Monocyte count as a predictor of cardiovascular mortality in older Korean people

Abstract

Background

white blood cells (WBCs) have been known to mediate the inflammatory process, which may be a pivotal mechanism for atherosclerosis and cardiovascular mortality.

Objective

we investigated which WBC subtypes increased cardiovascular mortality and explored its connection to coronary artery diseases in a prospective study among older Koreans.

Study design and subjects

this study was conducted from 2005 to 2011 as a part of the Korean Longitudinal Study on Health and Aging and included 439 men and 561 women over 65-year old.

Outcomes

the primary endpoints were all-cause and cardiovascular mortality.

Results

in the cox proportional hazard models, subjects in the higher tertiles of monocyte count were at a higher risk for cardiovascular mortality even in the fully adjusted model (2nd tertile hazard ratio = 2.51; 3rd tertile = 2.81). However, the total WBC, neutrophil and lymphocyte counts did not affect cardiovascular mortality. Logistic regression models revealed that subjects in the 3rd tertile of monocyte count had an increased risk for any coronary artery plaque, vulnerable plaque and calcified plaque (odds ratio = 1.80, 2.68, 1.59, respectively) but not for significant stenosis. Other WBC subtypes were not related to coronary artery diseases.

Conclusion

the results showed that a high monocyte count is a risk factor for cardiovascular mortality as well as coronary artery plaque formation.

Introduction

Chronic low-grade inflammation may be a pivotal mechanism for insulin resistance and atherosclerosis [1, 2]. Inflammation is characterised by increased production of cytokines and acute-phase reactants as well as activation of the inflammatory signalling pathway. Inflammatory cells dominate early atherosclerotic lesions and their effector molecules accelerate progression of the lesions. The resulting activation of inflammation can elicit cardiovascular diseases [3, 4]. In addition, inflammation on its own can affect insulin signalling and promote beta cell death, indirectly increasing the risk of Type 2 diabetes [1, 5, 6]. Furthermore, obesity, which provokes insulin resistance, is also associated with inflammation as fat tissue releases inflammatory cytokines [7].

Several proinflammatory markers appear to participate in the induction and maintenance of inflammation: high-sensitivity C-reactive protein (hsCRP), tumour necrosis factor-α, interleukin-6, resistin and monocyte chemotactic protein-1 [8, 9]. Furthermore, white blood cells (WBCs) also mediate the inflammatory process and interact with proinflammatory markers. In addition, previous epidemiological studies have shown that the WBC count is related to metabolic syndrome, cardiovascular diseases and the incidence of Type 2 diabetes. In addition, the WBC count has an important role in the development and progression of atherosclerosis [2, 4, 10, 11].

WBCs mainly consist of neutrophils, lymphocytes and monocytes. However, studies have not reached a consensus regarding which WBC subtype is the greatest contributor to atherosclerosis or cardiovascular mortality [12, 13]. For example, monocytes contribute to plaque formation and progression by giving rise to foamy macrophages and reactive oxygen species. However, both macrophages and lymphocytes secrete proinflammatory cytokines, which recruit circulating monocytes to the nascent lesions and lead to unstable coronary artery plaques. On the other hand, neutrophils are intimately involved with the adaptive process of infarct healing [14]. Therefore, studies are needed to identify which subtype contributes the most to cardiovascular mortality.

Thus, in this study, we investigated which WBC subtype contributed the most to cardiovascular mortality using a prospective cohort study of older Koreans. We also examined the related mechanisms including the link between differential WBC counts and coronary artery diseases.

Methods

Study population

This study was conducted as a part of the Korean Longitudinal Study on Health and Aging (KLoSHA), an ongoing, population-based, observational study among older residents of Seongnam, Korea [15]. A total of 439 men and 561 women over 65-year old were enroled in the baseline KLoSHA (September 2005). Subjects were invited to participate in this study through letters and telephone calls using age- and sex-stratified random sampling. Each subject's medical history, including smoking status, was assessed by trained nurses who were certified for this epidemiologic study and the assessment of older subjects. Comorbidities including diabetes, hypertension, cardiovascular disease and cerebrovascular disease were based on self-report, clinical diagnosis and medication use. The Institutional Review Board of the Seoul National University Bundang Hospital approved this study, and written informed consent was obtained from each subject (B-0912-089-005).

Measurement of anthropometric and biochemical parameters

Height and body weight were measured to the nearest 0.1 cm and 0.1 kg, respectively. The body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Blood pressure was measured three times between 7 AM and 9 AM in a relaxed state for at least 10 min. Hypertension was defined as blood pressure ≥140/90 mmHg or patients using anti-hypertensive agents. After a 12 h overnight fast, blood samples were drawn from the antecubital vein. Plasma was separated immediately with centrifugation (2,000 rpm for 20 min at 4°C), and biochemical measurements were obtained within 2 h. A complete blood cell count analysis, including the total and differential WBC counts, was performed using the XE-2100 (Sysmex, Kobe, Japan). The precision error of WBC count analysis was 2.7%. Fasting plasma concentrations for glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were measured enzymatically with a Hitachi 747 Chemical Analyser (Hitachi, Tokyo, Japan).

Study endpoints

All-cause mortality and cardiovascular mortality were the primary endpoints. Cardiovascular mortality included heart disease and stroke. The survival status of each individual and the cause of death were ascertained from the National Statistical Office (www.kostat.go.kr) before 31 December 2011. Cardiovascular disease-related deaths were identified by the ICD–10 codes I10–I11, I20–I25 and I61–64. The final date for follow-up was 31 December 2011.

Cardiac computed tomography data acquisition and analysis

At baseline, computed tomography (CT) angiography was performed with a 64-detector row CT scanner (Brilliance 64; Philips Medical Systems, Best, the Netherlands) with 64 × 0.625 mm section collimation, 420 ms rotation time, 120 kV tube voltage and 800 mA tube current with electrocardiographically gated dose modulation. An 80 ml bolus of iomeprol (Iomeron 400; Bracco, Milan, Italy) was intravenously, injected at a rate of 4 ml/s and followed by a 50 ml saline chaser. Images were initially reconstructed in the mid-diastolic phase (75% of the RR interval) of the cardiac cycle. Additional reconstructions were performed at other cardiac phases during retrospectively gated helical acquisitions if motion artefacts were observed. All images were analysed blindly and independently, by two experienced radiologists using a three-dimensional workstation (Brilliance; Philips Medical Systems). We analysed the plaque characteristics on a per-segment basis according to the modified American Heart Association classification [16]. All coronary segments larger than 1.5 mm in diameter were assessed for significant stenosis (more than 50% of diameter), plaque severity and plaque characteristics. Coronary artery stenosis was estimated by semiautomatically tracing the contrast material-enhanced portion of the coronary lumen at the maximal stenotic site and comparing the mean value for the proximal and distal reference sites [17]. Plaques were identified as structures larger than 1 mm² within or adjacent to the vessel lumen, which were clearly distinguishable from the lumen and surrounding epicardial fat. Plaque type was classified as follows: (i) plaques that contained calcified tissue that comprised >50% of the plaque area (attenuation >130 Hounsfield unit on native images) were classified as calcified (n = 202), (ii) plaques that contained calcified tissue that comprised <50% of the plaque area were classified as mixed (n = 151) and (iii) plaques without any calcium were classified as non-calcified lesions (n = 43) [17]. The vulnerable plaques designate a plaque at high risk of disruption leading to thrombosis, which is an inflamed thin-cap fibroatheroma (n = 34).

Statistical analyses

All data are expressed as the mean ± standard deviation. Baseline characteristics were compared using the Student's t-test for continuous variables or the χ² test for categorical variables. Cox proportional hazard regression analyses were used for the all-cause and cardiovascular mortality according to tertiles of the total and differential WBC counts: total WBC tertiles (<5,440, 5,440–6,769 and ≥6,770), neutrophil tertiles (<2,856, 2,856–3,754 and ≥3,755), lymphocyte tertiles (<1,860, 1,860–2,368 and ≥2,369), monocyte tertiles (<328, 328–448 and ≥449). We examined the hazard ratios (HRs) adjusted for covariates such as age, gender and BMI (Model 1), and the HRs adjusted for these factors as well as smoking, serum total cholesterol, serum triglycerides, diabetes mellitus, hypertension, cardiovascular diseases and cerebrovascular diseases (Model 2). For women, we excluded smoking variables because of the low number of smokers. Logistic regression analyses were performed for cardiac CT angiography findings according to the total and differential WBC count tertiles (see Supplementary data, Appendix 1, available at Ageand Ageing online). We analysed the Kaplan–Meier curves for cumulative cardiovascular disease-free survival according to the total and differential WBC count tertiles. Statistical significance was defined as P < 0.05. All analyses were performed with IBM SPSS Statistics for Windows v19.0 (IBM, Armonk, NY, USA) and Stata 11.0 (StataCorp LP, College Station, TX, USA).

Results

Baseline characteristics of the study participants are presented in Table 1. Decedents were older than survivors, and females tended to live longer than males. Decedents had a lower BMI, lower total cholesterol and lower triglyceride level than survivors. However, the serum hsCRP level was higher in decedents than in survivors. WBC and neutrophil counts were not significantly different between survivors and decedents, but the lymphocyte count was lower and the monocyte count was higher in decedents than in survivors. The prevalence of hypertension, diabetes mellitus, cardiovascular disease and cerebrovascular disease was not different between the two groups. WBC and neutrophil counts were not different between men and women. Monocyte count was higher in men than in women but lymphocyte count was higher in women than in men. Older subjects tended to have lower lymphocyte count, whereas WBC, neutrophil and monocyte counts were not associated with age. Regarding BMI, WBC and lymphocyte counts were positively correlated with BMI, and monocyte count showed no relationship with BMI (data not shown).

Table 2 shows the results of the cox proportional hazard models predicting all-cause mortality (n = 222) and cardiovascular mortality (n = 56). In the model adjusted for age, gender and BMI (Model 1), the monocyte count was the only significant predictor for all-cause mortality among WBC subtypes. However, the monocyte count was no longer significant after additionally adjusting for serum total cholesterol, serum triglycerides, diabetes mellitus, hypertension, cardiovascular diseases and cerebrovascular diseases (Model 2). A higher lymphocyte count was related to a lower all-cause mortality, but it lost significance in adjusted models. The total WBC and neutrophil counts did not increase the all-cause mortality risk. On the other hand, subjects within the higher tertiles of monocyte count were at a higher risk of cardiovascular mortality even in the fully adjusted model (HR = 2.51 for 2nd tertile and 2.81 for 3rd tertile). On the contrary, the total WBC, neutrophil and lymphocyte counts did not affect cardiovascular mortality (Table 2b). In Cox proportional hazard models for non-cardiovascular mortality, the monocyte count did not predict the non-cardiovascular mortality. However, the higher neutrophil count was related with higher risk for non-cardiovascular mortality (Table 2c). Accordingly, the monocyte count predicted only cardiovascular mortality independently of confounding factors including BMI. The logistic regression models examining the cardiac CT angiography findings according to the total and differential WBC count tertiles showed that subjects in the 3rd tertile for monocyte count were at an increased risk for any coronary artery plaque, vulnerable plaque and calcified plaque [odds ratio (OR), 1.80; 2.68; 1.59, respectively]. However, the monocyte count was not associated with significant stenosis. Similarly, the total WBC, neutrophil and lymphocyte counts did not increase the risk for significant stenosis or coronary artery plaques (Table 3).

Discussion

In community-dwelling, older Koreans, only monocyte count independently predicted cardiovascular mortality, but not non-cardiovascular mortality. However, this effect may be mediated by coronary artery plaque formation, findings that are supported by previous studies. For example, in the Atherosclerosis Risk in Communities Study, subjects in the highest quartiles of monocyte count had a higher rate of cardiovascular mortality than those in the lowest quartiles [18], and a similar study showed that the monocyte subtype was a promising predictor for cardiovascular disease [19]. Furthermore, in healthy persons, the monocyte count has been shown to be crucial in novel carotid plaque formation, which leads to cerebrovascular diseases [14, 20]. The central role of monocytes in the development and progression of atherosclerotic plaques has also been proved in experimental studies [21]. Activation of circulating monocytes and differentiation into lipid-laden macrophages are fundamental events in the formation of atherosclerotic lesions [21]. Monocytes also contribute to atherosclerosis by giving rise to reactive oxygen species and proinflammatory cytokines, which perpetuate the chronic inflammatory process [22].

In this study, the cardiac CT angiographic findings showed that the effect of monocytes seemed to be linked to plaque formation and not to significant arterial stenosis [20]. However, a meta-analysis demonstrated that raised neutrophil and lymphocyte counts were associated with a higher risk of Type 2 diabetes while monocyte counts were not [5]. The discrepancy between studies may be attributable to differences in age and ethnicity between the study subjects as well as differences in follow-up duration.

This study failed to show an association between total WBC count and mortality. In contrast, several studies have shown that the total WBC count is an independent predictor of cardiovascular events and all-cause mortality [11, 23]. For example, in the Women's Health Initiative Observational Study including 93,676 postmenopausal American women, an elevated baseline total WBC count was independently associated with cardiovascular events and death in older women after adjusting for traditional risk factors [11]. Furthermore, the Baltimore Longitudinal Study of Aging found that WBC count was almost linearly associated with cardiovascular mortality [24]. However, the results of the Tromsø Study showed no association between the total WBC count and carotid plaque, but did show that the monocyte count was a predictor for carotid plaque formation [14], results that are similar to those found in our study. In another study examining 9,996 Korean older people, the total WBC count predicted mortality risk, but the monocyte subtype provided the greatest predictive ability [13]. Because the total WBC is a composite of several WBC subtypes, its relation may be dependent on its subtype. Therefore, monocyte count may be a better and more specific marker of inflammatory activity in atherosclerosis (i.e. in older Korean persons) than total WBC.

Furthermore, no association was found between the neutrophil count and cardiovascular mortality in our study, whereas the neutrophil count was associated with non-cardiovascular mortality. On the other hand, several studies have shown that the neutrophil count is a strong predictor of cardiovascular events in the general population. For example, in the EPIC-Norfolk Prospective Population Study, an increased neutrophil and lymphocyte counts seemed to account for the higher risk for cardiovascular disease [25]. However, the role of neutrophils in developing atherosclerosis has rarely been examined. The main cellular components related to the formation of atherosclerotic lesions are monocytes and lymphocytes, but not neutrophils [26, 27]. Monocytes may have a specific role in initiating the formation of atherosclerotic lesions, with neutrophils possibly activated once the inflammatory process begins [14].

The results also showed that a low lymphocyte count increased all-cause mortality but not cardiovascular mortality. Previous studies have shown that, among older persons, a low lymphocyte count is related to poor nutrition [28], and an inverse relationship between lymphocyte and mortality exists [29]. Therefore, according to the concept of immune surveillance, a decreasing lymphocyte count may render an older person more vulnerable to disease and death.

Our study has some limitations. The total and differential WBC counts were measured only once. Hence, measurement errors may exist. In addition, our study was composed only of older Koreans. Therefore, the generalisability of our findings to other age or ethnic groups is limited. Furthermore, the 6-year follow-up duration might not have allowed sufficient time to observe mortality. Another limitation of our study was that we did not analyse the incidence of cardiovascular events because of a lack of data.

Nonetheless, the current study had several strengths. First, the study population was homogenous and accurately characterised community-dwelling, older Koreans. Second, we explored the total and differential WBC counts related to comprehensive outcomes including coronary CT findings and cardiovascular mortality emphasising the pivotal role of monocytes. Third, the mortality data in our study came from the National Statistical Office, which collects all mortality reports in South Korea. Therefore, the mortality data were nearly complete without loss to follow-up.

In this prospective, longitudinal cohort study of older Koreans, a high monocyte count was a risk factor for cardiovascular mortality. Evidence of this relationship was further supported by the association between the monocyte count and coronary artery plaque formation. Therefore, monocyte count can be used as an easy-to-measure, inexpensive and reliable parameter for cardiovascular risk among older Koreans.

Supplementary data

Supplementary data are available at Age and Ageing online.

Acknowledgements

We thank the study subjects who consented to participate the study.

Conflicts of interest

None declared.

Funding

This study was supported by the National Research Foundation grant funded by the Korea government (MEST) (No. 2006-2005410) and grants from the Korea Healthcare technology R&D Project of the Ministry for Health, Welfare & Family Affairs (A110948-1101-0000100) and from Seoul National University Bundang Hospital (02-2009-035).

References

Author notes