0 54.6 42.3
⫾ ⫾ ⫾ ⫾
5.3 10.0 5.4 12.9
.0453 .0332 .1506 .0329
CAD ⫽ coronary artery disease; PAD ⫽ peripheral artery disease analyzed by Mann-Whitney U test.
the relationship between interleukins and cardiovascular disease. An increased serum level of IL-6, which is wellknown as one of the major interleukins, was associated with myocardial infarction,19 and IL-1 and IL-6-stimulated CRP production.20 Moreover, elevation of serum IL-10 concentration was associated with decreased risk of coronary events in patients with unstable angina.21 More recently, serum IL-18 concentration has been watched with keen interest as a risk factor for stroke22 and coronary
heart disease.23 In the present study, we measured several already known inflammatory-related substances: hs-CRP and IL-6 as representative inflammatory markers, and sICAM-1 and sVCAM-1 as representative soluble cell adhesion molecules. The elevated serum levels of hs-CRP2 and IL-65 in patients with SOD were reasonable findings compatible with previous reports. A previous study sug-
Table 3. Relationship between IL-15 and other factors
**
IL-15
(pg/mL)
Correlation Coefficient
**
700 600 500 400 300 200 100 0 No
Mild
Severe
organ damage FIG. 2. Relationship between serum interleukin-15 (IL-15) concentration and cardiovascular complications. **P ⬍ .01 analyzed by Mann-Whitney U test.
Male/female Age BMI (kg/m2) Smoking index SBP (mm Hg) DBP (mm Hg) HR TC (mg/dL) HDL-C (mg/dL) FBG (mg/dL) HbA1c (%) Medication
0.029 0.017 0.073 0.037 0.030 0.006 0.039 0.088 0.055 0.023
P .3892 .5687 .7342 .1530 .4671 .5579 .9203 .4397 .0949 .3028 .7117 .0946
Abbreviations as in Table 1. Analyzed by single regression analysis, except for male/female and medication (Mann-Whitney U test).
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Table 4. Correlation with severe organ damage analyzed by multiple linear logistic regression analysis
Sex Age BMI (kg/m2) Current smoking SBP (mm Hg) DBP (mm Hg) TC (mg/dL) HDL-C (mg/dL) FBG (mg/dL) Medication IL-15 (pg/mL)
F
P
8.41 9.88 0.001 4.15 0.00 1.89 0.00 0.00 2.30 3.23 6.85
.0037 .0017 .9697 .1254 .9955 .1692 .9760 .9840 .1290 .0723 .0089
Abbreviations as in Table 1. Analyzed by multiple logistic regression analysis.
gested that serum level of sICAM-1 was associated with BP,3 which is one of the possible reasons why sICAM-1 level was higher in the MOD group. As a limitation of this study, we could not homogenize the baseline characteristics of the three groups and could not remove the influence of medication, especially antihypertensive drugs. In the present study, serum IL-15 concentration in patients taking antihypertensive drugs was increased compared to that in patients not taking antihypertensive drugs. This does not necessarily mean that administration of antihypertensive drugs increased the serum level of IL-15. Most of the patients with SOD were administered antihypertensive drugs, and the percentage of patients without antihypertensive drugs was relatively higher in the NOD group. This might be one of the reasons for the observation of increased IL-15 concentration in patients receiving antihypertensive drugs. To confirm the influence of antihypertensive drugs on serum level of IL-15, a cohort study comparing serum IL-15 concentration in patients before and after antihypertensive drug administration might be required. The present study showed a decreased level of IL-15 in patients with MOD compared with that in the NOD group, without statistical significance (P ⫽ .379), as shown in Fig. 2. This finding suggests that IL-15 might not be related to mild hypertensive complications, such as left ventricular hypertrophy and retinal artery stenosis, but only becomes elevated after the occurrence of cardiovascular disease. The present study investigated serum IL-15 concentration and cardiovascular disease in patients with essential hypertension, and showed that an elevated serum concentration of IL-15 in patients with essential hypertension was an independent correlative risk factor for SOD, including existent or preexistent cardiovascular disease. From this accumulated evidence, serum IL-15 concentration was only increased in patients with severe cardiovascular complications, whereas serum hs-CRP and IL-6 concentrations were associated with cardiovascular complications, serum
sICAM-1 concentration was associated with mild organ damage, and serum sVCAM-1 concentration was associated with only mild organ damage. Although there are many limitations in this study, these findings raise the possibility that IL-15 might not be related to hypertension and a substantial amount of this cytokine might only be detected when severe organ damage occurs and its detection would be the consequence of an inflammatory reaction. Study Limitations Although we examined several inflammatory markers in this study, the role of inflammatory reaction in the development of atherosclerosis might be quite complicated, with many other inflammatory substances involved in this phenomenon. In the present study, although serum IL-15 concentration was adopted as one of the independent factors by multiple linear logistic regression analysis, a larger case-controlled study might be required to confirm the independence of serum IL-15 concentration. As inflammatory cytokines and cardiovascular risk factors were modified by administration of renin-angiotensin inhibition, statins, or their combinations, we should more carefully conclude that additional study without administration would be required. This study was a cross-sectional, hospital-based study, which could not reveal whether IL-15 concentration might be a predictor of cardiovascular complications and it is unclear whether an elevated serum IL-15 concentration influences the occurrence of cardiovascular events. We should emphasize the importance of a larger cohort study to clarify and confirm the influence of serum IL-15 concentration on the occurrence of cardiovascular events and its mortality. However, the evidence that systemic IL-15 concentration may not be related to hypertension and could be raised when severe organ damage occurs, and that its detection would be the consequence of an inflammatory reaction, might be important in managing patients with essential hypertension.
Acknowledgments We are most grateful to Tomoko Ikeda, Seiko Kaji, and Kazuko Iwasa for their technical assistance.
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