Clinical and Population Studies

Plasma S100A8/A9 Correlates With Blood Neutrophil Counts, Traditional Risk Factors, and Cardiovascular Disease in Middle-Aged Healthy IndividualsSignificance

Ovidiu S. Cotoi, Pontus Dunér, Nayoung Ko, Bo Hedblad, Jan Nilsson, Harry Björkbacka, Alexandru Schiopu
https://doi.org/10.1161/ATVBAHA.113.302432
Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:202-210
Originally published December 11, 2013

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Abstract

Objective—The S100 alarmins A8, A9, and A8/A9, secreted by activated neutrophils and monocytes/macrophages, are involved in the pathogenesis of various inflammatory diseases. S100A8/A9 has previously been linked to atherogenesis and cardiovascular (CV) disease. We investigated whether S100A8, A9, and A8/A9 correlate with carotid artery disease and CV risk in apparently healthy individuals.

Approach and Results—We measured baseline S100A8, A9, and A8/A9 in 664 individuals aged 63 to 68 years, with no previous history of CV disease, randomly selected from the Malmö Diet and Cancer population cohort. We examined the correlations between S100 proteins and circulating cell populations, plasma cytokines, carotid artery disease, and incidence of CV events during a median follow-up period of 16.2 years. We found that plasma S100A8/A9 concentration is positively influenced by circulating neutrophil numbers, smoking, body mass index, glycosylated hemoglobin A1c, and low-density lipoprotein. High-density lipoprotein was negatively associated with S100A8/A9. S100A8/A9 and the neutrophil counts were positively correlated with intima-media area in the common carotid artery, independently of age, sex, and CV risk factors. S100A8/A9 and circulating neutrophils presented similar associations with the incidence of coronary events (hazard ratio [95% confidence interval] per 1 SD: 1.28 [1.03–1.59] and 1.26 [1.04–1.53], respectively) and CV death (1.34 [1.06–1.71] and 1.59 [1.33–1.90], respectively). These relationships were mainly supported by strong associations in women, which were independent of traditional risk factors. There were no independent relationships between S100A8 and S100A9, and CV disease.

Conclusions—Our study supports the value of S100A8/A9 as a potentially important link between neutrophils, traditional CV risk factors, and CV disease.

Introduction

Innate immunity plays a central role in the development of atherosclerosis and cardiovascular disease (CVD).1 Danger-associated molecular patterns or alarmins are structurally and functionally diverse intracellular molecules that are passively released during tissue damage and cellular necrosis. Alarmins activate pattern-recognition receptors on innate immune cells and stimulate phagocytosis of cellular debris and tissue repair.2 Alarmins can also be actively secreted from activated leukocytes and under pathological conditions amplify and maintain chronic inflammatory processes involved in the pathogenesis of autoimmune disorders and cancer.2 The involvement of alarmins in the pathogenesis of atherosclerosis has attracted increased interest in recent years and several members of this group, including heat shock proteins,3 high-mobility group box protein 1,4 and cathelicidin,5 have been shown to be proatherogenic. The S100 proteins A8 and A9 (also known as calgranulin A and B or myeloid-related proteins 8 and 14) are alarmins belonging to the S100 calcium-binding protein family. S100A8 and S100A9 are constitutively expressed in neutrophils and monocytes. In neutrophils, S100A8 and S100A9 represent ≈45% of all cytosolic proteins compared with only ≈1% in monocytes.6 S100A8 and S100A9 expression differs between subsets of human monocytes. Higher levels of S100A8 mRNA were detected in classical CD14++CD16– monocytes compared with their nonclassical CD14+CD16++ counterparts.7 S100A8 and S100A9 are secreted from activated neutrophils and monocytes/macrophages mainly as the S100A8/A9 heterodimer (also called calprotectin), which is considered to be the main biologically active compound. However, S100A8 and S100A9 also form homodimers, and previous reports suggest that these compounds may have biologically distinct functions.8

S100A8/A9 is an endogenous ligand of toll-like receptor 4 (TLR4) and of the receptor for advanced glycation end products (RAGE).9,10 S100A8/A9 stimulates recruitment and activation of neutrophils and monocytes and plays a pivotal role as innate immune mediator in various autoimmune and inflammatory diseases.8,10 Studies in S100A9 knockout mice have demonstrated that S100A8/A9 is actively involved in atherogenesis and in the vascular response to injury.11 Importantly, hyperglycemia-induced S100A8/A9 production in neutrophils stimulates further release of neutrophils and inflammatory monocytes from the bone marrow and impairs the regression of atherosclerotic plaques in mice.12 Serum S100A8/A9 was shown to correlate with the severity of coronary artery disease in patients with diabetes mellitus12,13 and to predict incident CV events in healthy postmenopausal women.14 In patients with myocardial infarction (MI), S100A8/A9 is locally expressed in infiltrating neutrophils and monocytes and is released into the systemic circulation from the site of the myocardial injury.15,16 Plasma S100A8/A9 levels are highly elevated in MI compared with stable and unstable angina, and S100A8/A9 correlates with the incidence of recurrent CV events in MI survivors.16,17

The purpose of our study was to elucidate whether plasma S100A8, S100A9, and S100A8/A9 reflect the severity of existing carotid artery disease and correlate with long-term CV risk in apparently healthy middle-aged individuals. We measured baseline concentrations of S100A8, S100A9, and S100A8/A9 in 664 subjects with no previous history of CVD and examined their correlations with circulating leukocyte populations, smoking, diabetes mellitus, body mass index (BMI), blood pressure, plasma lipids, glycemic control, plasma cytokines, carotid intima-media thickness (IMT), and IM area, as well as with the risk for incident CV events during a median follow-up period of 16.2 years.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Population Characteristics

During a median follow-up time of 16.2 years, 129 individuals experienced ≥1 acute CV event (Table 1). The cases had a significantly higher prevalence of diabetes mellitus, higher glycosylated hemoglobin A1c, higher blood pressure, and lower high-density lipoprotein cholesterol compared with controls (Table 1). The numbers of circulating neutrophils and monocytes were higher in cases. There was a tendency toward higher S100A8/A9 concentrations in cases, but the difference did not reach statistical significance.

View this table:
Table 1.

Baseline Characteristics of the Study Population

Correlations Between S100 Proteins, CV Risk Factors, Circulating Cell Populations, and Plasma Cytokines

To examine the associations between baseline concentrations of S100A8, S100A9, and S100A8/A9, CV risk factor burden, and circulating cell populations, we performed multivariate linear regression analyses with S100 proteins as dependent variables. Blood neutrophil counts presented the strongest association with S100A8/A9 (Figure 1 and Table 2). Other independent positive determinants of S100A8/A9 variability were age, BMI, glycosylated hemoglobin A1c, and low-density lipoprotein cholesterol, whereas high-density lipoprotein cholesterol was negatively correlated with S100A8/A9 (Table 2). Smokers had significantly higher plasma levels of S100A8/A9 (median [interquartile range]: 1632 [1246–2280] versus 1471 [1034–1980]; P=0.003) and significantly higher blood neutrophil counts (mean±SD: 4.51±1.27 versus 3.55±1.11; P<0.001) compared with nonsmokers. S100A8/A9 did not correlate with the circulating numbers of lymphocytes, platelets, total monocytes, or any of the monocyte subpopulations considered. S100A8 and S100A9 were positively associated with nonclassical CD14+CD16++ monocytes (Table 2). All other associations tested were not significant.

View this table:
Table 2.

Associations Between CV Risk Factors, Circulating Cell Populations, and S100 Proteins

Figure 1.
Figure 1.

Correlation between S100A8/A9 and blood neutrophil counts. Scatterplot demonstrating the association between plasma S100A8/A9 and circulating neutrophil numbers. The correlation coefficient and the P value are calculated using the Spearman test.

Furthermore, we tested how S100 proteins correlate with the concentrations of circulating cytokines and with each other in a Spearman correlation analysis. S100A8/A9 was positively correlated with interferon γ, tumor necrosis factor (TNF) α, interleukin-1β (IL-1β), and IL-10 (Table 3). S100A8 was positively correlated with interferon γ, TNF-α, IL-1β, IL-1, IL-8, IL-10, and IL-12p70. S100A9 was positively correlated with TNF-α and negatively with IL-1β. The levels of S100A8/A9 did not correlate with S100A8 but were negatively correlated with S100A9. There was a strong association between S100A8 and S100A9 (Table 3).

View this table:
Table 3.

Association Between S100 Proteins and Plasma Cytokines

S100A8/A9 and Circulating Neutrophils Are Associated With IMT and IM Area in the Common Carotid Artery

In a multivariate linear regression model corrected for age and sex, plasma levels of S100A8/A9 and neutrophil counts were positively associated with IMT and longitudinal IM area in the common carotid artery (Table 4, model A). In addition, carotid IMT was correlated with circulating monocyte numbers (Table 4, model A). The associations between S100A8/A9 and IM area, and, neutrophils and IM area remained significant after additional adjustment for CV risk factors (age, sex, smoking, diabetes mellitus, BMI, hypertension, low-density lipoprotein, high-density lipoprotein, and triglycerides; Table 4, model B). S100A8, S100A9, and blood lymphocyte numbers showed no associations with carotid IMT or IM area.

View this table:
Table 4.

Correlation Between S100 Proteins and Carotid Artery Disease

S100A8/A9 and Blood Neutrophil Counts Are Correlated With the Incidence of Coronary Events and CV Death

An acute coronary event (CE) occurred in 83 of the participants during follow-up, and there were 55 cases of stroke. CV disease was the main cause of death in 67 individuals (Table 5). We used Kaplan–Meier survival analyses with log-rank significance tests to examine the associations between baseline S100A8, S100A9, and S100A8/A9 and the incidence of CE, stroke, and CV death. Similar tests were performed for baseline numbers of circulating neutrophils because neutrophils are the leukocyte population with the strongest correlation with S100A8/A9. We found that the incidence of CE and CV death was significantly associated with baseline S100A8/A9 levels and neutrophil counts (Figure 2). In unadjusted Cox regression analyses, CV risk was most elevated among individuals with high levels of both variables. Compared with the combined bottom tertiles, the hazard ratio of study participants with baseline values within the top tertile of both variables was 2.72 (confidence interval, 1.19–6.21; P=0.018) for CE and 3.46 (confidence interval, 1.55–7.70; P=0.002) for CV death. The corresponding hazard ratios were 1.79 (confidence interval, 1.03–3.10; P=0.038) for CE and 2.88 (confidence interval, 1.53–5.43; P=0.001) for CV death when neutrophil tertiles were considered alone.

View this table:
Table 5.

S100 Proteins, Neutrophils, and CV Risk

Figure 2.
Figure 2.

A to D, Associations between S100A8/A9, neutrophils, and cardiovascular (CV) risk. Kaplan–Meier 1-minus event-free survival plots of the associations between tertiles of S100A8/A9, blood neutrophil counts, and the incidence of coronary events and CV death during follow-up. The P values for trend are calculated using the log-rank test.

In Cox regression models adjusted for age and sex, S100A8/A9 and neutrophil numbers presented significant associations with CE and CV death in the entire study population (Table 5, model A). Adjustment for diabetes mellitus, BMI, hypertension, and plasma lipids did not alter the associations between S100A8/A9 and CV death and between neutrophils and CE and CV death (not shown). However, after additional adjustment for smoking, only the association between baseline neutrophil counts and CV death remained statistically significant (Table 5, model B). The relationships between S100A8/A9, neutrophils, and the risk of CE and CV death were mainly supported by strong correlations in women, which were independent of traditional CV risk factors (Table 5). Neutrophil counts were correlated with the incidence of stroke in women, and the S100A8 homodimer was associated with the incidence of stroke and CV death in men, but these relationships lost statistical significance when adjusting for CV risk factors (Table 5). Finally, we found a negative correlation between S100A8 and CV death in women, valid only in the fully adjusted model.

Discussion

Our study provides important clinical evidence supporting the value of S100A8/A9 as a potential biomarker of neutrophil involvement in CVD. We show that S100A8/A9 and blood neutrophil counts in apparently healthy middle-aged individuals without previous CV disease are associated with the extent of carotid artery disease and with the long-term risk of CE and CV death. In addition, we demonstrate that plasma S100A8/A9 is correlated with neutrophil numbers and with smoking, obesity, dyslipidemia, and glycemic control, independently of blood neutrophil counts. These findings are in line with previous experimental and clinical studies demonstrating that smoking, hyperlipidemia, and hyperglycemia stimulate neutrophilia12,18,19 and S100A8/A9 production.12

Although the involvement of neutrophils in CVD has long been disregarded, recent experimental and clinical evidence has revealed that these cells may play an important role both in atherogenesis and as mediators of plaque vulnerability and rupture.20,21 The presence of neutrophils has been detected in mouse, primate, and human atherosclerotic plaques.1,20 In murine models of atherosclerosis, neutrophilia induced by hyperlipidemia or CXCR4 blockade promotes atherogene sis and is correlated with atherosclerotic plaque size.18,22 Conversely, deficiency of the neutrophil-secreted alarmin cathelicidin has recently been shown to delay lesion progression.5 The suggested mechanisms of neutrophil involvement in atherogenesis include endothelial activation, inflammatory monocyte recruitment, generation of reactive oxygen species, low-density lipoprotein oxidation, apoptosis, and secretion of matrix-degrading proteases.5,20,21,23 Here, we show that neutrophils are the only leukocyte population that significantly correlates with plasma S100A8/A9 in humans and that blood neutrophil numbers are the strongest independent determinants of S100A8/A9 concentration. Nagareddy et al12 have recently demonstrated that hyperglycemia triggers S100A8/A9 release from neutrophils. In turn, S100A8/A9 binding to RAGE on common myeloid progenitor cells stimulates the production of neutrophils and inflammatory monocytes in the bone marrow, leading to impaired regression of atherosclerotic plaques in diabetic mice.12 Similarly, hyperlipidemia stimulates neutrophilia through increased granulopoiesis and enhanced neutrophil release from the bone marrow.18 In humans, plasma S100A8/A9 correlates with insulin resistance and is increased in patients with type 2 diabetes mellitus and in nondiabetic obese individuals.24,25 Weight loss leads to decreased S100A8/A9 levels alongside improved glycemic control and insulin resistance.25 Importantly, we show that BMI, glycemic control, and plasma lipids significantly influence S100A8/A9 variability independently of neutrophil numbers, suggesting that these traditional CV risk factors might stimulate neutrophil and monocyte activation and S100A8/A9 release.

In recent years, there has been increased interest in the role of S100A8/A9 in CVD.6 Plasma S1008A/A9 has been shown to correlate with the extent of coronary and carotid artery disease in patients with diabetes mellitus,12,13 and elevated levels of S100A8/A9 in human carotid plaques are associated with a vulnerable plaque phenotype.26 S100A8/A9 activates the vascular endothelium and increases endothelial permeability.27,28 In addition, S100A8/A9 promotes neutrophil and monocyte recruitment into the tissues by upregulating surface CD11b expression and enhancing the binding affinity of CD11b to intercellular adhesion molecule 1, integrins, and fibrinogen.6,8 In hyperlipidemic mice, the absence of S100A8/A9 is associated with reduced macrophage recruitment and delayed atherosclerosis.11 In patients with MI, S100A9 mRNA transcripts are increased in circulating platelets14 and S100A8/A9-positive neutrophils and macrophages infiltrate both the occluding thrombus and the infarcted myocardium.15,16 S100A8/A9 is released early into the systemic circulation during the acute coronary event and correlates with markers of myocardial injury and neutrophil counts.15,16 In MI survivors, elevated S100A8/A9 levels at 30 days postevent are associated with a higher risk for recurrent CV events within the next 30-day period.17 The inflammatory activity of extracellular S100A8/A9 is mediated by the receptors TLR4 and RAGE present on various cell types.9,29 TLR4-mediated signaling is considered to be proatherogenic, and atherosclerosis progression is delayed in apolipoprotein E–knockout or low-density lipoprotein receptor–knockout mice deficient in TLR4 or its adaptor protein MyD88.30,31 S100A8/A9-induced TLR4 activation on macrophages triggers a positive autocrine feedback loop leading to increased production of inflammatory cytokines, chemokines, and reactive oxygen species.10 RAGE is locally expressed in human atherosclerotic plaques,32 and hyperlipidemic apolipoprotein E–deficient RAGE–/– double knockout mice develop significantly less atherosclerosis compared with their apolipoprotein E–deficient controls.9 Binding of S100A8/A9 to RAGE triggers intracellular signaling mediated through nuclear factor-κB and amplifies myelopoiesis, vascular inflammation, and postischemic cardiac dysfunction.9,33 We found S100A8/A9 to be positively correlated with the proinflammatory cytokines interferon γ, TNF-α, and IL-1β. TNF-α and IL-1β are mainly produced by monocyte/macrophages, and interferon γ is the signature cytokine of T helper type 1 cells. All these cytokines have been shown to be proatherogenic and to play important roles in CVD.1

We and others have previously reported important correlations between distinct populations of monocytes/macrophages and lymphocytes in apparently healthy individuals and the incidence of acute CV events.1,34–37 In the present study, we reveal a strong association between circulating neutrophils and CV death, independently of the traditional risk factors for CVD. These data are in line with a previously published study demonstrating that healthy individuals with high baseline neutrophil counts are at increased risk to experience an ischemic coronary event and to die during the first day after the event.38 Measurement of S100A8/A9 contributes to further risk stratification of individuals with neutrophil values within the top tertile because in our study CV risk was highest in participants with elevated values of both variables. The relationships between S100A8/A9 and CV death and between neutrophils and CE in the entire study population were independent of diabetes mellitus, BMI, hypertension, and plasma lipids but became nonsignificant after additional adjustment for smoking. Within our cohort, smokers had significantly increased S100A8/A9 and neutrophil levels. Smoking-induced enhanced neutrophilia in apparently healthy individuals has previously been reported, and smoking cessation led to rapid decrease in circulating neutrophils.19 These findings suggest that the increased levels of blood neutrophils and S100A8/A9 in smokers might contribute to the elevated incidence of acute CV events in this particular risk category. In women, the associations between S100A8/A9, neutrophils, and CV risk are stronger than in men and are independent of other CV risk factors. Similar findings have previously been reported by Healy et al14 in a population of apparently healthy postmenopausal women, with a median follow-up time of 2.9 years.

Our study has several limitations that should be taken into account. Despite revealing similar association patterns between S100A8/A9, neutrophils, and CVD, in line with previous experimental studies demonstrating a proatherogenic role of neutrophils and S100A8A/9, our data cannot prove causality. Further mechanistic studies are required to investigate whether S100A8/A9 directly mediates the effects of neutrophils on atherogenesis and on plaque rupture. In addition, we cannot rule out the importance of monocytes/macrophages, endothelial cells, and platelets as alternative sources of S100A8/A9. However, neutrophils outweigh monocytes by >10-fold in normal blood and contain much higher amounts of S100A8/A9. We did not find any associations between plasma S100A8/A9 and the numbers of circulating monocytes, monocyte subpopulations, or platelets at baseline, suggesting that most of the circulating S100A8/A9 is derived from neutrophils.

In conclusion, our findings suggest that S100A8/A9 might represent an important link between circulating neutrophils, traditional CV risk factors, and CVD and promote S100A8/A9 as a potential biomarker and mediator of neutrophil involvement in CVD. We demonstrate that blood neutrophil counts and plasma S100A8/A9 present similar association patterns with the extent of carotid artery disease and with the risk of CE and CV mortality in middle-aged individuals with no previous history of CVD. S100A8/A9 is of particular interest as a possible therapeutic target for the prevention of acute CV events because compounds that block the binding of S100A8/A9 to its receptors have been developed and are already approved for clinical testing in humans.39,40

Sources of Funding

This work was supported by grants from the Swedish Research Council, the Swedish Heart–Lung foundation, the Knut and Alice Wallenberg foundation, VINNOVA, Swedish Foundation for Strategic Research, the Albert Påhlsson Foundation, Skåne University Hospital foundation, CardioVax, and Söderberg foundations.

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302432/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    BMI
    body mass index
    CE
    coronary events
    CVD
    cardiovascular disease
    IM area
    intima-media area
    IMT
    intima-media thickness
    MI
    myocardial infarction
    RAGE
    receptor for advanced glycation products
    TLR
    toll-like receptor
    TNF
    tumor necrosis factor

  • Received August 23, 2013.
  • Accepted October 23, 2013.

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Significance

Our study reveals for the first time a strong association between circulating neutrophils and the concentration of the inflammatory protein S100A8/A9 in human plasma. In addition, plasma S100A8/A9 is significantly increased by smoking, dyslipidemia, and hyperglycemia. S100A8/A9 and the circulating neutrophils present similar associations with the extent of carotid artery disease and with the risk for future acute cardiovascular (CV) events in healthy middle-aged individuals with no previous history of CV disease. Our findings suggest that S100A8/A9 might represent an important link between neutrophils, traditional CV risk factors, and CV disease and promote S100A8/A9 as a potential biomarker and mediator of neutrophil involvement in CV disease. In mouse studies, S100A8A/A9 has previously been shown to be involved as a mediator in the pathogenesis of atherosclerosis and of post–myocardial infarction heart failure. Importantly, S100A8/A9 blockers have already been developed and are approved for clinical testing, opening up exciting new therapeutic opportunities for CV disease.

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  • Plasma S100A8/A9 Correlates With Blood Neutrophil Counts, Traditional Risk Factors, and Cardiovascular Disease in Middle-Aged Healthy IndividualsSignificance
    Ovidiu S. Cotoi, Pontus Dunér, Nayoung Ko, Bo Hedblad, Jan Nilsson, Harry Björkbacka and Alexandru Schiopu
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:202-210, originally published December 11, 2013
    https://doi.org/10.1161/ATVBAHA.113.302432
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