Neutrophil Activation Status in Stable Coronary Artery Disease

BACKGROUND: During the last years, neutrophils have emerged as important players in atherogenesis. They are highly activated in peripheral blood of patients with unstable angina. Moreover, a primed state of circulating neutrophils has been proposed in patients with stable angina. Our aim was to investigate the neutrophil activation status in patients with stable coronary artery disease (CAD) at conventional drug treatment. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty patients with stable CAD and 30 healthy controls were included using a paired design. The neutrophil expression of CD18 and high-affinity state of CD11b was analysed by flow cytometry before and after stimulation with chemoattractants. Also, the production of reactive oxygen species (ROS) was determined by chemiluminescence. During basal conditions, the neutrophil expression of CD18 or high-affinity state of CD11b did not differ between patients and controls. Chemoattractants (Interleukin-8 and Leukotriene B(4)) did not increase either the expression or the amount of high-affinity CD11b/CD18-integrins in CAD patients compared to controls, and had no effect on the production of ROS. On the other hand, the ROS production in response to C3bi-opsonised yeast particles and the neutrophils' inherent capacity to produce ROS were both significantly decreased in patients. CONCLUSION/SIGNIFICANCE: We could not find any evidence that neutrophils in patients with stable CAD were primed, i.e. more prone to activation, compared to cells from healthy controls. According to our data, the circulating neutrophils in CAD patients rather showed an impaired activation status. It remains to be elucidated whether the neutrophil dysfunction in CAD is mainly a marker of chronic disease, an atherogenic factor or a consequence of the drug treatment

Clinical and laboratory characteristics of patients and controls.

1)<p>Data are given as median (inter-quartile range).</p>2)<p>ACE-I, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blockade</p>3)<p>Measured at the accredited clinical chemistry laboratory at Linköping University Hospital, Sweden.</p>4)<p>CRP, C-reactive protein</p

Baseline expression of CD18 and high-affinity state CD11b on neutrophils in patients with stable CAD.

<p>Whole blood from patients (P) and individually matched control persons (C) were incubated at 37°C with (A,C) the MHM23-antibody or with (B,D) the CBRM1/5-antibody for 5 and 10 min, respectively, whereafter the samples were incubated on ice. Contaminating erythrocytes were removed by lysis, and antibody bound to CD18 or to CD11b-integrins displaying the high-affinity epitope was detected by FACS analysis. The cells were gated to identify the neutrophil population, and 10 000 cells were analyzed in each sample. Data are shown as one representative histogram plot (A,B) or as median of the mean fluorescence intensity (MFI) values (C,D) from 30 (A,C) and 25 (B,D) experiments per cohort done in triplicate. Mean value is indicated as a black hyphen. Wilcoxon signed-ranks test was used to determine significance between each patient and its individually matched control.</p

IL-8 and LTB₄ induced expression of CD18 on neutrophils from patients with stable CAD.

<p>Whole blood from patients (P) and individually matched control persons (C) were stimulated for 10 min at 37°C with (A,C) IL-8 (10 ng/mL) or (B,D) LTB₄ (100 nM), and the samples were co-incubated with the MHM23-antibody during the last 5 min of stimulation. The stimulation was stopped by incubation the samples on ice, and contaminating erythrocytes were removed by lysis. Antibody bound to CD18 was detected by FACS analysis. The cells were gated to identify the neutrophil population, and 10 000 cells were counted in each sample. Data are shown as one representative histogram plot (A,B) or as median of the mean fluorescence intensity (MFI) values (C,D) from 22 (A,C) and 30 (B,D) experiments per cohort done in triplicate. Mean value is indicated as a black hyphen. Wilcoxon signed-ranks test was used to determine significance between each patient and its individually matched control.</p

IL-8 and LTB₄ induced ROS production in neutrophils from patients with stable CAD.

<p>The production of ROS in response to (A–B) IL-8 (100 ng/mL), (C–D) LTB₄ (100 nM), or (E–F) PMA (50 nM), was measured in isolated neutrophils from patients (black dashed line and black dot, respectively) and individually matched controls (grey line and grey dot, respectively). Data are expressed as counts per min (cpm) and shown as one representative histogram plot (A,C,E) or as total ROS production peak value from 9–10 experiments per cohort (B,D,F). Mean value is indicated as a black hyphen. Wilcoxon signed-ranks test was used to determine significance between each patient and its individually matched control (** represents significant difference; <i>p</i><0.01).</p

A representative gating to identify the neutrophil population in the flow cytometry analysis.

<p>G: Granulocytes, M: Monocytes, L: Lymphocytes, FSC: Forward scatter, SSC: Side scatter.</p