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J Am Coll Cardiol, 2002; 40:34-42 © 2002 by the American College of Cardiology Foundation |
T polymorphism, plasma levels of the soluble endotoxin receptor CD14, their association with chronic infections and risk of stable coronary artery disease
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* Department of Internal Medicine II-Cardiology, University of Ulm Medical Center, Ulm, Germany
Department of Clinical Chemistry, University of Freiburg, Freiburg, Germany
Department of Epidemiology, University of Ulm, Ulm, Germany
Department of Epidemiology, the German Centre for Research on Ageing, University of Heidelberg, Heidelberg, Germany
Manuscript received January 16, 2002; revised manuscript received March 14, 2002, accepted March 27, 2002.
* Reprint requests and correspondence: Dr. Dietrich Rothenbacher, Department of Epidemiology, German Centre for Research on Ageing, Bergheimerstr. 20, D-69115 Heidelberg, Germany.
rothenbacher{at}dzfa.uni-heidelberg.de
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Abstract |
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BACKGROUND: It has been suggested that genetic variation of the CD14 receptor with increased CD14 gene expression might play a role in atherogenesis. A mechanistic link would consist in its contribution to the inflammatory response seen in this disease.
METHODS: We measured levels of sCD14 (µg/ml; ELISA) in 312 patients with angiographically proven CAD and stable angina pectoris, and in 477 age- and gender-matched healthy blood donors. CD14 genotype was determined by polymerase chain reaction. In addition, seropositivity to Chlamydia pneumoniae and Helicobacter pylori, a complete lipid profile and various sensitive systemic markers of inflammation were measured.
RESULTS: CD14 C(-260)
T genotype was not independently associated with increased risk of CAD after multivariable adjustments (odds ratio [OR] 1.34; 95% confidence interval [CI] 0.84 to 2.16). However, sCD14 plasma levels were higher in subjects with TT genotype compared with those with CT or CC genotype (p = 0.005). Plasma levels were not different between cases and controls (4.2 ± 1.3 µg/ml vs. 4.3 ± 1.3 µg/ml, p = NS). In multivariable logistic regression, the OR for the presence of CAD was 1.11 (95% CI, 0.65 to 1.91) if the top quintile of the sCD14 distribution was compared with the bottom quintile. There was no consistent association between seropositivity to either C. pneumoniae or H. pylori, or both, and sCD14 levels and between sCD14 levels or CD14 genotype and the various markers of inflammation.
CONCLUSIONS: These results do not confirm an independent relationship between CD14 genotypes or plasma levels of sCD14 and risk of stable CAD in this population.
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Lipopolysaccharide component of gram-negative bacteria cell wall is considered to be responsible for several pathologic conditions in which these microorganisms are involved. One way by which LPS could initiate the atherosclerotic lesion is by formation of complexes with lipoproteins that are transported from the circulation into the arterial wall and initiate an inflammatory response (11). A further potential mechanism consists in the activation of monocytes/macrophages. Lipopolysaccharide-activated macrophages produce proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6 (12–14) and growth factors (15). Lipopolysaccharide induces expression of adhesion molecules in endothelial cells (ECs) (16,17) and also stimulates smooth muscle cell (SMC) proliferation and migration by release of platelet-derived growth factor from monocytes (18). Moreover, it promotes procoagulant activity by induction of tissue factor expression in monocytes and ECs (19,20). In addition, LPS can induce oxidative modification of low-density lipoprotein (LDL), a major mediator in atherogenesis (11); it increases the levels of LDL and very low-density lipoprotein and decreases high-density lipoprotein (HDL) levels (21). Lipopolysaccharide is a potent stimulus for inducible nitric oxide (NO) synthase activity, leading to the formation of large amounts of NO, which can cause endothelial dysfunction and disruption (22).
Lipopolysaccharide is internalized by cells through the CD14 receptor (23,24). Recent data indicate that HSP60 also activates mononuclear cells and monocyte-derivated macrophages through CD14 signalling, sharing this pathway with bacterial LPS (25). CD14 receptor is expressed in considerable amounts by mature monocytes, macrophages and activated neutrophil granulocytes. In these cells, CD14 is anchored in the cell membrane (membrane-bound CD14 [mCD14]). In addition, the soluble form (sCD14) can be found in plasma in response to LPS stimulation, where two major isoforms coexist (26). Lipopolysaccharide binds to the membrane-bound CD14 on monocytes and macrophages and activates these cells. Soluble CD14 plays an important role in the LPS-mediated activation of cells lacking membrane-bound CD14, so-called "CD14-negative cells" (endothelial, epithelial and SMCs). In addition, these CD14-negative cells are activated indirectly by cytokines from LPS-stimulated monocytes (27). But sCD14 can also activate monocytes independently of LPS (28). Thus, both mCD14 and sCD14 are competing for LPS (29) and are able to bind it (30). Changes in CD14 expression and plasma sCD14 levels seem to be associated with an increasing number of disorders like septicemia (31,32) or periodontitis (33,34). In these cases, increased levels of sCD14 may be due to chronic exposure to LPS. But expression of mCD14 is also increased in patients with noninsulin-dependent diabetes mellitus, correlates significantly with serum concentrations of C-reactive protein (CRP) (35), and the density of CD14 was found to be increased in patients with acute myocardial infarction (AMI) (36). However, to our knowledge, no data have been published on sCD14 levels in a large group of patients with stable angina pectoris compared with healthy subjects.
Recently, research in this field has focused on the gene of the CD14 receptor. A polymorphism in the CD14 gene has been reported to be associated with increased risk of AMI (37–39). It was also suggested that the C(-260)T change in the promoter region affects the level of CD14 gene expression (37). However, this polymorphism has been found to be associated with AMI in only two small case-control studies (37,39) and in subgroup analysis in another retrospective study (38), but it was not associated with future AMI, in one larger prospective study (40), and with risk of ischemic cerebrovascular disease, in a Japanese population (41).
Thus, because results of several studies are controversial, we sought to determine the effect of the CD14 polymorphism and of plasma levels of sCD14 on coronary artery disease (CAD) risk in a large case-control study. We further wanted to link the CD14 genotype to sCD14 plasma levels in patients with CAD and in healthy controls and test whether seropositivity to Chlamydia pneumoniae, or Helicobacter pylori, or both, is associated with increased sCD14 levels. Finally, we were interested in the relationship between CD14 polymorphism and plasma levels of sCD14, and sensitive systemic markers of inflammation, all reflecting established predictors of coronary risk.
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Methods |
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The case group was referred to the Department of Cardiology at the University of Ulm Medical Center for elective coronary angiography. A total of 312 consecutive patients of German nationality aged 40 to 68 years with clinically stable, angiographically confirmed CAD (>50% luminal diameter stenosis of at least one major coronary artery), diagnosed only within the previous two years to reduce the likelihood of survival bias, were included. Patients with acute ischemic syndromes within the previous four weeks, on anticoagulant therapy, with acute infectious diseases or with evidence of malignant diseases, possibly associated with an acute phase reaction, were excluded.
The control group consisted of 476 subjects who were occasional blood donors at the local Red Cross center serving the University hospitals of Ulm. All controls had no history of definite or suspected CAD and did not report infections or surgery within the previous four weeks. Participation rate was 78% in eligible patients and 84% in eligible controls.
Frequency matching for age and gender was performed, and a case-control ratio of 1:1.5 was intended. All subjects underwent standardized interviews conducted by trained interviewers. Participants were asked about medical history including specific questions related to physician diagnosed hypertension, diabetes and gastroduodenal disease. Furthermore, current medication, sociodemographic characteristics and lifestyle habits were recorded.
Laboratory methods
Venous blood was drawn in the morning under standardized conditions, and a complete blood cell count was done (Coulter STKS chamber, Coulter Co., Krefeld, Germany). Within 30 min, the remaining blood was centrifuged at 3,000 g for 10 min, immediately aliquoted and frozen at –70°C until analysis. Plasma level of sCD14 have been determined by a commercial sandwich-type ELISA (IBL, Hamburg, Germany), using an oligoclonal capture antibody, followed by an enzyme-tagged monoclonal detection antibody. This ELISA measures only free sCD14 and not sCD14 bound in any complex with LPS. The two isoforms described for sCD14 are equally well recognized. Specific anti-H. pylori IgGs were measured by use of a commercial ELISA (H. pylori-IgG-ELISA; Medac, Wedel, Germany). IgG antibodies against Chlamydia were measured by ELISA using recombinant chlamydial LPS (42) (Medac, Wedel, Germany), and a titer 
100 was defined as seropositive.
The following markers of inflammation and hemostasis were also determined by ELISA: IL-6 and TNF-alpha (Quantikine, R & D Systems, Wiesbaden, Germany), intercellular adhesion molecule (ICAM)-1 (Diaclone, Besancon, France), plasminogen-activator inhibitor (PAI)-1 activity (Immuno, Heidelberg, Germany), D-dimer (Dimertest Gold EIA, Agen Biomedical Ltd., Acacia Ridge, Australia) and von Willebrand factor (vWF) (Haemochrom, Essen, Germany). In addition, CRP determinations were done by an immunoradiometric assay (range 0.05 to 10 mg/l) calibrated with the WHO reference standard 85/506 (43). Fibrinogen was measured by immunonephelometry (Dade Behring, Marburg, Germany) and according to the Clauss method. Serum amyloid A (SAA) was also determined by immunonephelometry (Dade Behring, Marburg, Germany), and, finally, measurement of plasma viscosity was done in a Harkness Coulter viscometer (Coulter Electronics, Luton, United Kingdom). Interassay coefficients of variation were 15.7% for sCD14, 7% for IL-6, 17.9% for TNF-alpha, 14.2% for ICAM-1, 12% for CRP, 7.4% for SAA, 5% for fibrinogen, 11% for PAI-1, 7.2% for D-dimer, 15.8% for vWF and 2% for plasma viscosity. Total and HDL cholesterol concentrations were determined by routine enzymatic methods. Lipoprotein (Lp) (a) and apoproteins were determined by immunoturbidimetry on a WAKO R-30 automated analyzer. All laboratory analyses were done in a blinded fashion.
Analysis of the C(-260) T polymorphism of the CD14 gene
Genomic DNA has been isolated from white blood cells by standard method (44). Polymerase chain reaction (PCR) was performed at total volume of 20 µl (100 ng of genomic DNA, 10 pmol of each primer, 250 µmol/l of each dNTPs and 1 U TaqDNA polymerase in the provided reaction buffer (Boehringer Mannheim). The promoter of CD14 receptor gene was amplified by the primers CDP-1, 5"-ATCATCCTTTTCCCACACC-3" and CDP-2, 5"-AACTCTTCGGCTGCCTCT-3" under the following conditions: an initial denaturation at 95°C for 3 min followed by 35 cycles at 95°C for 30 s, 1 min of annealing at 60°C, 30 s of extension at 72°C and a final extension time of 5 min at 72°C. The PCR product was digested 3 h at 37°C with 10 U HaeIII restriction enzyme (New England Biolabs, Beverly, Massachusetts). The DNA fragments were separated by electrophoresis through a 2% NuSieve agarose gel containing 0.5 µg/ml of ethidium bromide and were visualized under UV light.
Angiographic evaluation
Coronary angiography was performed among cases by the Judkins method. Three different scores were used to evaluate the angiographic severity and extension of CAD: the number of stenosed (>50% reduction of luminal diameter) or occluded vessels (one- to three-vessel disease), the quantitative extension score (1 to 15 segments) according to the guidelines of the American Heart Association and the qualitative and quantitative evaluation by the Gensini score (45). Scoring of all coronary angiograms was done visually by a single experienced observer who was blinded to clinical and laboratory data. The intraclass correlation coefficient for intrarater reliability was 1.0 (one- to three-vessel disease score), 0.79 (tertiles of the extension score) and 0.85 (tertiles of the Gensini score).
Statistical methods
Demographic and clinical characteristics were compared in a descriptive way. Differences in the allele and genotype distribution among cases and controls were compared with values predicted by the Hardy-Weinberg equilibrium. Furthermore, we used a linear regression model to calculate the age- and gender-adjusted mean values for sCD14 in patients and controls, and according to CD14 genotypes. The same method was used to assess the relationship of age- and gender-adjusted plasma levels of sCD14 according to serostatus of C. pneumoniae and serostatus of H. pylori, and the combination of both, in patients and controls.
The association of CD14 genotype distribution with presence of CAD was assessed by a chi-square test. Soluble CD14 levels were measured and divided in quintiles according to the distribution in controls. Furthermore, we used unconditional logistic regression to assess the independent association of sCD14 distribution (quintiles) and CD14 genotypes with CAD, while simultaneously controlling for body mass index (BMI), duration of school education, cigarette smoking (pack-years), alcohol consumption, history of hypertension and history of diabetes mellitus and HDL cholesterol values.
Blood, plasma and serum parameters are reported as means (arithmetic or, if skewed in normality plot, geometric). To assess the association of sCD14 (upper quintile) and CD14 genotype with markers of inflammation and hemostasis and lipid parameters, we calculated age- and gender-adjusted mean values for these markers for patients and controls separately and the respective p value for a difference.
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Results |
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Table 2 shows mean values and distribution of sCD14 and the allele and genotype frequencies of the CD14 gene among cases and controls. Cases had, in general, similar mean plasma values of sCD14 (4.21 ± 1.3 µg/ml vs. 4.28 ± 1.3 µg/ml, p = 0.51, after adjustment for age and gender). Allele and genotype frequencies in cases and controls did not show a significant departure from the Hardy-Weinberg equilibrium (p = 0.81 and p = 0.94, respectively). Furthermore, there was no difference among cases and controls with respect to CD14 genotypes (p = 0.74, adjusted for age and gender).
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T polymorphism. The OR for CAD associated with being in the top quintile of the sCD14 distribution compared with the bottom quintile was 1.06 (95% confidence interval [CI], 0.66 to 1.70) after adjustment for age and gender and 1.11 (95% CI, 0.65 to 1.91) after adjustment for other covariates. It increased only slightly after additional adjustment for HDL (OR, 1.14; 95% CI, 0.65 to 2.01).
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The OR for CAD associated with bearing the TT genotype was 1.17 (95% CI, 0.77 to 1.78) after adjustment for age and gender and increased to 1.34 (95% CI, 0.84 to 2.16) after adjustment for other covariates. Again, additional adjustment for HDL did not change the estimate appreciably (OR, 1.35; 95% CI, 0.82 to 2.23).
In 305 of 312 patients with angiographically determined CAD, the severity of CAD was evaluated by three different scores. No association between either sCD14 plasma levels (top quintile vs. combined lower four quintiles) or CD14 genotypes and any of the three scores applied was found (data not shown).
Plasma concentrations of CRP, fibrinogen, D-dimer, plasma viscosity, leukocyte count, PAI-1, Lp(a), SAA, vWF, IL-6, TNF-alpha and ICAM-1 were, in general, higher in patients compared with controls; however, there was no clear and consistent difference with regard to the sCD14 distribution (Table 6), taking into account the large number of variables tested, and the CD14 C(-260)T polymorphism (Table 7), neither in patients nor in controls. The only exception was CRP, which was slightly elevated in subjects being in the top quintile of the sCD14 distribution compared with the combined four lower quintiles, in patients and in controls, after adjustment for age and gender.
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Discussion |
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T gene polymorphism with angiographically confirmed CAD. In addition, there was no consistent association of sCD14 with seropositivity to C. pneumoniae, H. pylori and a combination of both. Furthermore, sCD14 plasma levels, or CD14 C(-260)T gene polymorphism, were not related with a variety of sensitive systemic markers of inflammation and hemostasis or lipid parameters. Thus, these data are in contrast to several reports in the literature and do not suggest an important role for CD14 C(-260)T gene polymorphism or sCD14 plasma levels as risk markers for stable CAD.
Association between CD14 genotype and CAD.
Three recent retrospective studies have reported a positive association between CD14 C(-260) T gene polymorphism and risk of AMI or CAD (37–39), whereas our study and one large prospective study (40) found no association. Several reasons may be responsible for these discrepant findings. First, in the study by Unkelbach et al. (38), the positive association reported was confined to post-hoc analysis of small low-risk subgroups, whereas in the total group of patients no such association was seen. Second, in the study by Hubacek et al. (37), the control group was rather small (n = 135), and the frequency of the TT genotype was considerably lower compared with our study and three others (38–41). Furthermore, surprisingly, conventional risk factors were not different between cases and controls in this study. Third, numbers in the study by Shimada et al. (39) carried out in a Japanese population were also small (83 controls and 128 patients with angiographically documented CAD), and a positive association was only seen in those with a recent AMI (n = 81) but not in those with stable CAD (n = 47). The TT genotype was more than twice as frequent in this ethnically different group compared with all other reports in Caucasians. In contrast with these at least partly positive studies, in our large, carefully controlled case-control study, no strong independent association between CD14 C(-260)T gene polymorphism and risk of CAD could be demonstrated in multivariable analysis, although a weak relationship cannot be ruled out. Thus, our data are in accordance with the only so far published prospective study by Zee et al. (40) who also found no evidence of a significant association between CD14 allele and relative risk of future AMI.
Association between sCD14 plasma levels and CAD
Mean levels of the monocyte-specific activation marker sCD14 in cases and controls were not different in our study. As a consequence, the OR for the presence of CAD in the top quintile of the sCD14 distribution was not significantly increased in multivariable analysis, compared with the bottom quintile. Only one study so far has reported on the potential association between plasma levels of sCD14 and CAD. In this relatively small study (46), sCD14 levels in patients with stable CAD were not different from controls but were 41% higher in plasma from patients with unstable angina, suggesting increased monocyte activation. Thus, although in both studies no association was found between sCD14 plasma levels and chronic, stable CAD, there may still be an important relationship with unstable or acute disease.
Association between CD14 genotype and sCD14 plasma levels
We found slightly higher plasma levels of sCD14 in the TT genotype, compared with CT or CC genotype in patients (p = 0.03), and a tendency to elevated values was seen in controls (p = 0.11). Although no such data are available from other studies in patients with CAD, similar results have been reported in patients with ischemic cerebrovascular disease with plasma levels of the same magnitude (43). Such small differences (0.4 to 0.5 µg/ml), however, are unlikely to be clinically relevant.
Association between CD14 genotype, sCD14 plasma levels and sensitive systemic markers of inflammation
Assuming that genetic variation of the CD14 receptor with increased CD14 gene expression might play a role in atherogenesis, a mechanistic link would consist in its contribution to the inflammatory response seen in this disease (1). Such effect could result in either increased levels of sCD14 receptor in plasma but might also be demonstrated by increased levels of sensitive systemic markers of inflammation, which have been shown to be independently related to coronary disease in prospective studies. We have measured a large variety of such markers and were unable to detect any meaningful relationship with CD14 genotype. Most of these markers are known to increase after stimulation of various cell types, including macrophages, SMCs or ECs by LPS. Furthermore, with the exception of CRP, no other inflammatory marker was associated with plasma levels of sCD14 (top quintile vs. combined lower four quintiles). Taking into account the large number of markers tested, such finding may have occurred by chance alone.
Strengths of this study
We carefully selected cases and controls and excluded patients with acute coronary syndromes in order to eliminate the possibility of an elevation of the measured parameters as a result of an acute phase response. In addition, we carefully recorded the conventional risk factors and investigated their relationship to sCD14 and CD14 C(-260)T genotype in order to assess their potential for confounding and adjusted for them in the final analysis by means of multivariate methods. In contrast with other studies, we measured a large variety of sensitive systemic markers of inflammation to evaluate a potential contribution of the genetic variation of the LPS receptor or sCD14 plasma levels to the inflammatory response known to represent an integral part of the atherosclerotic process.
Study limitations
Several potential limitations of our study have to be considered. We used a case-control design; therefore, the temporal relationship of the serum parameters and disease is difficult to establish. Selection and survival bias may represent another problem. However, because the hospital of the University of Ulm Medical Center is the only hospital serving the city of Ulm and, besides being a tertiary care center, it also serves as a primary care center. Furthermore, the study comprised all consecutive patients referred for coronary angiography within a defined time period. To limit selective survival we restricted the case group to those with a diagnosis of CAD within less than two years. Although controls came from the same geographic region as patients, they were probably healthier than population-based controls. Furthermore, asymptomatic CAD in these subjects cannot be ruled out completely because no electrocardiogram or coronary angiogram could be obtained in controls. However, the prevalence of CAD in an asymptomatic middle-age population appears to be low (47), and selection of controls among subjects who undergo coronary angiography for various reasons possibly introduces an even more severe bias. This, however, cannot explain the lack of a significant association that emerged after control for confounding factors. Furthermore, although this study did not have the power to detect a weak association between sCD14 levels, or the CD14 C(–260)T polymorphism and CAD, it had a power of 80% to detect an OR of 1.63 (associated with being in the upper quintile of sCD14) and of 1.59 (associated with the TT genotype), respectively, at 
= 0.05.
Conclusions
Our results do not confirm an independent and clinically relevant relationship of CD14 gene polymorphism with risk of CAD. The lack of an association between sCD14 plasma levels and risk of stable CAD or markers of inflammation and between CD14 genotype and the inflammatory response constitute novel findings that contribute to the ongoing discussion on inflammatory pathways in atherosclerosis. Therefore, it seems unlikely that, in the population studied, sCD14 plasma levels or CD14 C(-260)T genotype are promising tools for risk assessment of stable CAD.
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Acknowledgments |
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Footnotes |
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References |
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and matrix metalloproteinase expression. Circulation. 1998;98:300–307T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation. 1999;99:3218–3220T gene polymorphism and the risk of myocardial infarction. Atherosclerosis. 2001;154:699–702[CrossRef][Medline]
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