Coronary leukocyte activation in relation to progression of coronary artery disease

Abstract

Leukocyte activation has been linked to atherogenesis, but there is little in vivo evidence for its role in the progression of atherosclerosis. We evaluated the predictive value for progression of coronary artery disease (CAD) of leukocyte activation markers in the coronary circulation. Monocyte and neutrophil CD11b, neutrophil CD66b expression and intracellular neutrophil myeloperoxidase (MPO) in the coronary arteries were determined by flow cytometry in patients undergoing coronary angiography. The primary outcome included fatal and nonfatal myocardial infarction or arterial vascular intervention due to unstable angina pectoris. In total 99 subjects who were included, 70 had CAD at inclusion (26 patients had single-vessel disease, 18 patients had two-vessel disease and 26 patients had three-vessel disease). The median follow-up duration was 2242 days (interquartile range: 2142–2358). During follow-up, 13 patients (13%) developed progression of CAD. Monocyte CD11b, neutrophil CD11b and CD66b expression and intracellular MPO measured in blood obtained from the coronary arteries were not associated with the progression of CAD. These data indicate that coronary monocyte CD11b, neutrophil CD11b and CD66b expression and intracellular MPO do not predict the risk of progression of CAD.

Keywords

coronary artery disease / inflammation / integrin / myeloperoxidase / leukocyte activation

Introduction

Classical risk factors used to predict cardiovascular risk include hyperlipidemia, hypertension, smoking, obesity and diabetes mellitus [ 1]. In the past decades, it has become evident that atherosclerosis is an inflammatory disease [ 2]. Therefore, attention has shifted to inflammatory markers as predictors of future cardiovascular events. Total leukocyte count, C-reactive protein (CRP) and the third component of complement (C3) are predictors of cardiovascular risk [ 3– 5]. In this respect, besides leukocyte count, also the level of leukocyte activation may be an interesting marker. Leukocytes can become activated by several stimuli, such as lipids and glucose [ 6– 10]. Activated neutrophils and monocytes express integrins on their cell surface, facilitating the binding of these leukocytes to the intact endothelium [ 11, 12]. One of these integrins, CD11b, is present on the surface of activated monocytes and neutrophils and binds to intercellular adhesion molecule-1 on endothelial cells [ 11]. It has been demonstrated in vitro that increased expression of CD11b on monocytes and neutrophils is accompanied by enhanced leukocyte adhesion to the endothelium [ 13– 15]. CD66b is a marker of neutrophil degranulation [ 16]. Myeloperoxidase (MPO) is an important enzyme in neutrophil host defense, as it inactivates bacterial toxins [ 17]. Activated neutrophils release MPO from their granules and therefore, intracellular MPO levels are a negative marker of neutrophil activation, with low levels representing increased activation [ 17]. When bound to the endothelium, activated leukocytes migrate to the subendothelial space. Monocytes bind modified lipoproteins and form foam cells, and the development of the atherosclerotic plaque is initiated [ 2].

Several lines of evidence suggest that increased leukocyte activation is linked to the presence of ischemic heart disease and peripheral artery disease [ 18– 21]. The expression of monocyte and neutrophil CD11b is higher in patients with multiple risk factors for atherothrombosis than in those with none or only one risk factor [ 22]. Intracellular MPO is reduced in patients presenting with acute coronary syndromes [ 23]. Finally, increased leukocyte activation has been associated with the presence of microvascular diabetic complications [ 24, 25].

We have previously described an inflammatory gradient of intracellular MPO in patients with stable coronary artery disease (CAD), with the highest level of neutrophil activation in the coronary arteries, suggesting focal inflammation in CAD [ 26]. In healthy controls, this gradient was observed as well, although levels of neutrophil activation were lower than in patients with CAD [ 26]. For monocyte CD11b, neutrophil CD11b and CD66b expression, this gradient was not present [ 26].

Although the current literature underlines an association between leukocyte activation and CAD, no studies have evaluated the use of leukocyte activation markers CD11b and CD66b and intracellular MPO as possible indicators of future coronary events. The aim of the current study was to assess whether levels of leukocyte activation in the coronary circulation may help to predict which patients are at risk of progression of CAD, in patients undergoing elective coronary angiography (CAG).

Methods

Study design and study population

The design of the study has been described extensively and published elsewhere [ 26]. The study was designed as a prospective follow-up study. Briefly, subjects who visited the outpatient clinic of the Department of Cardiology, Sint Franciscus Gasthuis, Rotterdam, The Netherlands, between July 2007 and September 2008 and who were scheduled to undergo a diagnostic CAG were invited to participate. Their indications for CAG were typical chest pain and a positive cycle ergometry.

Exclusion criteria were the presence of inflammatory disorders such as rheumatoid arthritis, systemic lupus erythematosus and infections, a plasma CRP level above 10 mg/L, and disorders of kidney, liver and thyroid function. The study was conducted according to the Helsinki Declaration. The Institutional Review Board of the Sint Franciscus Gasthuis Rotterdam and the regional independent medical ethics committee of the Maasstad Hospital Rotterdam approved the study. The study was registered at clinicaltrials.gov under clinical trial number NCT02376738. All participants gave written informed consent.

On the day of the angiography, anthropometric measures, the use of medication and cardiovascular history were recorded. Shortly before CAG, venous blood was obtained from a peripheral vein of the forearm. During the angiography, blood samples were obtained from each coronary artery. In addition, blood was collected from the femoral artery and midway from the abdominal aorta. The first 2 ml were discarded to avoid contamination with contrast. Subsequently, blood samples were collected in tubes containing EDTA (1 mg/ml) and kept on ice until processed for determination of leukocyte activation markers. CAG images were scored by an independent cardiologist.

Scoring of coronary events

The primary outcome was defined as progression of CAD, evaluated by an independent investigator at approximately six years follow-up. This endpoint included fatal and nonfatal myocardial infarction and any arterial vascular intervention that had not already been planned at the time of inclusion (e.g., coronary bypass, percutaneous coronary intervention, peripheral vascular surgery or angioplasty/stenting).

Analytical methods

Parameters for renal and liver function, glucose, CRP, total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides were determined using Synchron LX-20 analyzers (Beckman Coulter, Brea, CA, USA) according to standard procedures in our laboratory. Low-density lipoprotein (LDL) cholesterol values were calculated using the Friedewald formula. Apolipoproteins AI and B were determined by rate nephelometry using an IMMAGE analyzer (Beckman Coulter). Blood cell counts were determined using LH750 analyzers (Beckman Coulter, Miami, FL, USA).

Leukocyte activation markers

The expression of leukocyte activation markers on the cell surface was determined using fluorescent labeled monoclonal antibodies (Immunotech Coulter, Marseille, France). Antibodies for CD66b were labeled with fluorescein isothiocynate (FITC) and were diluted 10 times from their stock concentration. Antibodies for CD11b were labeled with phycoerythrin (PE) and were diluted 40 times. Antibodies for CD45 labeled with PE-Texas Red (ECD) were used in order to be able to differentiate leukocytes from erythrocytes and platelets. Twenty microliters of blood from an EDTA-anticoagulated blood sample were added to 2.5 µl of each CD66b-FITC, CD11b-PE and CD45-ECD. Cells were incubated for 15 min in the dark at room temperature. Erythrocytes were lysed by adding 300 µl of ice-cold isotonic erythrocyte lysing solution (NH₄Cl 0.19 mol/L; KHCO₃ 0.01 mol/L; Na₂EDTA•2H₂O 0.12 mol/L, pH 7.2) for 15 min.

Antibodies against MPO (Beckman Coulter) were FITC-conjugated. Membrane permeabilization was performed with Intraprep (Beckman Coulter). To each blood sample of 20 µl, 2.5 µl of titrated anti-MPO (2 times diluted) was added. Cells were incubated for 15 min in the dark at room temperature.

A Coulter Epics XL-MCL flow cytometer with a 488 nm Argon ion laser and EXPO 32 software were used for measurement and analysis. Fluorescence intensity of each cell was expressed as the mean fluorescence intensity (MFI), given in arbitrary units (au). Lymphocytes, monocytes and granulocytes were identified based on their side scatter and the level of CD45 on their cell surface.

Patients were considered to have high leukocyte activation if the level of monocyte CD11b, neutrophil CD11b or CD66b expression was above, or intracellular MPO was below, the median of the total group.

Statistical analysis

Data are given as mean±standard deviation (SD) for continuous variables with a normal distribution, and median (interquartile range (IQR)) for continuous variables with a skewed distribution (triglycerides, CRP, leukocyte count, C3, monocyte CD11b expression, neutrophil CD11b and CD66b expression and MPO). Differences in leukocyte activation between groups was tested with the Mann-Whitney-U test.

A Cox-regression analysis was performed to study the impact of leukocyte activation markers on the progression of CAD, using a two-step approach. First, a univariate analysis was performed with several known cardiovascular risk factors as factor, using progression of CAD as event, and time to event as the time to coronary event since CAG at inclusion. The presence of CAD at CAG at inclusion was found to predict progression of CAD, while age (above or below the median of 65 years) and gender did not. In the second step, leukocyte activation markers in the coronary arteries were added to the Cox-regression model with the presence of CAD entered into the model and the individual markers entered as additional variables. A P-value<0.05 (two-sided) was regarded as statistically significant. All statistical analyses were performed using PASW statistics version 22.0 (IBM SPSS Statistics, New York, USA).

Results

General characteristics

A total of 99 subjects were included. Their baseline characteristics are listed in Table 1. The median duration of follow-up was 2242 days (IQR: 2142‒2358). At the time of inclusion, 37 (37%) patients had a history of CAD. In 70 patients (71%), CAD was established by CAG at inclusion, resulting in 33 new diagnoses of CAD: 26 patients had single-vessel disease, 18 patients had two-vessel disease and 26 patients had three-vessel disease. In the remaining 29 patients, no significant coronary stenosis was established by CAG, and none of these patients had a history of clinical CAD.

Presence of CAD at inclusion predicts progression of CAD

During follow-up, 13 patients (13%) showed progression of CAD: 10 patients developed unstable angina pectoris requiring intervention (percutaneous coronary intervention or bypass surgery) and 3 patients developed fatal myocardial infarction. A comparison between the baseline characteristics of those with progression of CAD, and those remaining free of progression, is given in Table 1. All patients with progression of CAD had significant coronary stenosis by CAG at inclusion. In a univariate analysis, only the presence of CAD, established by CAG at inclusion, predicted the progression of CAD (Log-rank test P = 0.014).

Coronary leukocyte activation and risk of progression of CAD

In a Cox-regression analysis, none of the investigated leukocyte activation markers in the coronary circulation contributed significantly to the predictive power of progression of CAD in addition to CAD at inclusion (Table 2). Monocyte CD11b, neutrophil CD11b and CD66b and MPO in the other vascular regions (peripheral vein, femoral artery and abdominal aorta) did not increase the predictive power of progression of CAD either (data not shown).

Discussion

To the best of our knowledge, this is the first prospective study investigating the value of coronary monocyte CD11b, neutrophil CD11b and CD66b expression and intracellular MPO in the prediction of future coronary events. In the present study, in patients scheduled for CAG due to typical chest pain or positive cycle ergometry, these leukocyte activation markers measured in the coronary circulation, or in other vascular regions, did not predict the risk of CAD progression.

We have previously published the baseline values of the investigated leukocyte activation markers in the coronary arteries in this study population [ 26]. While MPO was significantly lower in patients with CAD, the expression of CD11b and CD66b in the coronary circulation was similar in patients with and without CAD [ 26].

Several studies have pointed at a role for leukocyte activation in the development of CAD. Neutrophil infiltration has been associated with acute coronary syndromes, and autopsy studies have shown that ruptured plaques contained more neutral endopeptidase positive neutrophils than eroded plaques [ 27]. In survivors of non-ST elevation myocardial infarction, plasma MPO was associated with short-term risk (<30 days) of recurrent acute coronary syndrome and myocardial infarction, but this association was lost after 180 days [ 28]. In a large prospective study among patients without previous CAD, plasma MPO in peripheral blood was associated with increased risk of future coronary heart disease in men but not in women [ 29]. In addition, the level of neopterin, a marker of monocyte activation, in the peripheral circulation was an independent predictor of future cardiovascular events in patients with and without CAD, indicating the presence of systemic leukocyte activation in CAD [ 30, 31].

The effect of reducing leukocyte activation on CAD has been investigated in animal studies. In rats, treatment with antibodies selectively blocking CD11b or CD18 reduced leukocyte adhesion to endothelial cells [ 13]. Treatment with anti-CD11b or CD18 reduced myocardial infarct size after regional myocardial ischemia in dogs [ 14, 32]. However, studies on the effect of these blocking antibodies on myocardial infarction size or on risk of future coronary events in humans are lacking.

The lack of association with the presently investigated leukocyte activation markers may possibly be explained by the nature of these markers. The integrins CD11b and CD66b can bind to various selectins on the endothelial surface [ 12– 15, 33]. Moreover, activated neutrophils transfer MPO to endothelial cells via the CD11b integrin, contributing further to endothelial cell activation and leukocyte adhesion [ 34]. Therefore, the measurement of these integrins on the leukocyte surface in blood samples may in fact be an underestimation of the true level of leukocyte activation, since activated leukocytes expressing these integrins, adhere to the endothelial surface and may be missed by blood sampling.

Some limitations of the present study are the relatively small number of patients included, the low number of reported coronary events, the relatively short duration of follow-up and the heterogeneity of the included patients. Our study group comprised patients with and without a history of CAD, and many of the study subjects already received intensive treatment for primary or secondary intervention purposes. All of the study subjects with progression already had significant coronary sclerosis at inclusion, and the number of patients without clinical coronary sclerosis at inclusion was low. Therefore, no conclusions can be drawn regarding the value of coronary leukocyte activation in the prediction of future CAD in healthy subjects. A strength of the present study is that this is the first study to investigate coronary leukocyte activation in the progression of CAD in vivo in humans.

In conclusion, expression of monocyte and neutrophil activation markers CD11b and CD66b, and intracellular MPO in the coronary arteries was not associated with the risk of progression of CAD.

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