One of the numerous inflammatory molecules detected in atherosclerotic plaques is interleukin-12 (IL-12) 1. It is a heterodimeric cytokine composed of the p35 and p40 subunits, and the major human cell types that produce IL-12 are antigen-presenting cells, such as monocytes/macrophages and dendritic cells 2. IL-12 induces interferon-γ production in these cells, regulating innate responses, and determines the type and duration of adaptive immune response. Therefore, IL-12 plays a central role in coordinating innate and adaptive immunity 3.
IL-12 protein appears to play a multifaceted role in atherogenesis 1. It induces T-cell recruitment into the atherosclerotic plaque and may impair plaque stability 4,5. The in-vitro data suggest that, in atherosclerotic plaques, the balance between the productions of IL-12 and IL-10 governs the level of immune-mediated tissue injury 6.
The IL-12 concentration seems to be elevated in unstable coronary artery disease (CAD) and acute myocardial infarction (AMI), but remains at the level of control individuals in patients with stable angina 7. Paradoxically, the concentrations of Th1-type proinflammatory cytokines have been reported to be lower in the combined CAD group with or without concomitant myocardial infarction (MI) compared with the patients in the non-CAD control group admitted to hospital with chest pain 8, suggesting that the regulation of IL-12 is complex also in vivo.
Over the past few years, a series of circulating biomarkers have been identified as indicators of the level of the instability of atherosclerotic plaques. These new markers not only serve as diagnostic tools for the identification of patients with unstable CAD or AMI, but they may also help in identifying high-risk patients before serious acute events. Some markers are used clinically for risk stratification in stable ischaemic heart disease (IHD) as well as in monitoring the condition of patients with acute coronary syndrome 9. To understand the role of the inflammatory markers in IHD and use them successfully, the associations of the markers with risk factors of atherosclerosis and other personal determinants need to be known. Indeed, there is a report on the relationship of C-reactive protein (CRP) with some well-known risk factors of atherosclerosis 10, but less information is available on the association of cytokines with risk factors of atherosclerosis and other diseases.
The objective of the present study was to explore how the plasma IL-12 concentration is related to the major risk factors of atherosclerosis and to other personal clinical and biochemical determinants in stable IHD patients. Because of the inflammation in atherosclerosis plaques, the hypothesis was that plasma IL-12 concentration is associated positively with the inflammatory markers and the risk factors of atherosclerosis.
Materials and methods
The present study group included patients with doctor-diagnosed, well-documented IHD on the basis of hospital records. The IHD patients were recruited for a project (HIPPU) on air pollution and inflammation 11. The inclusion and exclusion criteria of the patients were the same as those in the AIRGENE project 12. The patients signed an informed consent and the trial was approved by the ethical committee of the Kymenlaakso Hospital District. The patients were nonsmokers at the time of recruitment and during the study, and did not have any chronic inflammatory disease. Also, their blood concentrations of common inflammatory markers such as erythrocyte sedimentation rate (ESR), hsCRP and leucocytes were within the normal clinical range. The current cohort included 31 men and 20 women (50–80 years, median age 71 years; see Supplement 1, Supplemental digital content 1, http://links.lww.com/CAEN/A8: recruitment). At the start of the trial, the patients completed a questionnaire on their health status, medication and lifestyle, including questions similar to those used in the AIRGENE project 11,12. They also underwent basic laboratory tests and routine ECG recording.
All the blood samples were obtained preferably after fasting for 10 h or more. Samples were not included in the study if the patient had symptoms or signs of an acute inflammatory disease at the time of blood sampling. The present study compares the basic laboratory and clinical data and the plasma IL-12 concentration (the first available IL-12 measurement of an accepted visit) of the patients of the HIPPU study, obtained 0–58 days after the collection of the basic data.
Venous blood samples were drawn into tubes used routinely for each laboratory test [ethylenediaminetetraacetic acid (K2-EDTA)] tubes for plasma (303149 Veno VT; Oriola-KD Inc., Espoo, Finland) and whole-blood measurements; lithium heparin or citrate for plasma measurements; plasma tubes containing a glycolysis inhibitor for glucose determination; serum tubes for the allergy screen; sedimentation rate tubes at the Kymenlaakso Hospital Services (Carea, Kotka, Finland), which is part of the external quality control system of Labquality Inc. (Helsinki, Finland) and has a quality manual according to the standard SFS-EN ISO/IEC 17025. Starting at the blood collection, the EDTA tubes for IL-12 and homocysteine assays were placed on ice, centrifuged in the cold within 30 min and the plasma samples were stored at −20°C until analysed. IL-12 was determined using an immunologic ELISA method (OptEIA; Becton Dickinson, San Jose, California, USA) at the National Institute for Health and Welfare (Kuopio, Finland). The detection limit of the method was 5 pg/ml and the total coefficient of variation between pairwise IL-12 samples placed in different tubes by the same venepuncture was 29% (N=19).
After centrifugation, the following plasma determinations were carried out using an automatic chemical analyser (Aero set; Abbott Diagnostics, Abbott Park, Illinois, USA) in the Kymenlaakso Hospital Services using reagents produced by Abbott Diagnostics: cholesterol (enzymatic determination, Cholesterol List No. 7D62-20), low-density lipoprotein (LDL) cholesterol (direct measurement, Sentinel Cholesterol LDL Liquid List No. 6K28-02), high-density lipoprotein (HDL) cholesterol (Sentinel Cholesterol HDL Liquid List No. 3K28-02), triglycerides (enzymatic determination, Triglyceride List No. 7D74-20), glucose (hexokinase method, Glucose List No. 7D66-20), creatinine (kinetic, Jaffe, Creatinine List No. 7D64-20) and high-sensitivity CRP (hsCRP, immunoturbidimetric method, Sentinel CRP Vario List No. 6K26-02; detection limit 0.1 mg/l). Haemoglobin A1c was analysed using the Integra 800 automatic chemical analyser (Roche Diagnostics Inc., Roche Finland, Espoo, Finland) using the whole-blood application of Roche (Cat. No. 20753521 322), calibrated with the reference method of the International Federation of Clinical Chemistry (Cat. No. 20755664 322). Blood cell count was performed using an automatic haematology analyser (Cell-Dyn 4000; Abbott Diagnostics), ESR using an automatic instrument (Sedimatic; ILS Laboratories Scandinavia Ltd, Helsinki, Finland) and plasma natriuretic peptide (BNP) using an immunochemical method (Axsym; Abbott Diagnostics). The serum allergy panel was immunochemical (ImmunoCAP fluorescent enzyme immunoassay, Phadiatop Combi; Pharmacia Diagnostics, Uppsala, Sweden), and determination of plasma homocysteine was carried out using a luminoimmunometric assay (IMMULITE 2000 Homocysteine Cat. No. L2KHO2, IMMULITE 2000 analyser; DPC Diagnostic Products Corporation, Los Angeles, California, USA). ECG was analysed both by an automatic routine method [Cardio Control; Welch Allyn (UK) Limited, Buckinghamshire, UK] and by two Kymenlaakso Hospital physicians using the Minnesota coding independent of each other 13.
Health status determinants
Sex, age, BMI, doctor-diagnosed AMI, last MI within the past 5 years, chest pain symptoms, arrhythmias, cardiac insufficiency, other cardiac problems, elevated blood pressure, asthma, hay fever, arthrosis, other chronic disease, cardiac pacemaker, diabetes (type 2, no one had type 1 diabetes), respiratory wheezing during the past 12 months, respiratory wheezing during the past 12 months not associated with flu, regular coughing during night or day in wintertime and ECG were health status determinants.
The association of IL-12 was studied using the following drugs: anti-inflammatory (ATC codes, anatomical–therapeutic–chemical codes, B or M 01AC06, M01AE01 or 03, M01AH05, N02BE01), statin (ATC C10AA01-07) and antithrombotic (warfarin, ATC B01AA03) medication, and medication for arterial thrombosis (clopidogrel, ATC B01AC04).
Number of smoking years, alcohol consumption, use of omega-fish oil, use of other additional nutrients, self-evaluation of health status (compared with individuals of the same age), capability to undergo intense stressing activities without angina pectoris, days per week with mildly stressing activities were the lifestyle determinants.
The associations of the IL-12 concentration with biochemical determinants were determined by trend analysis, using linear regression (Excel, Windows; Microsoft Oy Suomi, Espoo, Finland), but the association of the allergy test results with IL-12 was analysed by analysis of variance. The associations of BMI and age with the IL-12 concentration were determined by regression analysis. Results from other health status and personal determinants, and the lifestyle determinants, were divided into relevant fractions on the basis of the questionnaires, for example, yes or no answers. Subsequently, the IL-12 concentrations of the fractions were compared using analysis of variance (Excel, Windows).
Power analyses were carried out for the determinants that were not analysed by regression analysis: 13 patients per group were sufficient to show a statistically significant difference of 500 pg/ml in the median IL-12 between groups (P<0.05, power 0.80) and nine patients to show a difference of 600 pg/ml 14.
The arithmetic mean±SD IL-12 concentration in the plasma of the patients was 599±450 pg/ml (range 136–1944 pg/ml). IL-12 was associated inversely with the plasma concentrations of triglycerides (P=0.001; Fig. 1) and homocysteine (P=0.04; Fig. 2). In contrast, the IL-12 concentration was associated directly with the concentration of HDL cholesterol (P=0.03; Fig. 3). IL-12 tended to be inversely, but not statistically significantly, associated with hsCRP, ESR and the concentration of leucocytes and neutrophils (P=0.19–0.23) and also with haemoglobin A1c and plasma creatinine (P=0.14 and 0.18, respectively; see Supplement 2, Supplemental digital content 2, http://links.lww.com/CAEN/A9). IL-12 concentration was not associated with other biochemical determinants (see the Materials and methods section). IL-12 was significantly lower in patients with a positive allergy test (368±85 pg/ml, mean±SD, N=13) compared with nonallergic patients (678±76 pg/ml, N=38, P=0.01), but the power of statistical testing was not sufficient to exclude a false-positive result (Supplement 2, Supplemental digital content 2, http://links.lww.com/CAEN/A9).
IL-12 was significantly higher in patients with cardiac pacemakers and significantly lower in patients with cardiac insufficiency (P=0.002 and 0.03, respectively; Supplement 3, Supplemental digital content 3, http://links.lww.com/CAEN/A10), but again the power of statistical testing was not sufficient to exclude a false-positive result. IL-12 concentration was not associated with the other health status determinants or with age, sex, BMI, medication or lifestyle determinants of the patients (Supplement 3, Supplemental digital content 3, http://links.lww.com/CAEN/A10).
The present study is, to our knowledge, the first to report how a large number of personal and biochemical risk factors of atherosclerosis are related to the IL-12 concentration in the plasma of stable IHD patients. Despite specimen handling in the cold and for a minimum time, the coefficient of variation of the IL-12 method for duplicate samples was 29%, which is in agreement with previous reports 15–17. A smaller methodological variation could probably have enhanced the associations observed.
It has been shown previously that, when compared with the control group, IL-12 concentrations are significantly higher in patients with stable angina pectoris 18. Our results are in agreement with this as the range of plasma IL-12 concentrations in the present IHD patients (136–1944 pg/ml) was clearly much higher than those in healthy control individuals of previous studies using the same IL-12 analysis method. In the largest of these control cohorts (90 participants), the range of plasma IL-12 concentration was 1–132 pg/ml and the median was 26 pg/ml 19–22.
The present results, using data only from the first IL-12 measurement in each patient (N=51), showed mean±SD plasma levels (599±450 pg/ml) that were rather similar to those in our previous study 11, which was carried out using a much larger database among the same patients [mean±SD of personal medians of 2–12 consecutive IL-12 measurements 474±335 pg/ml (N=583) during 6 months of intensive monitoring of ambient air quality and personal pollutant exposure]. Thus, the present study has a relatively small number of patients, but the strict inclusion criteria probably enabled us to select a rather homogenous group of IHD patients with respect to stability of the disease. Also, the previous analysis of the much larger database of repeated measurements lends credibility to the current results 11.
The present results suggest that, contrary to the hypothesis, IL-12 concentration was associated inversely with two known risk factors of coronary atherosclerosis, that is, the plasma concentrations of triglycerides and homocysteine in stable IHD patients 23,24. The lack of association (and the trend of inverse association) of IL-12 with hsCRP, ESR and the number of inflammatory cells suggests that higher IL-12 does not imply inflammation. In IHD patients with low concentrations of the risk factors triglycerides and homocysteine (i.e. in mild stable IHD), there is increased IL-12 production, which is a sign of proinflammatory activity. Of course, the observed inverse association does not necessarily mean any direct causal relationship. However, this inverse association implies that high plasma concentrations of triglycerides or homocysteine as such are not associated with proinflammation.
According to the negative association with triglycerides, a moderately elevated IL-12 concentration may be a good sign in stable IHD patients, which is in agreement with the previous finding that frailty is associated with decreased IL-12 production in older individuals 25. In the present study, there were other trends of lower IL-12 concentration being associated with signs of impaired health; for example, the trends of inverse association of IL-12 with blood haemoglobin A1c concentration, plasma creatinine concentration and with cardiac insufficiency. A common consequence of CAD is cardiac insufficiency, which leads to an expanded plasma volume. Therefore, dilution may be one of the mechanisms decreasing the plasma IL-12 concentration in chronic heart failure 26,27.
However, the situation is more complex with plasma HDL, which has been documented to become proinflammatory when systemic inflammation is present 28–30. Modification of the protein components can convert HDL from an anti-inflammatory into a proinflammatory particle, and coronary atherosclerosis is one of the states that may promote proinflammatory HDL. It remains unclear as to why the plasma concentration of HDL was directly associated with the concentration of a proinflammatory molecule, IL-12, in the stable IHD patients in the present study. As systemic inflammation was not involved, this may be an anti-inflammatory signal in response to elevated IL-12 production.
Our IL-12 results are similar to those of the study of Martins et al.8, in which the concentrations of the proinflammatory and Th1-type cytokines IL-12 and IL-18 were, paradoxically, lower in the combined CAD group with or without concomitant MI than in the non-CAD control group with chest pain 8. This previous finding, and the presently observed inverse association of IL-12 with triglycerides and homocysteine, may be explained as follows: when the atherosclerotic process and progression of inflammation are more severe in IHD patients, the proinflammatory status tends to withdraw and lead to systemic inflammation. Our findings, therefore, suggest that a low IL-12 concentration in the plasma of stable IHD patients may indicate that coronary atherosclerosis is progressing. However, the observed trend (P=0.14) between lower IL-12 and improved capability to undergo intense or moderately stressing activities without angina pectoris does not support this hypothesis.
One of the mechanisms that makes IL-12 a predominantly proinflammatory cytokine may be downregulation of IL-12 by inflammatory or other risk factors. For example, CRP treatment has been reported to suppress the production of the proinflammatory and inflammatory cytokines (including IL-12) and suppress IL-10 secretion in activated human monocyte-derived macrophages, thus altering the balance between anti-inflammatory and proinflammatory factors, which is pivotal in atherothrombosis 31,32. Data on the cross-regulatory roles of the elements of the inflammation chain have been published in the study of Uyemura et al.6. Interestingly, IL-12 was the quickest and the most strongly responding inflammatory factor (mean concentration up by 227%) among these stable IHD patients, who were investigated during an unexpected 12-day period of transboundary transport of forest fire smoke from Russia, which also multiplied the local ambient air fine particle concentrations at the end of our previously reported 6-month study 11.
It is somewhat surprising that despite considerable documentation linking the LDL and cholesterol concentrations to the initiation and progression of atherosclerosis 33, these biomarkers were not associated with the IL-12 concentration. A logical explanation for this could be the use of cholesterol-lowering medication (statins) in 90% of the patients: statins inhibit cholesterol synthesis and reduce the LDL concentration, which narrows the difference between the lower and higher quartiles of these risk factors 34. Second, statins have been reported to alleviate inflammatory and atherosclerotic processes in the vascular endothelium 34,35. Third, statins cause a modest increase in the HDL concentration 36, which may have contributed toward the currently observed direct association of IL-12 with HDL.
According to previous reports on patients with acute or unstable IHD, in whom the inflammatory response is strong, the IL-12 response differs from the findings in the stable IHD 7,18,37. In patients whose IHD is not in a stable state, the IL-12 concentration has been reported to be elevated, compared especially with controls who do not have the proinflammatory process of stable or unstable IHD.
The present results suggest that IL-12 can be inversely associated with the plasma concentrations of two known risk factors of coronary atherosclerosis, that is, triglycerides and homocysteine, in stable, statin-medicated IHD patients. The lack of association of IL-12 with hsCRP, ESR and the number of inflammatory cells in blood suggests that moderately elevated IL-12 did not imply ongoing systemic inflammation in these patients. Although increased IL-12 production is a sign of proinflammatory activity, it was directly associated with the plasma HDL cholesterol concentration. Overall, moderately elevated plasma IL-12 concentration may even be a good sign in stable IHD patients.
The authors thank Ms Reetta Tiihonen, Ms Sini Herrala, Ms Ulla Purtilo and the staff of the Laboratory of Clinical Chemistry of the Kymenlaakso Hospital Services, the Kymenlaakso University of Applied Sciences and the National Institute for Health and Welfare for technical help.
This work was supported by the Finnish Funding Agency for Technology and Innovation (Tekes/EAKR; 70078/04, Diary number 2229/31/04); the Kymenlaakso Hospital District; the Cities of Kotka and Hamina; Kotkan Energia Oy; Sunila Oy and Stora Enso Oyj.
The study complies with the ethical guidelines of the Declaration of Helsinki. An informed consent was obtained from the patients, and the study protocol was approved by the ethics committee of the Kymenlaakso Hospital District (reference number 5/2005).
Conflicts of interest
There are no conflicts of interest.
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