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Defining the Proinflammatory Phenotype Using High Sensitive C-Reactive Protein Levels as the Biomarker

Laboratory for Atherosclerosis and Metabolic Research, University of California Davis Medical Center (I.J., S.D.), Sacramento, California 95817; and Department of Surgery, University of Washington (G.O.), Seattle, Washington 98104

Address all correspondence and requests for reprints to: Dr. Ishwarlal Jialal, Laboratory for Atherosclerosis and Metabolic Research, University of California Davis Medical Center, 4635 2nd Avenue, Sacramento, California 95817. E-mail: ishwarlal.jialal{at}ucdmc.ucdavis.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Inflammation is pivotal in atherosclerosis. The prototypic marker of inflammation is C-reactive protein (CRP). Numerous studies have confirmed that high CRP levels in normal volunteers predict cardiovascular events.

Objective: The objective of this study was to define proximal and associated abnormalities of the proinflammatory phenotype using CRP levels as the biomarker.

Design and Subjects: Two groups of normal, healthy subjects, selected by stringent criteria from an initial cohort of 252, were studied over the period of 12 months. Group 1 included subjects with consistently low CRP (<0.004 µM or <0.5 mg/liter; low CRP group; n = 15). Group 2 included subjects with consistently high CRP (>2.0 or >0.016 µM to <10 mg/liter or <0.085 µM; high CRP group; n = 13).

Main Outcome Measures: Fasting blood (50 ml) was obtained, and the following parameters were assayed: high sensitivity CRP, fibrinogen, lipid profile, insulin, whole blood cytokines after stimulation with lipopolysaccharide (LPS; 100 ng/ml for 24 h), soluble cell adhesion molecules, plasminogen activator inhibitor-1, CD40, CD40 ligand, leptin, adiponectin, monocyte chemoattractant protein-1, IL-8, matrix metalloproteinase-3 (MMP-3), and MMP-9. Genomic DNA was obtained from peripheral blood leukocytes, and the TNF- –308 genotype was determined.

Results: The median CRP levels were 0.0018 µM (0.21 mg/liter) and 0.031 µM (3.7 mg/liter) for the low and high groups, respectively. High CRP subjects were older and had significantly higher body mass indexes, triglycerides, insulin, homeostasis model assessment, and leptin levels compared with low CRP subjects. The markers of inflammation, plasminogen activator inhibitor-1, MMP-9, fibrinogen, and vascular cell adhesion molecule-1 levels were significantly higher in the high compared with the low CRP group. LPS-stimulated levels of whole blood IL-1ß, IL-6, and TNF were significantly higher, and IL-4 levels were significantly lower in the high CRP group. After age- and body mass index-adjusted analysis of covariance, only plasma MMP-9 levels and LPS-stimulated whole blood IL-1ß and TNF levels were significantly higher in the high CRP group. The frequency of the rare A allele at TNF- –308 was equivalent in high and low CRP groups.

Conclusions: A phenotype characterized by increased plasma inflammatory mediators as well as increased LPS-stimulated whole blood TNF- and IL-1ß levels is associated with high plasma CRP levels. This systemic inflammatory phenotype may contribute to vascular inflammation or may reflect inflammation in vessels or at other sites.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
INFLAMMATION IS PIVOTAL in all phases of atherosclerosis, from the initiation of the fatty streak to the culmination of acute coronary syndromes (1, 2). An important downstream marker of inflammation and tissue injury is C-reactive protein (CRP) (3, 4, 5, 6, 7). CRP is a member of the pentraxin family, and it is comprised of five noncovalent promoters arranged symmetrically around a central core (8). In humans, it is a nonglycosylated protein, and the gene has been mapped to chromosome 1. Current concepts suggest that CRP secretion is largely under the transcription control of IL-6; however, IL-1 and TNF also contribute to the synthesis of CRP (9, 10). Because the half-life of CRP is approximately 19 h in both health and disease, it is a very stable downstream marker of the inflammatory process (3, 4, 5, 6, 7, 8).

Numerous studies from various parts of the world have clearly established that CRP predicts future risks for cardiovascular disease in apparently healthy persons independently of established major risk factors in the majority of the studies (3, 4, 5, 6, 7). To date, CRP has been shown to predict myocardial infarction, coronary artery disease (CAD), death, stroke, peripheral arterial disease, and sudden death. Furthermore, in the Women’s Health Study, CRP was shown to be additive to low-density lipoprotein cholesterol, the total cholesterol/high-density lipoprotein cholesterol ratio, and the Framingham 10-yr risk score in predicting future cardiovascular events. Also in this population, it was shown to predict future events in patients with the metabolic syndrome. Thus, given the importance of CRP as a risk marker, the aim of this study was to define proximal and associated abnormalities using CRP as the biomarker. To this end, normal individuals were entered into the study after stringent exclusion criteria to better appreciate the proximal and associated abnormalities that constitute the proinflammatory phenotype. The major impetus of this study was to define additional novel candidate genes that could also be used in future studies in assessing cardiovascular disease risk.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
All volunteers gave informed consent to participate in this study. Of 252 volunteers screened over the course of 1 yr, 46 subjects met our stringent selection criteria. Of these, subjects who did not remain consistently low (one of three CRP measurements >0.004 µM or >0.5mg/liter; n = 7) or consistently high (one of three CRP measurements >0.085 µM or >10 mg/liter or <0.0169 µM or <2 mg/liter; n = 11) were excluded. Two groups of apparently normal, healthy subjects were studied over a period of 12 months. Group 1 included subjects who were followed up for 1 yr, consistently had levels of CRP below 0.004 µM (on three separate occasions over the 1-yr period), and were clinically free of disease and/or infection or trauma at the time of sample collection (low CRP group; n = 15). Group 2 included subjects who were also followed up for 1 yr, consistently had levels of CRP between 0.0169–0.085 µM (2.1–9.9 mg/liter on three separate occasions), were clinically free of disease and/or infection or trauma at the time of sample collection, and were in a steady state (high CRP group; n = 13). The two levels of CRP were chosen in accordance with several prospective studies (3, 4, 5, 6, 7, 8) that showed that levels of high sensitivity CRP (hsCRP) less than 0.004 µM (0.5 mg/liter) are associated with a low risk of CAD, whereas levels of 0.0169 µM (2 mg/liter) or greater are associated with a high risk for CAD. Because CRP levels greater than 0.085 µM (10 mg/liter) are suggestive of macroinflammation, this was chosen as the upper limit in the high CRP group. Other selection criteria for the subjects were as follows: nonhypertensive; nonsmokers; nondiabetics; no evidence of impaired fasting glucose (6.1–6.9 mM); no high intensity exercisers; not taking antioxidant or vitamin supplementation; no chronic disease or gastrointestinal problems; no recent infection, trauma, or surgery; not pregnant or lactating; no bleeding diathesis; normal complete blood count; normal renal and liver function; alcohol intake less than 1 ounce/d; and not taking hypolipidemic drugs, thyroid drugs, nonsteroidal antiinflammatory drugs, oral contraceptives, or anticoagulants. Postmenopausal women were excluded because estrogen replacement therapy elevates hsCRP levels.

After obtaining informed consent, personal and family medical histories were obtained from both groups of volunteers by questionnaires at screening. Blood pressure was measured as the average of three seated readings using an automated oscillometric device. Weight was recorded using scales, and height was measured using a portable stadiometer. Waist circumference was also obtained at the time of the blood sampling.

Fasting blood (50 ml) was obtained from volunteers, and the following parameters were assessed: hsCRP, fibrinogen, lipid profile, insulin, whole blood cytokines, soluble cell adhesion molecules, plasminogen activator inhibitor-1 (PAI-1), CD40, CD40 ligand, adipocytokines, (leptin and adiponectin), chemokines [monocyte chemoattractant protein-1 (MCP-1) and IL-8], matrix metalloproteinase-3 (MMP-3), and MMP-9. Levels of MMP-3, MMP-9, IL-8, MCP-1, and soluble CD40 were assayed by sandwich ELISA (11). The hsCRP levels were quantified by a latex-enhanced immunonephelometric assay (12). Fibrinogen was assayed by immunonephelometry. Insulin, leptin, and adiponectin levels were assessed by RIA using reagents from Linco Research, Inc. (St. Charles, MO). Soluble TNF receptor and IL-1 receptor antagonist were measured by ELISA. Whole blood was activated with lipopolysaccharide (LPS; 100 ng/ml for 24 h). The supernatant was collected and stored at –70 C for analyses of proinflammatory (IL-1ß, TNF-, and IL-6) and antiinflammatory (IL-4 and IL-10) cytokines by sandwich ELISA (13). All ELISAs had intraassay coefficients of variation less than 5%, and RIAs had intraassay coefficients of variation less than 9%. All ELISAs were performed using a single lot of reagent to minimize variability.

Genomic DNA was extracted from buffy coat cells using the QIAamp DNA Blood Midi Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions, and DNA was stored at –20 C until amplified. Fragments containing the GA single nucleotide polymorphism (SNP) at the –308 position in the TNF- promoter were amplified from genomic DNA by PCR using Taq DNA polymerase (Roche, Indianapolis, IN). All amplifications were performed in a PTC 200 thermal cycler (MJ Research, Watertown, MA) using previously described thermal profile, reaction conditions, and primer sequences (available on request from the author) (14).

Genotypes were determined by Pyrosequence analysis on a PSQ 96 Pyrosequencer (Pyrosequencing AB, Westborough, MA) and PSQ 96 SNP software (version 1.2 AQ). Each SNP was assayed with a specific primer sequence [forward (biotinylated), 5'-AGG CAA TAG GTT TTG AGG GCC AT-3'; reverse, 5'-TCC TCC CTG CTC CGA TTC CG-3'; Pyrosequencing primer (reverse), 5'-GGC TGA ACC CCG TCC-3'], which enabled the scoring of heterozygotes and alternate homozygotes. All genotypes were confirmed by repeat pyrosequencing. We previously determined pyrosequencing to be accurate in comparison with dye terminator sequencing for this and other SNPs (14).

Statistics

Statistical analyses were performed using SAS (SAS Institute, Cary, NC) by the General Clinical Research Center biostatistician. Parametric data were compared using paired t tests, and nonparametric data were compared using Wilcoxon signed-rank tests. Variables were examined for their effects as risk factors and confounders and were included in the final model. The significance value was set at P < 0.05. Multiplicity of testing was considered, but was thought to be overly conservative; hence, exact P values are provided. Covariates included age and body mass index (BMI). Log transformations were performed for skewed variables such as CRP and cytokines. Allele gene frequencies were estimated by gene counting. Categorical data were presented as numbers and percentages. Continuous data were presented as medians and interquartile ranges (25th to 75th percentiles). Categorical data were compared by ² analysis, and continuous data were compared by Mann-Whitney U test.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Over a 1-yr period, on a random basis, 28 individuals entered the study. To be entered into the low or high CRP group, in addition to the stringent exclusion criteria, the individuals had to have three CRP measurements performed over a 1-yr period that fell within the same range, i.e. less than 0.04 µM (0.5 mg/liter) or between 0.0169–0.085 µM (2–9.9 mg/liter). Table 1 shows the baseline characteristics of the study volunteers and the metabolic profile. Compared with the low CRP group, the high CRP group individuals were older and had significantly increased BMI and waist circumference. There were no differences between the two groups with respect to blood pressure, lipid profile, and plasma glucose. However, both insulin levels and a measure of insulin resistance, the homeostasis model assessment index, were significantly increased in the high CRP group. Two adipokines, adiponectin and leptin, were also measured in this study. Although adiponectin levels were not different between the two groups, leptin levels were significantly higher in the individuals with high CRP. In addition, there were no significant differences in aspartate aminotransferase, alanine aminotransferase, urate, and creatinine between the two groups. Furthermore, subjects in the two groups did not differ in their exercise level, alcohol intake, or vegetable/fruit intake. Using age and BMI as covariates, none of the above differences persisted. Table 2 illustrates the proximal and associated biomarkers that were obtained in plasma/serum. As shown in Table 2, there was no difference in the chemokines MCP-1 and IL-8 between the two groups. However, levels of E-selectin, vascular cell adhesion molecule-1, and PAI were significantly increased in the high CRP group. Furthermore, levels of MMP-9 were significantly increased, whereas MMP-3 and myeloperoxidase levels were not different. Both soluble CD40 and CD40 ligand levels were not different. Fibrinogen levels were significantly higher in the high CRP group compared with the low CRP group. Although plasma levels of soluble TNF receptors I and II were not different in the two groups, IL-1 receptor antagonist levels were significantly lower in the high CRP group. Using age and BMI as covariants, only the increase in MMP-9 in the high CRP group continued to be significant. Table 3 shows the cytokines assayed after LPS activation of whole blood. There were significant increases in IL-1 ß, IL-6, and TNF-, and there was a significant decrease in IL-4. After adjustments for age and BMI as covariates, the only abnormalities that were still significant included higher levels of IL-1ß and TNF- in the high CRP group.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Several lines of evidence point to the critical role of inflammation in all stages of atherosclerosis (1, 2). The prototypic marker of inflammation is CRP. Numerous prospective studies in different populations have shown that high levels of CRP predict future cardiovascular events, even in apparently healthy individuals (3, 4, 5, 6, 7, 8, 20). This has led to the recent position statement by the American Heart Association-Centers for Disease Control, recommending cutoff levels of CRP less than 0.0085, 0.0085–0.025, and more than 0.025 µM (<1.0, 1.0–3.0, and >3.0 mg/liter) for low, average, and high risks for subsequent cardiovascular events (21).

In addition to it being a risk marker, recent data from several laboratories strongly suggest that CRP is proatherogenic and prothrombotic (4). The proinflammatory, proatherogenic effects of CRP that have been documented in endothelial cells include the following: decreased endothelial nitric oxide synthase and prostacyclin and increased endothelin-1, cell adhesion molecules, MCP-1, IL-8, and PAI-1. In monocyte-macrophages, CRP induces tissue factor secretion, increases reactive oxygen species and proinflammatory cytokine release, promotes monocyte chemotaxis and adhesion, and increases uptake of oxidized low density lipoprotein. Also, CRP has been shown in vascular smooth muscle cells to increase inducible nitric oxide production, increase nuclear factor-B and MAPK activities, and, most importantly, up-regulate angiotensin type 1 receptor, resulting in increased reactive oxygen species and vascular smooth muscle cell proliferation.

Thus, given the importance of CRP as a risk marker/mediator, we have attempted to define proximal and associated abnormalities using CRP as the biomarker. In this study we show that after age and BMI adjustment, the most significant abnormalities in biomarkers of inflammation that cluster with high CRP levels include increased leukocytic IL-1ß, TNF-, and serum MMP-9.

CRP is a stable biomarker of inflammation. The appeal of CRP as a potential clinical tool is increased by the widespread availability of standardized, high-sensitivity assays and its relatively long half-life of 19 h. Furthermore, CRP does not appear to have significant circadian variation, and plasma levels are relatively stable over time. For instance, the correlation between two measurements taken 5 yr apart in the Cholesterol and Recurrent Events Trial (52) trial was 0.6, a value similar to that observed for lipid parameters. Although hsCRP is an acute phase reactant and, as such, is subject to marked and rapid shifts secondary to intercurrent illness, Ockene et al. (22) demonstrated that there is considerable stability in the measurement of hsCRP, and this compares favorably with that of total serum cholesterol. Furthermore, Yusuf et al. in the INTERHEART study (23) reported the effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries. In the present study we also accounted for these variables, because we did not include smokers, diabetics, or hypertensives, and subjects did not report any differences in exercise levels, alcohol intake, or vegetable/fruit consumption in the two groups studied. The analytic component of hsCRP variability is relatively small, with between-subject variation accounting for most of the observed variability. With regard to risk assessment, if the value on two occasions 1 month apart is in the same category, i.e. less than 0.0085, 0.0085–0.025, or more than 0.025–0.085 µM (<1, 1–3, or 3–10 mg/liter), this can be taken as reliable evidence with regard to low, average, and high risks for subsequent cardiovascular disease. However, if the CRP level is greater than 0.085 µM (10 mg/liter), then CRP cannot be used to assess cardiovascular risk and other active inflammatory processes (e.g. trauma, infection, etc.) should be excluded. In the present study, subjects were followed up for 1 yr, and CRP levels were measured on three occasions and were always less than 0.04 µM or less than 0.5 mg/liter (low CRP group) or between 0.0169–0.085 µM or 2–10 mg/liter (high CRP group). Having defined our populations using CRP as a biomarker, we set out to define proximal and associated abnormalities of the proinflammatory phenotype.

The two groups of subjects differed in BMI and age, and although BMI is strongly associated with high CRP levels, some studies have suggested a relationship of CRP with age (24, 25, 26, 27). Thus, although there was no significant correlation between age and CRP levels in our population (r = 0.28; P = 0.15), age and BMI were used as covariates in the statistical analyses. Furthermore, leptin levels were significantly increased in subjects with high CRP. Previously, leptin levels have been shown to be independently associated with CRP even after adjustment for age, gender, BMI, waist to hip ratio, smoking, and alcohol consumption (28). However, the association of leptin with CRP failed to persist after adjustment for BMI in our unique study population.

With regard to biomarkers of inflammation in plasma, vascular cell adhesion molecule-1, E-selectin, PAI-1, MMP-9, and fibrinogen were significantly increased in the high CRP group; however, these were all related to BMI, and only MMP-9 was significantly increased in the high CRP group after adjustment. Recently, Nomoto et al. (29) have shown that in patients with acute coronary syndrome, levels of hsCRP and MMP-9 are significantly elevated, and there is a significant correlation in these biomarkers in all subjects. Previously, Ferroni et al. (30) have shown that serum MMP-9 levels may represent a novel marker of inflammation in patients with known CAD and might provide an index of plaque activity in this clinical setting. Recently, CRP has been shown to induce the gelatinase activities of MMP-2 and MMP-9 in human umbilical vein endothelial cells (31), implying a cause-effect relationship. Also, in monocytes, CRP induces MMP-1 and not tissue inhibitors of MMP-1 (32).

Among the pro- and antiinflammatory cytokines, there were significant increases in LPS-activated whole blood release of IL-1, TNF, and IL-6 and a significant decrease in the IL-1 receptor antagonist, IL-4, in the high CRP group compared with the low CRP group. After adjustment for age and BMI, only IL-1ß and TNF- remained significantly higher. Current concepts suggest that CRP secretion is largely under the transcription control of IL-6; however, IL-1ß and TNF- also contribute to the synthesis of CRP. The combination of IL-1ß and IL-6 appeared to be the most potent stimulator of CRP from hepatocytes, a finding that we have extended to vascular aortic endothelial cells (9, 10, 33). Although the IL-6 level was significantly elevated in the high CRP group, it was strongly associated with BMI. Obesity is a strong risk factor for the metabolic syndrome and cardiovascular disease (34). Previous studies have also shown that IL-6 is increased in obesity (35, 36, 37), and recently, Ghanim et al. (36) have shown that mononuclear cells from obese subjects secrete increased amounts of IL-6, TNF, and MMP-9, all of which are strongly associated with BMI. Also, Carey et al. (38) have recently shown elevated levels of CRP and IL-6 in patients with type 2 diabetes mellitus, and these were strongly related to fat mass, but not to insulin responsiveness. Calabro et al. (39) have shown that inflammatory cytokines, IL-1, IL-6, and TNF, promote CRP production in human coronary artery smooth muscle cells. Our group has shown that macrophage-conditioned media induce CRP expression in human aortic endothelial cells, mainly via IL-1 and IL-6 (40). The locally produced CRP could directly participate in atherothrombosis.

TNF is a proinflammatory cytokine, mainly produced by macrophages (41). TNF is released at the site of inflammation and up-regulates adhesion molecules, activates endothelial cells, induces IL production, and increases vascular permeability (41). TNF is expressed in neointimal formation and atherosclerotic plaques, but not in the normal vessel wall (42), and circulating levels of TNF are elevated in patients with CAD (43). The genes for TNF- are located in tandem with the major histocompatibility class III region in a highly polymorphic region on the short arm of chromosome 6 (44). A point mutation of G to A at position –308 in the promoter region of the TNF gene (TNFA2) is associated with increased production of TNF. This finding combined with the pivotal role of inflammation and CAD have instigated studies on the association between TNFA gene polymorphism and CAD (45, 46, 47, 48, 49). These studies have yielded conflicting results; some researchers have found associations between the TNFA2 allele and subgroups of patients with CAD (47, 48), but these reports are contradicted by others (44). In a Brazilian population of 684 asymptomatic individuals, when serum concentrations of hsCRP were distributed into population quartiles, there was no significant difference in hsCRP serum concentration with regard to the CRP gene G1059C polymorphism. However, there was a tendency for higher hsCRP serum levels in individuals harboring the TNFA2 allele (TNF-308) in quartile 4 (mean hsCRP level, 0.081 µM or 9.6 mg/liter). These findings suggest an association between a functional genetic variant of the TNF- gene and hsCRP levels; these were evident only in individuals older than 48 yr (50). In the present study, although strong independent associations were observed for TNF levels between the high and low CRP groups, and the allele frequency for TNF-308 polymorphism was present in 31% of the population, we failed to observe any significant association between the polymorphism and CRP levels. This may be due to several factors, such as the limited sample size, younger individuals than in the study by Araujo et al. (50), and lower median levels of CRP (0.031 µM or 3.7 mg/liter). Also, TNF is induced by CRP in macrophages (51); thus, SNPs in the TNF gene might not be expected to cluster with high CRP levels.

In conclusion, we show that levels of LPS-activated whole blood IL-1, TNF, and serum MMP-9 are significant proximal and/or associated biomarkers of the proinflammatory phenotype. Because there are no data to suggest that MMP-9 triggers CRP synthesis, and in vitro studies have shown that CRP induces MMP activity, one can infer that CRP most likely induces MMP-9 and in this way could result in plaque instability. With respect to IL-1 and TNF, no firm conclusions can be drawn, because both cytokines induce hepatic CRP synthesis, and CRP in monocytes induces IL-1 and TNF secretion. It will be interesting to determine the roles of IL-1 and TNF in mediating the documented atherothrombotic effects of CRP. These studies provide major insights into defining additional novel candidate genes that could also be used in future studies for assessing cardiovascular disease risk. Furthermore, it is interesting that in the present study, high CRP levels were associated with obesity, insulin resistance, and inflammation; this evidence supports including the assessment of CRP in diagnosis of the metabolic syndrome.

	Footnotes

 
Presented in part at the Fifth Annual Conference of Arteriosclerosis, Thrombosis, and Vascular Biology, San Francisco, CA, 2004. This work was supported by National Institutes of Health Grants K24-AT-00596, RO1-HL-074360, and RR 0119975.

First Published Online May 17, 2005

Abbreviations: BMI, Body mass index; CAD, coronary artery disease; CRP, C-reactive protein; hsCRP, high sensitivity CRP; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; PAI-1, plasminogen activator inhibitor-1; SNP, single nucleotide polymorphism.

Received January 12, 2005.

Accepted May 11, 2005.

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