Atherosclerosis 229 (2013) 338e347

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Flow cytometry and gene expression proling of immune cells of the carotid plaque and peripheral blood
Zohara Sternberg a, *, Husam Ghanim b, Kristen M. Gillotti c, Joseph D. Tario Jr. c, Frederick Munschauer a, Richard Curl d, Sonya Noor d, Jihnhee Yu e, Julian L. Ambrus Sr. a, Paul Wallace c, Paresh Dandona b
a

Stroke Center, Kaleida Medical Center, Buffalo, NY, USA Diabetic Center of Western NY, Kaleida Medical Center, Buffalo, NY, USA Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY, USA d Department of Vascular Surgery, Kaleida Medical Center, Buffalo, NY, USA e Department of Biostatistics, State University of NY at Buffalo, Buffalo, NY, USA
b c

a r t i c l e i n f o
Article history: Received 15 January 2013 Received in revised form 8 April 2013 Accepted 26 April 2013 Available online 14 May 2013 Keywords: Carotid stenosis Cell adhesion Cell surface markers Gene expression Immunophenotyping Mononuclear cells Thrombosis

a b s t r a c t
Objectives: The relative contribution of the local vs. peripheral inammation to the atherothrombotic processes is unknown. We compared the inammatory status of the immune cells of the carotid plaque with similar cells in peripheral circulation of patients with advanced carotid disease (PCDs). Methods: Mononuclear cells (MNCs) were extracted from carotid endarterectomy (CEA) samples by enzymatic digestion and subsequent magnetic cell sorting. The cell surface antigenic expressions, and mRNA expression levels were compared between CEA MNCs and peripheral MNCs, using ow cytometry and RT-PCR techniques. Results: The percentages of resting MNCs were lower, and activated MNCs, particularly monocytes, were higher in the CEAMNCs, as compared to the peripheral MNCs. The percentages of activated T cells and B cells were higher in the peripheral MNCs of PCDs, than in healthy controls (HCs), but the percentages of activated monocytes did not differ between the two groups. The expression levels of both proinammatory/pro-thrombotic (P38, JNKB-1, Egr-1 PAI-1, MCP-1, TF, MMP-9, HMGB-1, TNF-a, mTOR) and anti-inammatory (PPAR-g, TGF-b) mediators were signicantly higher in the CEA MNCs as compared to the peripheral MNCs. Furthermore, MMP-9 and PPAR-g expression levels were higher in the peripheral MNCs of PCDs than HCs. Conclusion: The inammatory status is higher in the immune cells of the carotid plaque, as compared to those cells in the peripheral blood. The altered expression levels of both pro-inammatory/prothrombotic and anti-inammatory mediators in the milieu of the plaque suggest that the balance between these various mediators may play a key role in carotid disease progression. Published by Elsevier Ireland Ltd.

1. Introduction Advanced carotid atherosclerosis accounts for 30e40% of cases of ischemic stroke in the general population, and it leads to functional impairment and death [1]. Immune-inammatory and pro-

thrombotic mechanisms play key roles in the pathology of atherosclerosis, including the progression of the plaque, and its instability leading to rupture [2]. Morphological studies of carotid atherosclerotic plaque show the presence of a rich inltrate, consisting of monocytes-

Abbreviations: ATF, atrial brillation; BP, blood pressure; BMI, body mass index; CV, cardiovascular; CEA, carotid endarterectomy; CDP, carotid disease patients; CVD, cerebrovascular disease; CAD, coronary artery disease; DM, diabetes mellitus; JNK, Jun N-terminal kinase; Egr, early growth response; ERK, extracellular regulated kinase; HC, healthy control; HF, heart failure; HMGB, high-mobility group protein B; mTOR, mammalian target of rapamycin; MRA, magnetic resonance angiography; MMP, matrix metalloproteinase; MAP, mitogen activated protein; MNC, mononuclear cell; MCP, monocyte chemotactic protein; NF-KB, nuclear factor kappa B; P, peripheral; PAD, peripheral arterial disease; PMT, photomultipler tube; PPAR, peroxisome proliferator-activated receptor; PAI, plasminogen activator inhibitor; TLR, toll-like receptor; TGF, transforming growth factor; TF, tissue factor; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule. * Corresponding author. Jacobs Neurological Institute, Buffalo, NY, USA. Tel.: 1 716 859 4235; fax: 1 716 859 2430, 1 716 859 7573. E-mail address: zs2@buffalo.edu (Z. Sternberg). 0021-9150/$ e see front matter Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.atherosclerosis.2013.04.035

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macrophages and lymphocytes, in the plaques of subjects who subsequently developed stroke [3]. The inltrated T cells are often autoreactive, capable of inducing the procoagulant TF in macrophages [4]. Furthermore, the oxidized LDL-stimulated macrophages/ monocytes of the plaque could activate signaling pathways, such as MAP kinases and transcription factors Egr-1 and AP-1, resulting in the subsequent upregulation of the pro-inammatory cytokine TNF-a [2]. In addition, the binding of the ligand, HMGB-1, to the pattern recognition receptors, TLR-2 or TLR-4, could also activate the transcription factor NF-kB [5], promoting TNF-a release from activated monocytes [6,7]. TNF-a is capable of increasing the expression levels of macrophage activation marker, MCP-1 [8], and matrix proteinases [9], facilitating plaque destabilization and rupture. Blocking the activity of TNF-a, or disrupting the expression of its genes, diminishes the development of atherosclerosis in apoE/ mice [10,11]. In addition, the activation of serine/threonine kinase mTOR (the mammalian target for the antibiotic rapamycin), downstream the TLRs, also promotes atherosclerosis [12]. Unlike the detrimental effects of the activated T cells and monocytes, the B type immune cells are atheroprotective. A reduction in B cells, via splenectomy, or the adoptive transfer of spleen cells reduces atherosclerotic lesion development in apoE/ mice [13]. Nevertheless, a recent study shows that B cell depletion reduces atherosclerosis [14]. Immunohistochemical studies show the presence of B cells in the endarterectomy samples, as well as the upregulation of antiinammatory mediators such as the cytokine TGF-b [15]. Via suppressing MCP-1 expression, TGF-b is capable of limiting atherosclerosis [16]. In addition, the nuclear transcription factor, PPAR-g, also plays an anti-inammatory role in the pathogenesis of atherosclerosis [17], via suppressing PAI-1transcription factor [18], and reducing TNF-a production in stimulated macrophages [19]. A gene array study reports dysregulation in the mRNA expression levels of 48% genes in the smooth muscles and endothelial cells of the carotid plaque, as compared to normal arteries [20]. Furthermore, microdissected carotid plaque samples demonstrate differentially expressed patterns of genes in macrophage-rich regions as compared to the smooth muscle cell-containing regions of the plaque [21]. The inammatory status of the plaque correlated with the histological and morphological features related to plaque vulnerability [21]. Patients with carotid disease also demonstrate a higher than normal systemic inammation, indicated by higher TNF-a and MCP-1 serum levels [22]. However, very few studies have measured inammation simultaneously in the carotid plaque and peripheral blood. These studies show higher intra plaque levels of the inammatory markers IL-6 [1] and MMP-9 [23], as compared to their levels in the blood. Due to their critical role in inammatory processes, we separated MNCs from endarterectomy samples of patients with carotid disease, and compared their inammatory status with MNCs derived from the peripheral blood, using ow cytometry and RTPCR techniques. 2. Material and methods 2.1. Population A total of forty (40) patients with advanced carotid disease (PCDs) (23 M, 17 F), ages 70.3 9.5 years, who were enlisted to undergo carotid endarterectomy (CEA) for extracranial high-grade internal carotid artery stenosis (75e99% stenosis indicated by Doppler sonography and/or MRA) were recruited from Buffalo

General Hospital, Buffalo, NY. The time delay between symptoms to the carotid endarterectomy ranged between 1 and 10 days depending on the severity of the symptoms. For asymptomatic patients, the average delay between diagnosis and the surgical intervention was three months. Patients sample was compared with 32 (18 M, 14 F) agematched (68.3 9.2 years) healthy controls (HCs). The HCs were recruited from the Buffalo General Hospital Volunteers ofce and among the hospital staff. Exclusion criteria for HCs included reports of any self and/or family history of vascular disease (CAD, CVD, PAD), renal disease, and DM. All investigations were approved by the Institutional Review Board of the University of Buffalo, and informed consent was obtained from PCDs before scheduled surgery, and from HCs.

2.2. Measurements Due to the relatively limited number of MNCs in the CEA samples, 16/40 CEA samples were devoted to ow cytometry studies, and 16/40 to gene expression proling. The other 8 CEA samples were unsuitable for analysis for one reason or another. Among the HC blood samples, 12 MNCs samples were used for ow cytometry, and 20 others for gene proling. Using specic cell surface markers, the levels of resting (T cells, monocytes, B cells) and activated (HLADR, macrophages, plasma cells) MNCs were determined in 16 CEA samples, and compared with similar cells in the peripheral circulation of PCDs and HCs. In addition, the mRNA expression levels of the proinammatory/pro-thrombotic and anti-inammatory mediators were compared between CEA MNCs and peripheral MNCs of PCDs. The pro-inammatory/pro-thrombotic mediators included MAP kinases, P38, JNKb-1, ERK-1, and the serine/threonine kinase mTOR; the pattern recognition receptors, TLR-2 and TLR-4; the ligand to the pattern recognition receptors, the transcription factor, HMGB-1, and the transcription factor Egr-1; the coagulation factors, PAI-1 and TF; the marker of plaque vulnerability, MMP-9 and the cytokine TNF-a; and the chemokine, MCP-1. The anti-inammatory mediators included the transcription factor, PPAR-g and the cytokine, TGFb-1. Furthermore, the expression levels of selected genes, including HMGB-1, Egr-1, PAI-1, MMP-9, TNF-a, MCP-1, and PPAR-g were compared between peripheral MNCs of 17 PCDs and 20 HCs.

2.3. Reagents The following uorochrome-conjugated anti-human monoclonal antibodies to cell surface receptors were used to determine the activation state of the MNCs subpopulations. The CD45-APC (purchased from BD) was used to gate MNCs (T cells, monocytes and B cells). The CD3-FITC (purchased from R&D Systems), HLADRPerCP (purchased from BD) were used to tag the resting and activated T cells respectively. The CD14-PerCP (purchased from BD) and CD68-FITC (purchased from Invitrogen) were used to tag the resting and activated monocytes/macrophages respectively. The CD20-PE and CD69-FITC (purchased from BD) were used to tag the resting and activated B cells/plasma cells respectively. CD45-APC antibody-covered microbeads (Miltenyi Biotec) was used to separate CEAMNCs. Additional reagents were Ficoll-Hypaque and human IgG (Sigma Aldrich), endotoxin-free Blenzyme III (combination of collagenase and thermolysin) and Porcine elastase II (Roche Applied Biosciences). Primers for the genes under study were commercially available.

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2.4. Sample processing Blood samples from both PCDs and HCs were drawn in EDTA tubes. Patients blood was drawn from an indwelling catheter on the day of surgery and processed within an hour. Venous blood was obtained from HCs and was processed similarly. CEA surgical samples were taken from patients and immediately transferred on ice to the laboratory for processing. All samples were processed by a single research scientist. 2.5. Immune cell isolation 2.5.1. Peripheral blood The peripheral blood was diluted 1:1 with PBS (pH 7.3) and layered on Ficoll, spun at 400 g for 30 min to obtain the peripheral blood MNCs. The cell pellets were washed three times with the Hanks Buffered Salt Solution (HBSS). 2.5.2. CEA samples The available ultrasound/MRA tests results were limited to the degree of stenosis, but did not provide information on the plaque morphology. Therefore, CEA samples were graded by the naked eye for the degree of calcication, fattiness, and thrombosis. The grading ranged from 1 to 5 (1 being the lowest, and 5 being the highest). Subsequently, CEA samples were weighted, washed twice, and meticulously cleared of peripheral blood and thrombotic materials. To release plaque inltrating immune cells, the plaques were minced into paste and digested with 60 mg/ml Blenzyme III and 100 mg/ml elastase for an hour in 37  C in a rotating incubator. The volume of the buffer was adjusted to the weight of the CEA samples (10 ml buffer/g of tissue). The digestion was stopped by the addition of HBSS, which included 0.5% BSA and 2 mM EDTA. The resulting cell suspension was ltered through serial lters from coarse to ne 30 microns). The cell pellets was washed twice with the same solution and resuspended in 1 ml of the buffer. Subsequently, MNCs were separated from endothelial and smooth muscle cells by a positive selection, using CD45 antibody-covered microbeads, according to the manufacturers instructions. In order to minimize MNCs activation, the magnetic cell sorting was conducted in a cold room. 2.6. Quality control tests 1. Preliminary studies were conducted to determine whether magnetic cell sorting activates MNCs, by receptor crosslinking. Ficoll-derived peripheral MNCs of 7 HCs were divided to two equal parts. One part was subjected to magnetic cell sorting, similar to the protocol applied to separate MNCs from the CEA samples, whereas the other part was not. The effect of magnetic cell sorting on MNCs activation was assessed using cell surface antibodies to activated T cells and monocytes. 2. In order to exclude the possibility of digestive enzymes activating MNCs, Ficoll-derived peripheral MNCs from two patients were randomly selected and equally divided into two parts. One part was treated with Blenzyme and elastase, similar to the protocol applied to digest CEA samples, while the other part was not. 2.6.1. Immunophenotyping The protocol for immunophenotyping has been described in details in Suppl I. 2.6.2. RT-PCR technique Total RNA was isolated using a commercially available RNAqueous(R)-4PCR Kit (Ambion, Austin, TX). RT-PCR was performed using Stratagene Mx3000P QPCR System (La Jolla, CA), Sybergreen

master mix (Qiagen, CA) and gene specic primers (Invitrogen). All values were normalized to the expression of a group of housekeeping genes including actin, ubiquitin C and cyclophilin A. 2.7. Statistical analysis All statistical analyses were performed using SPSS 14.0 for Windows (SPSS Institute, Chicago, Illinois, USA). Pairwise t-test was carried out to determine the differences in the percentages of resting and activated cells, and differences in gene expression levels, between CEA MNCs and peripheral MNCs. Differences between PCDs/ HCs were analyzed by independent t-test. The Holms sequentially rejective algorithm was applied to correct for multiple comparisons [24], and P-values of 0.01 were considered signicant. Spearman correlation was used to investigate the association between MNCs activation state, plaque morphology (thrombosis, calcication, fattiness), and patients clinical characteristics (age, BMI, systolic and diastolic BP, plasma glucose), and between expression levels of inammatory-related mediators, plaque morphology, and patients characteristics. 3. Results 3.1. Patients characteristics Table 1 presents the characteristics of the PCDs and HCs. In addition to the carotid disease, a signicant number of PCDs presented with other CV related diseases, including CAD, PAD, HF and ATF. In addition, CV drugs were commonly used by PCDs. As many as 100% of PCDs were on antihypertensive/diuretics, 97.5% were on antiplatelets/anticoagulants, and 75% were on hypolipidemic drugs. Furthermore, 42.5% of PCDs were on anti-diabetic drugs. However, a signicantly lower percentage of HCs were on antihypertensives/diuretics (41.6%), antiplatelets/anticoagulants (16.6%), hypolipidemics (16.6%), and antidiabetic (0%) drugs. The carotid disease was symptomatic in 32.5% (13/40) of PCDs. CEA samples were dened as symptomatic if symptoms were consistent with stroke or transient ischemic attack. 3.2. CEA characteristics The mean SD weight of the obtained CEA samples was 0.92 0.4 g, ranging from 0.3 to 1.9 g. The amount of mRNA
Table 1 Clinical characteristics of PCDs and HCs. PCDs Clinical characteristics Number of subjects Age (years) BMI (kg/m2) Plasma glucose Systolic BP/diastolic BP (mmHg) Symptomatic carotid plaque (%) CV drug usage Antihypertensives/diuretics (%) Antiplateletes/anticoagulants (%) Hypolipidemics (%) Antidiabetics (%) Other CV diseases CAD (%) PAD (%) HF (%) ATF (%) 40 (23 M, 17 F) 70.3 9.5 29.9 6.2 132.5 44.2 146.2 24.9/74.42 10.1 32.5 (13/40) 100 97.5 75 42.5 (17/40) 57.5 30 10 5 HCs 32 (18 M, 14 F) 68.3 9.2 26.9 4.4 NT NT NA 41.6 16.6 16.6 0 NT NT NT NT

Abbreviations: ATF: atrial brillation, BMI: body mass index, CV: cardiovascular, CAD: coronary artery disease, HC: healthy control, HF: heart failure, PAD: peripheral artery disease, PCD: patient with carotid disease.

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extracted from plaque ranged from 0.4 to 2.0 mg depending on the weight and cellularity of the plaque. 3.3. Quality control tests The magnetic cell sorting had no signicant effect on either T cells or monocytes activation states. Approximately 13.4 2% of the total T cells were activated after magnetic cell sorting, as compared to 10.7 0.1% without this treatment. Similarly, 4.8 0.02% of monocytes were in activated states compared to 7.7 0.03% without this treatment. Furthermore, the percentage of activated T cells, which comprise the majority of the cells in the plaque, was similar in the presence (13.6 0.6%) and absence (12.0 1.2%) of Blenzyme and elastase treatments (P-values > 0.05). Our results agree with those of an earlier study reporting no effect of enzymatic digestion on cells antigenic properties [25]. 3.4. Flow cytometry studies Fig. 1 presents the mean SEM percentages of resting (Fig. 1A) and activated (Fig. 1B) T cells, monocytes, and B cells in the CEA samples, and in the peripheral blood of PCDs and HCs. The resting cells are dened as: (Marker Positive Events e Activated Cells/Marker Positive Events)*100, whereas activated cells are dened as: (Activated Cells/Activated Cells Resting Cells)*100, as described in Suppl 1.

Percentages (resting cells)

100 90 80 70 60 50 40 30 20 10 0

CEA
* *

PCD HC

The percentages of resting T cells were near-signicantly lower in the CEA MNCs, than in the peripheral MNCs of PCDs (70.5 4.5% vs. 80.9 3.2%, P 0.03) (P < 0.01 is considered statistically signicant), but the percentages of peripheral resting T cells did not differ signicantly between PCDs and HCs (85.0 4.1%). In addition, the percentages of resting monocytes were signicantly lower in the CEA MNCs, than in the peripheral MNCs of PCDs (52.4 11.9% vs. 88.7 3.7%, P 0.006), but the percentages of peripheral resting monocytes did not differ between PCDs and HCs (91.1 2.8%). The percentages of resting B cells were signicantly lower in the CEA MNCs, than in the peripheral MNCs of PCDs (79.0 7.3% vs. 98.8 0.2%, P < 0.001), but the percentages of peripheral resting B cells did not differ between PCDs and HCs (99.5 0.09%) (Fig. 1A). The percentages of activated T cells were near-signicantly higher in the CEA MNCs, than in the peripheral MNCs of PCDs (30.4 5.1% vs. 20.8 3.4%, P 0.07), but the percentages of peripheral activated T cells were not statistically different between PCDs and HCs (15.1 3.8%). In addition, the percentages of activated monocytes were signicantly higher in the CEA MNCs, than in the peripheral MNCs of PCDs (37.75 4.7% vs. 8.9 2.3% P < 0.001), but the percentages of peripheral activated monocytes were similar between PCDs and HCs (7.57 1.9%). Furthermore, the percentages of activated B cells were higher in the CEA MNCs, than in the peripheral MNCs of PCDs (17.9 5.2% vs.1.1 0.2%, P < 0.001). In addition, the percentages of the peripheral activated B cells were higher for PCDs, than HCs (0.40 0.09%, P 0.004) (Fig. 1B). Fig. 2 presents ow cytometric histograms of cells in the peripheral blood (Fig. 2a) and the carotid plaques (Fig. 2b). The ow cytometric acquisition and analysis of leukocyte populations are described in details in the legend to Fig. 2. 3.5. Gene proling studies of CEA MNCs and peripheral MNCs of PCD Fig. 3 and Table 2 compare (pair-wise comparison) the mean SEM of the mRNA expression levels of the pro-inammatory/prothrombotic and anti-inammatory mediators between CEA MNCs and peripheral MNCs of PCDs. The pro-inammatory/pro-thrombotic mediators included P38, JNKb-1, ERK-1 and mTOR (Fig. 3A); TLR-2 and TLR-4 (Fig. 3B); HMGB-1 and Egr-1 (Fig. 3C); PAI-1 and TF (Fig. 3D); MMP-9 and TNF-a (Fig. 3E); and MCP-1 (Fig. 3F). The antiinammatory mediators included PPAR-g (Fig. 3G) and TGFb-1 (Fig. 3H). The expression levels of P38 (0.184 0.02 vs. 0.116 0.01, P 0.004), JNKB-1 (0.239 0.02 vs. 0.122 0.01, P < 0.001), and mTOR (0.156 0.02 vs. 0.11 0.01, P 0.006) were signicantly higher in the CEA MNCs, than in the peripheral MNCs, but after adjusting for multiple comparisons (P values of 0.01 was considered signicant), the differences in ERK-1 expression levels were not statistically signicant (0.135 0.01 vs. 0.111 0.01, P 0.043) (Fig. 3A). In addition, differences in the mRNA expression levels of TLR-2 (0.089 0.01 vs. 0.07 0.02, P 0.34) and TLR-4 (0.218 0.02 vs. 0.184 0.02, P 0.29) between CEA MNCs and peripheral MNCs were not statistically signicant (Fig. 3B). The expression levels of HMGB-1 (2.81 0.29 vs. 0.20 0.01, P < 0.001) and Egr-1 (1.03 0.13 vs. 0.13 0.03, P < 0.001) were signicantly higher in the CEA MNCs, than in peripheral MNCs of PCDs (Fig. 3C). Similarly, the expression levels of PAI-1 (2.48 0.4 vs. 0.156 0.029, P < 0.001) and TF (6.15 1.2 vs. 0.177 0.07, P < 0.001) were signicantly higher in the CEA MNCs, than in peripheral MNCs of PCDs (Fig. 3D). The expression levels of MMP-9 (3.97 1.0 vs. 0.165 0.05, P < 0.001) and TNF-a (0.8 0.14 vs. 0.10 0.01, P < 0.001) were signicantly higher in the CEA MNCs, than in the

T cells

Monocytes Cell subpopulations

B cells

A
Percentages (activated cells)
50 45 40 35 30 25 20 15 10 5 0

CEA

PCD HC

T cells

Monocytes Cell subpopulations

B cells

Fig. 1. Comparison between resting and activated MNCs of the carotid plaque and peripheral circulation. The Figure presents the mean SEM percentages of resting (A) and activated (B) T cells, monocytes, and B cells in the CEA MNCs and peripheral MNCs of PCDs and HCs. P  0.01 was considered signicant. *, Signicant differences in the percentages of MNCs subpopulations between CEA samples and peripheral MNCs of PCDs. y, Signicant differences between the percentages of MNCs subpopulations in the peripheral MNCs of PCDs and HCs. Abbreviations: A: activated, CEA: carotid endarterectomy, PCD: patients with carotid disease, HC: healthy control, R: resting.

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Fig. 2. Representative ow cytometric data from cells in the peripheral blood (a) and the carotid plaques (b). The acquisition and analysis of leukocyte populations in patient and normal samples from peripheral blood and carotid plaques were performed by ow cytometry, using uorescently-labeled antibodies with specicity for T cells (anti-CD3), monocytes (anti-CD14), and B cells (anti-CD20). Activation states for each of these subsets were determined by evaluation of HLA-DR on T cells, CD68 on monocytes, and CD69 on B cells. To analyze the samples, the scatter characteristics of cellular populations were identied using a bivariate histogram of FSC vs. SSC (Panel A). An elliptical region (R1) was used to circumscribe the leukocyte populations for both Peripheral (a) and CEA (b) samples, using the peripheral blood sample as a reference. Thereafter, scatter-inclusive cells (R1) were gated to a histogram of SSC vs. APC CD45 (Panel B). A rectangular region (R2) was used to discriminate CD45 events from CD45 events. Cells that were scatter-inclusive and CD45 were subsequently evaluated for the activation state of each respective measured population. For T cells, R2 events were gated to a bivariate histogram of CD3 vs. HLA-DR (Panel C); for monocytes, R2 events were gated to a bivariate histogram of CD14 vs. CD68 (Panel D), and for B cells, R2 events were gated to a bivariate histogram of CD20 vs. CD69 (Panel E). Quadstat regions were placed on each of the aforementioned plots to distinguish between marker-positive events that were resting (R3), and marker-positive events that were activated (R4).

peripheral MNCs of PCDs (Fig. 3E). Similarly the expression levels of MCP-1 were signicantly higher in the CEA MNCs, than in peripheral MNCs of PCDs (25.87 4.2 vs. 0.113 0.02, P < 0.001) (Fig. 3F).

Furthermore, the expression levels of PPAR-g (3.26 0.47 vs. 0.156 0.03, P < 0.001) (Fig. 3G) and TGF-b (0.166 0.01 vs. 0.123 0.01, P < 0.001) (Fig. 3H) were signicantly higher in the CEA MNCs, than in peripheral MNCs of PCDs.

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Fig. 3. Inammatory-related mediators in the CEA MNCs and peripheral MNCs of PCDs. The Figure compares (pair-wise) the mean SEM mRNA expression levels of the proinammatory and anti-inammatory mediators between CEA MNCs (n 16) and peripheral MNCs (n 16) of PCDs. The pro-inammatory mediators included MAP kinases, P38, JNKb-1, ERK-1, and the serine/threonine kinase mTOR (A); the pattern recognition receptorsTLR-2 and TLR-4 (B); the ligand to the pattern recognition receptor, the transcription factor HMGB-1, and the transcription factor Egr-1 (C); the coagulation factors PAI-1 and TF (D); the marker of plaque vulnerability, MMP-9, the pro-inammatory cytokine TNF-a (E); and the pro-inammatory chemokine, MCP-1 (F). The anti-inammatory mediators included the transcription factor PPAR-g (G), and the cytokine TGF-b (H). The asterisks show signicant differences in the mRNA expression levels between CEA MNCs and peripheral MNCs of PCDs. P  0.01 was considered signicant. Abbreviations: CEA: carotid endarterectomy, HMGB: high-mobility group protein B, JNK: c-Jun N-terminal kinase, EGR: early growth response, ERK: extracellular regulated kinase, mTOR: mammalian target of rapamycin, MAP: mitogen activated protein, MMP: matrix metalloproteinase, P38: mitogen-activated protein kinase, MNC: mononuclear cell, MCP: monocyte chemotactic protein, PCD: patient with carotid disease, P: peripheral, PPAR: peroxisome proliferator-activated receptor, PAI: plasminogen activator inhibitor, TLR: tall like receptor, TGF-b: transforming growth factor beta, TF: tissue factor, TNF: tumor necrosis factor.

Regression analysis revealed an association for mTOR (r 0.706, P 0.005) and TGF-b (r 0.583, P 0.002) expression levels, between CEA MNCs and peripheral MNCs of PCDs. 3.6. Peripheral MNCs of PCDs and HCs Fig. 4 and Table 3 compare the mean SEM mRNA expression levels of the pro-inammatory mediators HMGB-1 and Egr-1 (Fig. 4A); PAI-1 and MMP9 (Fig. 4B); TNF-a, and MCP-1 (Fig. 4C); and the anti-inammatory mediator, PPAR-g (Fig. 4D) between peripheral MNCs of PCDs and HCs. The mRNA expression levels of HMGB-1 (0.236 0.01 vs. 0.233 0.014) and Egr-1 (0.370 0.09 vs. 0.215 0.05) were not signicantly different between peripheral MNCs of PCDs and HCs (Fig. 4A). However, MMP-9 expression levels were signicantly higher in the peripheral MNCs of the PCDs than in HCs (0.394 0.11 vs. 0.08 0.01, P 0.002), but no signicant group differences in PAI-1 expression levels were observed (0.313 0.07 vs. 0.255 0.05) (Fig. 4B). The MCP-1 expression levels tended to be

lower in the peripheral MNCs of PCDs than in HCs (0.191 0.05 vs. 0.243 0.03, P 0.08), but no group differences in TNF-a expression levels were observed (0.119 0.02 vs. 0.120 0.028) (Fig. 4C). Furthermore, the expression levels of PPAR-g were higher in the peripheral MNCs of the PCDs than in HCs (0.183 0.03 vs. 0.090 0.02, P 0.01) (Fig. 4D).

3.7. Correlation studies A higher plaque weight was associated with the symptomatic plaque (r 0.62, P 0.010). Furthermore, a signicant relationship was observed between plasma glucose levels, and MCP-1 expression levels in the peripheral MNCs of PCDs (r 0.568, P 0.001). No other signicant associations between the expression levels of inammatory mediators (either in the CEA MNCs or peripheral MNCs), and patients clinical characteristics were observed. In addition, the percentages of resting and activated MNCs did not correlate with plaques characteristics or with patients clinical

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Fig. 3. (Continued)

characteristics. Values of lipid prole were not available for correlation studies. 4. Discussion We separated MNCs from endarterectomy samples and compared their inammatory states with similar cells in the
Table 2 Comparison of the mRNA expression levels of the pro-inammatory and antiinammatory mediators between CEA MNCs and peripheral MNCs of PCDs. CEA-MNC Pro-inammatory P38 JNK-1 ERK-1 mTOR TLR-2 TLR-4 HMGB-1 Egr-1 PAI-1 TF MMP-9 TNF-a MCP-1 mediators 0.184 0.02 0.239 0.02 0.135 0.01 0.156 0.02 0.090 0.01 0.218 0.02 2.81 0.29 1.03 0.13 2.488 0.45 6.58 1.20 3.97 1.07 0.80 0.14 25.87 4.2 Peripheral-MNC 0.116 0.122 0.111 0.110 0.077 0.184 0.205 0.130 0.156 0.177 0.165 0.10 0.11 0.02 0.01 0.01 0.01 0.02 0.02 0.01 0.03 0.03 0.07 0.05 0.01 0.02 P-value 0.004 <0.001 0.043 0.006 0.34 0.29 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Anti-inammatory mediators 3.26 0.47 PPAR-g 0.166 0.01 TGF-b

0.297 0.14 0.123 0.01

P < 0.01 is statistically signicant. Abbreviations as in Fig. 3.

peripheral blood of patients suffering from advanced carotid disease, by ow cytometry and RT-PCR techniques. Our results show that most MNCs in the CEA samples were T cells and monocytes, whereas signicantly smaller percentages were B cells. These results are consistent with morphological studies showing the presence of T cells, monocytes, and B cells in the carotid plaque using CD3, CD14 and CD20 cell surface antibodies respectively [26]. However, a recent ow cytometry study, which used CD19 antibody as the B cells surface marker, did not observe a signicant B cell labeling in the carotid plaque [25]. This discrepancy may stem from the nature of CD19 being expressed mainly in the early pro-B cell stage, whereas the CD19 expression is down-regulated upon B cell maturation to plasma cells. In addition, we observed higher percentages of activated MNCs, especially activated monocytes, in the CEA MNCs, as compared to the peripheral MNCs. These observations are consistent with the results of our genetic study showing higher expression levels of the pro-inammatory markers in the CEA MNCs, as compared to their expression levels in the peripheral MNCs. For a number of inammatory mediators, including TF and MMP-9, increases in the mRNA expression levels in the CEA MNCs approximated 100 times that of the peripheral MNCs, suggesting heightened immune cells-derived inammatory responses in the milieu of the plaque [1]. Moreover, it suggests that these mediators may play critical roles in the progression of carotid disease, including plaque rupture and thrombosis. The increases in the expression levels of the inammatory mediators were not limited to the pro-inammatory/pro-thrombotic

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1.2 1.0 0.8

P-PCD P-HC
0.6

P-PCD P-HC

Arbitrary Units

Arbitrary Units

0.6 0.4 0.2

0.4

0.2

0.0
0.0

A
1.4 1.2

HMGB-1

Egr-1

TNF-alpha

MCP-1

P-PCD P-HC

0.4

Arbitrary Units

1.0 0.8 0.6 0.4 0.2

0.3

Arbitrary Units

P-PCD P-HC

0.2

0.1

0.0
0.0

PAI-1

MMP-9

PPAR-gamma

Fig. 4. Inammatory-related mediators in the peripheral MNCs of PCDs and HCs. The Figure compares the mean SEM mRNA expression levels of the proinammatory transcription factors, HMGB-1 and Egr-1 (A); the coagulation factor, PAI-1, and the marker of plaque vulnerability, MMP9 (B); the cytokine TNF-a and the chemokine MCP-1 (C); and the anti-inammatory transcription factor, PPAR-g, between peripheral MNCs of PCDs (n 17) and HCs (n 20). The asterisks show the signicance of the differences in the expression levels of inammatory-related markers between peripheral MNCs of PCDs and HCs. P  0.01 was considered signicant. Abbreviations as in Fig. 3.

ones. The anti-inammatory mediators, such as PPAR-g and TGF-b, were also signicantly elevated in the CEA MNCs, as compared to their expression levels in the peripheral MNCs. The increase in PPAR-g in the CEA MNCs also approximated 100 times that of the peripheral MNCs. Our results collectively suggest that the balance between pro-inammatory/pro-thrombotic mediators and antiinammatory mediators may modulate plaque progression.
Table 3 Comparison of the mRNA expression levels of the pro-inammatory and antiinammatory mediators between the peripheral (P) MNCs of PCDs and HCs. PCD (P-MNCs) Pro-inammatory mediators HMGB-1 0.236 0.01 Egr-1 0.37 0.09 MMP-9 0.394 0.11 PAI-1 0.31 0.07 MCP-1 0.191 0.05 0.119 0.02 TNF-a Anti-inammatory mediator 0.183 0.03 PPAR-g HC (P-MNCs) 0.233 0.169 0.08 0.255 0.243 0.120 0.01 0.03 0.01 0.05 0.03 0.02 P-value 0.79 0.12 0.002 0.79 0.089 0.54 0.01

0.09 0.02

P < 0.01 is statistically signicant. Abbreviations as in Fig. 3.

However, it should be noted that inammatory mediators such as p38 [27,28] and PPAR-g [17,29] have the potential to exert both a proatherogenic and an anti-atherogenic role. Nevertheless, the increase in p38 and other MAP kinases has been shown to accompany the progression of carotid atherosclerosis in humans [30]. Due to a relatively small sample size, this study did not analyze results in patients subgroups. Therefore, future studies, involving a larger cohort, should examine immune inammatory status in symptomatic/asymptomatic and in diabetic/non-diabetic patients. One of the interesting observations in this study was the signicant increase in the expression levels of HMGB-1 in the immune cells of the carotid plaque. HMGB-1, also known as amphoterin, is a transcription factor, and a key cytokine with a potential role in nucleosome formation [31]. However, upon release from activated macrophages, HMGB-1 has the potential to promote key processes involved in the initiation and progression of atherosclerosis, including inammation [32] and smooth muscle cell proliferation [33]. HMGB-1expression levels are enhanced in the atherosclerotic lesions, as compared to the levels in the normal arteries [33]. In addition, HMGB-1 reduction is associated with a reduced atherosclerotic plaque size and favorable changes in the plaque

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morphology in mice lacking RAGE, the receptor for advanced glycation end-products [34]. In addition to the RAGE, signaling through TLR-2, TLR-4 also contributes to HMGB-1-induced inammatory responses [6]. We did not measure RAGE expression levels, but TLR-2 and TLR-4 are known to be the main receptors mediating HMGB-1-induced proinammatory signaling in activated monocytes [5]. However, these two receptors were not upregulated in the CEA MNCs. It is possible that HMGB-1 signaling through TLR-2 and TLR-4, in MNCs, occurs in the early stages of the carotid lesion development, whereas these receptors are downregulated in advanced carotid lesions [5]. In addition, the percentages of activated monocytes, known to be critical in the pathology of atherosclerosis [35], were similar between peripheral MNCs, and except for MMP-9 and PPAR-g, which showed differences, the expression levels of other inammatory markers were not signicantly different between PCDs and HCs. One factor that could lower the levels of activated monocytes, and the expression levels of inammatory markers in the peripheral MNCs of PCDs is the aggressive treatment of this group with a combination of CV drugs, including antihypertensives, antiplatelets, and hypolipidemics. In addition, many patients were using multiple classes of CV drugs. Many CV drugs possess antiinammatory properties, capable of reducing peripheral inammation [36e38]. Similarly, anti-diabetic drugs reduce immune cell activation and the production of inammatory mediators [22,39]. Nevertheless, despite the chronic use of CV drugs, these patients presented with severe carotid stenosis, suggesting the complexity of factors, which are involved in the progression of the carotid plaque. Whether CV drugs inuence MNCs subpopulations differently warrants further investigations. However, unlike their levels in the peripheral circulation, the signicantly higher percentages of activated monocytes in the CEA samples suggest that CV drugs may be less effective in reducing activated monocytes in the advanced carotid plaque [40]. Furthermore, the relative contribution of the resting vs. activated MNCs to the inammatory processes of the carotid plaque is not well known. The results of an in vitro study show [41] that the pro-inammatory and plaque destabilizing cytokine, IL-17, is produced by both resting T cells and B cells as well as by activated monocytes and activated B cells. Similarly, both resting and activated T cells could stimulate endothelial cells, promoting the release of pro-atherogenic mediators, such as E-selectin and VCAM1 [42]. In addition, MNCs are heterogeneous in nature. For example, different subsets of T cells are found in atherosclerotic plaque, including Th1 cells, Th2 cells, dendritic cells, and T regulatory cells, to mention a few. Based on the nature of their cytokine production, Th1 cells are thought to promote atherogenesis, whereas Th2 cells inhibit it [43]. Furthermore, dendritic cells are known to promote atherosclerosis through pathways involving VCAM1 [44], whereas CD4 regulatory T cells augment TGF-b production, exerting anti-inammatory and anti-atherogenic activities [45]. Similarly, more than one monocyte subtype may be involved in atherogenesis. Monocytes heterogeneity depends on a set of chemokine receptors expressed on these cells, which dictates their migration properties, their role in plaque formation, and their accumulation in the plaque in the form of macrophages [46,47]. Furthermore, differences in B cells subpopulations have been observed, and their ability to either promote or suppress atherogenic processes have been documented [48,49]. Variations in MNCs sub-populations and subtypes, the interactions among the different cells, and their derived mediators

are few of the complex factors contributing to the progression of the carotid plaque. Whether pharmacological treatment and the reduction of systemic inammation prevents or slows down the progression of the advanced carotid disease warrants further investigations. This knowledge will have a signicant impact on future patients treatment. 4.1. Study limitations All our patients were using CV drugs; therefore, we could not examine the possible effects of these drugs on immune inammatory cells. In addition, MRA or ultrasound data were not available for HC group, and therefore, one could not exclude the existence of carotid and or coronary artery disease in this group. Funding source This study was supported in part by a grant from Jog for the Jake, a local philanthropic Foundation. Disclosure Authors report no conict of interest. Acknowledgment The authors thank the nurses in the surgical department of Buffalo General Medical Center for their help in providing endarterectomy samples. Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.atherosclerosis.2013.04.035. References
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