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【摘要】
Objective- We investigated the association of several chemokines with the risk of stable coronary heart disease (CHD) in a large case-control study after adjustment for other established risk factors. Furthermore, we analyzed their correlation with various acute-phase proteins, inflammation-associated cytokines, and an adhesion molecule.
Methods and Results- We included 312 patients aged 40 to 68 years with angiographically confirmed and stable CHD and 472 age- and gender-matched controls in this study. The main outcome measure was the odds ratio (OR) for CHD associated with increased levels of interferon (INF)-inducible protein of 10 kd (IP-10), interleukin (IL)-8, regulated on activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammation protein 1 (MIP-1 ), or eotaxin determined by rigidly evaluated sandwich ELISAs. Serum levels of IP-10 and IL-8 were higher, and serum levels of RANTES were lower in CHD patients when compared with age- and gender-matched controls. In addition, values in the second and top tertile of IP-10 and IL-8 were associated with an increased OR for CHD when compared with values in the bottom tertile [OR for IP-10 (top tertile) was 2.62 (95% CI, 1.79 to 3.85) in the age- and gender-adjusted model and 1.93 (95% CI, 1.23 to 3.04) in the fully adjusted model, and for IL-8, the OR was 1.77 (95% CI, 1.20 to 2.59) and 1.53 (95% CI, 0.98 to 2.39), respectively]; increased RANTES values were associated with a lower OR for CHD [OR, 0.67 (95% CI, 0.47 to 0.96) and 0.61 (95% CI, 0.40 to 0.94)]. Furthermore, positive correlations of IP-10 and IL-8 with several acute-phase proteins or inflammation-associated cytokines were evident, and positive correlations for IP-10 plasma viscosity and intercellular adhesion molecule 1 were also present.
Conclusions- The current study suggests that there may be no universal upregulation of chemokines in CHD-associated inflammation but different upregulation of IP-10 and IL-8 versus downregulation of RANTES; there was no clear disease association for MCP-1, MIP-1, or eotaxin.
In this large, case-control study including patients with angiographically defined stable CHD, we found evidence of a differential expression of chemokines associated with the risk for CHD.
【关键词】 chemokines coronary heart disease inflammation casecontrol study
Introduction
Cardiovascular diseases still are the leading cause of disability and death in the United States and other developed countries. 1,2 Half of all cases are directly attributable to coronary heart disease (CHD). Meanwhile, convincing evidence suggests that CHD is an inflammatory process, 3 and a variety of inflammatory and other biochemical markers potentially related to atherogenesis have been identified. 4,5 Factors triggering this immunologic response and the underlying mechanistic links are, however, yet unclear.
Chemokines are small molecular weight proteins that cause chemoattraction and activation of leukocytes, and they play an important role in immune reactivity. 6 They have been identified as key mediators of inflammation and other pathophysiological states. 7,8 Because subendothelial accumulation of various inflammatory cells is a main characteristic in various steps of atherogenesis, it is likely that chemokines attract and activate leukocytes in the diseased vessels. Their role in primary atherogenesis, however, is rather unclear. A better understanding of their involvement would not only provide new insights into disease etiology but might may also lead to new therapeutic avenues, possibly to neutralizing specific chemokine activity, and, thus, interfere with the disease process.
We analyzed data of a case-control study in patients with stable CHD to investigate the association of serum concentrations of interferon (INF)-inducible protein of 10 kd (IP-10), interleukin 8 (IL-8), regulated on activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammation protein 1, (MIP-1 ), and eotaxin with the risk of CHD after careful adjustment for other established risk factors. Furthermore, we investigated the correlation of chemokine levels with various acute-phase proteins, inflammation-associated cytokines, and intracellular adhesion molecule (ICAM) 1.
Methods
Patients and Controls
German speaking patients (n=312) aged 40 to 68 years who underwent coronary angiography at the Cardiology Department of the University of Ulm Medical Center between April 1996 and November 1997 and who showed at least one coronary stenosis of more than 50% of the luminal diameter were included in the study. The main exclusion 2 years ago, unstable angina pectoris, acute myocardial infarction within the past 4 weeks, infection within the past 3 weeks, malignant disease, and anticoagulant therapy within the past 2 weeks.
The control group consisted of 472 subjects who were occasional blood donors at the local Red Cross center serving the University Hospitals of Ulm. None of the controls had a history of definite or suspected CHD, and they did not report infections or surgery within the previous four weeks. Participation rate was 78% in patients and 84% in controls.
Frequency matching for age and gender was performed and a case:control ratio of 1:1.5 was intended. All of the subjects underwent standardized interviews conducted by trained interviewers. The primary objective of the study was to assess the effect of various infectious agents on CHD risk (for details see refs 9, 10 ) and to investigate the role of other emerging risk factors (for examples, see refs 11-13 ). All of the subjects gave written informed consent, and the study was approved by the ethics committee of the University of Ulm.
Laboratory Methods
Venous blood was drawn in the morning under standardized conditions. Details for the following markers of inflammation and hemostasis (which were used as covariates in the current analysis only) have been described elsewhere: 13 IL-6, tumor necrosis factor, ICAM-1, plasminogen-activator inhibitor (PAI)-1 activity, von Willebrand factor, C-reactive protein (CRP), fibrinogen, serum amyloid A, and plasma viscosity.
Analyses of Chemokines
IP-10, IL-8, RANTES, MCP-1, MIP-1, and eotaxin were determined by ELISA (R&D Systems). The sandwich ELISAs were established to meet the following criteria: linearity of signal for the standard curve between optical density 0.05 and 2.0, difference between expected and measured signal in spiking experiments <15%, mean intra-assay variation <20%, mean interassay variation <20%, and loss of signal after freezing and thawing of sera 3 times <20%. An interference of heterophile antibodies was not observed. We analyzed 90% of sera in single measurements and 10% of the sera in duplicate. All of the laboratory analyses were done in a blinded fashion.
Statistical Methods
We calculated age- and gender-adjusted mean concentrations by general linear regression for each chemokine separately and determined their distribution according to tertiles. Because all of the chemokine values were considerably skewed, geometric means were calculated after logarithmic transformation. Furthermore, we used unconditional logistic regression to assess the association of a chemokine value in the middle and top tertile (compared with the bottom tertile) with CHD, while simultaneously controlling for age and gender and additionally controlling for body mass index, duration of school education, cigarette smoking (pack-years), alcohol consumption, history of hypertension, history of diabetes mellitus, and high-density lipoprotein cholesterol values.
In addition, the mean concentrations of CHD-associated chemokines were determined according to various levels of sociodemographic and other cardiovascular risk factors (in control subjects only) and quantified by 2 test. Finally, a Spearman correlation coefficient was calculated for the chemokines and various acute phase proteins (CRP), serum amyloid A, fibrinogen, PAI-1, inflammation-associated cytokines (tumor necrosis factor and IL-6), and von Willebrand Factor, plasma viscosity, and ICAM-1. All of the analyses were carried out with the SAS statistical software package (SAS Institute, Inc.).
Results
In total, 784 subjects were enrolled in the study (312 patients with stable CHD and 472 age- and gender-matched controls) and had chemokine values measured. The main characteristics of the study population have been described in a previous article. 13 In brief, CHD patients more often had a lower school education compared with control subjects, and established cardiovascular risk factors, such as smoking, a high body mass index, a history of high-blood pressure, and a history of diabetes were more unfavorably distributed in patients compared with controls.
Table 1 shows age- and gender-adjusted geometric mean serum concentrations of the various chemokines in CHD patients and controls. IP-10 values (531.4 pg/mL versus 380.1 pg/mL; P <0.0001) and IL-8 values (17.1 pg/mL versus 13.9 pg/mL; P =0.0004) were statistically significantly higher, and RANTES values were lower (44.0 ng/mL versus 48.2 ng/mL; P =0.016) in CHD cases compared with control subjects. There were no statistically significant differences for MCP-1, MIP-1, or eotaxin.
TABLE 1. Age- and Gender-Adjusted Mean Serum Concentrations * (With Bottom and Top Tertile Cut Points) of IP-10, IL-8, RANTES, MCP-1, MIP-1, and Eotaxin in CHD Cases and Controls
The independent association between high concentrations of chemokines and CHD was then quantified by means of unconditional logistic regression analysis (see Table 2 ). Chemokine values in the second and third tertile of IP-10 and IL-8 were associated with an increased odds ratio (OR) for CHD when compared with the bottom tertile. This increased risk was somewhat stronger in the partially adjusted model (adjusted for age and gender) as compared with the full adjustment. The OR for IP-10 was 2.62 (95% CI, 1.79 to 3.85) in the age- and gender-adjusted model and 1.93 (95% CI, 1.23 to 3.04) in the fully adjusted model when subjects in the upper tertile were compared with these in the bottom tertile; the respective figures for IL-8 were ORs at 1.77 (95% CI, 1.20 to 2.59) and 1.53 (95% CI 0.98 to 2.39). In contrast, the ORs for RANTES were lower if in the second or third tertile, both in the partly and fully adjusted models [OR, 0.67 (95% CI, 0.47 to 0.96) and 0.61 (95% CI, 0.40 to 0.94)]. No statistically significant increase in the OR for CHD was seen for increased concentrations of MIP-1, MCP-1, or eotaxin. By contrast, MCP-1 was inversely associated with the OR for CHD after full adjustment for covariates.
TABLE 2. Association of Single Chemokines with Risk of Stable CHD
We then calculated mean concentrations of the CHD-associated chemokines according to levels of various cardiovascular risk factors in control subjects only (see Table 3 ). There was no association with age within the investigated age range in this 40-to-68-year-old population. However, there were differences with respect to gender (for IP-10 and IL-8), alcohol consumption (for IL-8), smoking status (for IP-10), for physical activity (IL-8), and for history of diabetes and hyperlipidemia (RANTES).
TABLE 3. Mean Concentrations of Various Chemokines in Serum According to Risk Factors of CHD and Other Sociodemographic Variables (in Control Subjects Only)
Finally, we analyzed the correlation between the chemokines and established laboratory risk markers of CHD (see Table 4 ). We found positive correlations of IP-10 and IL-8 with several acute-phase proteins or inflammation-associated cytokines; for IP-10, a very strong correlation with plasma viscosity and ICAM-1 was also seen. For RANTES, only a statistically significant association with viscosity was seen. No statistically significant associations were seen for MCP-1, MIP-1 (except with viscosity r =0.09; P =0.01), or eotaxin.
TABLE 4. Spearman Rank Correlations Between Various Chemokines and Established Laboratory Risk Markers of CHD
Discussion
In this large case-control study including patients with angiographically defined stable CHD, we observed a clear association between increased serum levels of IP-10 and IL-8 and decreased serum levels of RANTES and the risk of CHD, which persisted after the adjustment for conventional CHD risk factors, including a history of diabetes. In addition, we could also demonstrate that IP-10 and IL-8 correlated with several acute-phase proteins or inflammation-associated cytokines, which are central in the pathogenesis of atherosclerosis; for IP-10, such correlations were also seen with plasma viscosity and ICAM-1.
More than 40 chemokines have been identified within the past few years, 6 and their ability to attract and activate leukocytes in various tissues has prompted many researchers to investigate the role of several chemokines in various stages of atherogenesis. 6-8 Furthermore, many cell types, such as endothelial cells and smooth muscle cells, also express chemokine receptors, 14 and, therefore, chemokines may, beside determining cell trafficking, also be involved in other aspect of tissue homeostasis.
IL-8 is a glycoprotein with well-established proatherogenic activity within vessel lesions. 15 It has been found to increase the endothelial adhesiveness for monocytes, 16 and it is a mitogen and chemoattractant for vascular smooth muscle cells. 17 In one previous study, high-serum levels have been found in Chinese subjects with unstable angina pectoris and acute myocardial infarction. 18 We report here elevated serum levels of IL-8 also in Whites with stable CHD. There was a positive correlation of IL-8 levels with proinflammatory cytokines and PAI-1 activity, which may link the activity of IL-8 to other laboratory markers of CHD risk.
In addition, IL-8, IP-10, and MCP-1 have also been linked to lesions in animal models of atherosclerosis, 19 and activated T cells seem to be present throughout all stages of atherosclerotic lesion development. 20 Within the atherosclerotic plaque, macrophages, endothelial cells, and smooth muscle cells express IP-10 among others substances. 21 IP-10 and IL-8 stimulate cell proliferation, cell migration, and the inflammatory response of human smooth muscle cells. 22 IP-10 augments INF- production from T-helper 1 cells and, thus, may foster the inflammatory response within the vessel wall. IP-10 has also been described to be increased in a small group of patients (n=15) with coronary artery disease compared with other coronary artery disease patients. 23
High-MCP-1 serum values were associated with an increased risk of death or MI during a 10-month follow-up in a large study in patients with acute coronary syndromes. 24 Because MCP-1 recruits monocytes to sites of inflammation and into the infarct zone, 14 it may mainly play a role in the early stages of atherosclerosis or in acute events; because we excluded all of the patients with acute events, this may explain why we did not find increased values in patients compared with controls. Rather, we noted a negative association between MCP-1 and CHD after multivariable adjustment. This finding, however, may be spurious. A recent study reported that although there may be a transient increase in circulating chemokine levels after coronary angioplasty, there was no difference in the levels of circulating MCP-1 or eotaxin in subjects with and without atherosclerosis. 25 This contrasting association between IP-10 and IL-8 versus MCP-1 may be because of a different role in immunoregulation. The chemokine MCP-1 preferentially promotes T-helper 2-type reactivity, which prevails in many antibody-mediated diseases. 26,27
IP-10 and IL-8 preferentially contribute to the activation of immune cells involved in destructive cellular immunity, also described as T-helper 1-type immune reactivity. 28,29 Therefore, MCP-1 serves functions that are in part antagonistic to those of IP-10 and IL-8. This does not preclude a disease-promoting role of MCP-1 in other stages of atherosclerosis. However, upregulation of an immune mediator during disease development can be because of an agonistic or an antagonist function.
RANTES may play a role in the involvement of platelets in native lesion formation, 8 and this may be the reason why we did not find increased levels in CHD patients when compared with controls. It may be of special importance in the early stages of atherosclerosis, and the deposition of RANTES has been shown to trigger enhanced recruitment of monocytes. 30
Systemically measurable markers of low-grade inflammation are important predictors of CHD risk 4 and are increased in patients with stable CHD. 11 A large number of acute-phase reactants have been found to be consistently associated with CHD risk, 31 indicating an important role in the pathogenesis of CHD, although the underlying mechanisms are still unclear. In particular, CRP has been shown to be an important predictor of CHD in various clinical and epidemiological studies. 32,33 CRP is an acute-phase reactant produced in response to IL-6 stimulation. 34 Among other effects, CRP induces the expression of ICAM-1, an adhesion molecule that regulates attachment and transmigration of leukocytes across the vascular endothelium, 35 an important early step in the pathogenesis of atherosclerosis. The correlation of IP-10 and IL-8 with most of these established CHD risk markers now implies that, in particular, these chemokines might represent the link to the underlying inflammatory response or vascular cell changes and may have a direct role in atherogenesis. In this context, it should be noted that despite a general upregulation of acute phase proteins, only a minority of chemokines analyzed were found to be elevated in CHD. This suggests that the immune activation seen does not represent a nonspecific inflammatory response but may exhibit a specific quality.
The present study has several limitations, which should be addressed. CHD was defined invasively by coronary angiography in cases, but, for ethical reasons, no coronary angiogram could be obtained in controls. Although we excluded controls with a history or characteristic symptoms of CHD, the presence of asymptomatic CHD cannot be definitely ruled out; however, the prevalence of asymptomatic CHD cases seems to be rather low in a middle-aged population. Furthermore, the choice of blood donors can be considered as suboptimal, because they might be healthier than the target population that the cases were drawn from. We tried to minimize this potential bias by carrying out multivariate adjustments for a variety of covariates. However, as the observed association decreased from the partially to the fully adjusted model (especially when looking on IP-10 and IL-8), this might be an indication that these chemokines could represent a possible intermediate step in the pathway between the included risk factors (eg, factors related to the metabolic syndrome) and the risk of CHD. Furthermore, as always in case-control studies in which exposure and outcome are collected at one point in time, it is difficult to assess the time sequence of the described associations, and, therefore, it is highly desirable to replicate our results in prospective studies.
In summary, despite these limitations, the current study provides the first evidence that there is no universal upregulation of chemokines in CHD-associated inflammation but differential upregulation of IP-10 and IL-8 versus downregulation of RANTES. There was no clear disease association for MCP-1, MIP-1, or eotaxin.
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作者单位：Department of Epidemiology (D.R.), The German Centre for Research on Ageing, University of Heidelberg; German Diabetes Clinic (S.M.-S., C.H., H.K.), German Diabetes Center at Heinrich Heine University Duesseldorf; and the Department of Internal Medicine II-Cardiology (W.K.), University of Ulm Medica