Published online before print November 8, 2007, doi: 10.1161/ATVBAHA.107.154807
© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Accumulation of Myeloperoxidase-Positive Neutrophils in Atherosclerotic Lesions in LDLR–/– Mice
From the Departments of Clinical and Experimental Immunology (M.v.L., A.D., M.S., M.J.v.d.G., P.H., J.W.C.T.), Molecular Genetics (M.J.J.G., M.P.J.d.W.), and Pathology (M.J.J.G.), Cardiovascular Research Institute Maastricht, Maastricht University; and the Department of Pathology and Laboratory Medicine (P.H.), Medical Biology Section, University Medical Center Groningen, Groningen, the Netherlands.
Correspondence to M.P.J. de Winther, Department of Molecular Genetics, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. E-mail dewinther{at}gen.unimaas.nl
Abstract
Objective— Atherosclerosis is a chronic inflammatory disease in which the immune system plays an important role. Neutrophils have not been thoroughly studied in the context of atherogenesis. Here, we investigated neutrophils in the development of murine atherosclerotic lesions.
Methods and Results— LDLR–/– mice were given a high-fat diet for different time periods and subsequently atherosclerotic lesions were studied by immunohistochemistry. Staining with anti–Ly-6G monoclonal antibody, a specific marker for neutrophils, revealed a marked accumulation of neutrophils during atherosclerosis development. Neutrophils were observed in the lesion, attached to the cap, and in the arterial adventitia. In addition, at some sites, neutrophil accumulation colocalized with endothelial E-selectin expression. Immunofluorescence double staining with anti-myeloperoxidase and anti–Ly-6G antibodies demonstrated the presence of myeloperoxidase in atherosclerotic lesions and its colocalization with neutrophils. After introducing the high-fat diet, levels of circulating myeloperoxidase in plasma strongly increased, with a peak at 6 weeks and a subsequent decrease to almost normal levels after 16 weeks of diet.
Conclusions— We here demonstrate for the first time the presence of neutrophils and myeloperoxidase in murine atherosclerotic lesions. As a major cell type in inflammatory responses the neutrophil may also be an important mediator in the development of atherosclerosis.
We identified myeloperoxidase-positive neutrophils in mouse atherosclerotic lesions. Although neutrophils were not detected in early lesions, they were abundantly present in more advanced stages. In addition, circulating myeloperoxidase levels were strongly increased by high-fat feeding of the mice. Therefore, neutrophils should be considered as a potential important cellular mediator in atherogenesis.
Key Words: atherosclerosis • neutrophil • myeloperoxidase • immunohistochemistry • LDLR–/– mice
Atherosclerosis is a multifactorial chronic inflammatory disease with prominent involvement of the immune system. Although incompletely understood, atherogenesis is most likely the result of a complex interplay between various immune and nonimmune cell types. The most abundant immune cells in atherosclerotic lesions are monocyte-derived macrophages and T cells.1,2 These cells are therefore considered the immunologic key players in atherogenesis and have been extensively studied. In contrast, neutrophils, the principal cellular component of the innate immune system, have received little attention, and their histological presence in atherosclerotic lesions has not been described in detail before.
Neutrophils are professional phagocytes, whose main function is to sense and destroy pathogenic organisms. In addition, they are the most prominent leukocytes in acute inflammatory reactions and contribute to host tissue injury in a number of inflammatory conditions, including ischemia-reperfusion injury, sepsis, and vasculitis.3 Importantly, being the key cellular component of the acute inflammatory response, neutrophils are thought to contribute to the initiation and shaping of the immune response.4 For example, neutrophils generate chemokines that recruit monocytes and dendritic cells and can determine whether macrophages differentiate to a predominantly pro- or antiinflammatory phenotype.
Although not extensively investigated, indirect evidence exists implicating a role for neutrophils in the process of atherogenesis. First, atherosclerotic mice deficient in P-selectin or E-selectin show decreased lesion formation.5,6 Because selectins play a critical role in neutrophil attachment to and rolling along the endothelium, it might be postulated that neutrophils play a role in atherosclerosis. In human and mouse models of atherosclerosis, distinct infiltration of neutrophils has been demonstrated in ruptured lesions and culprit lesions suggesting a role for these cells in the destabilization of atherosclerotic lesions.7,8 Furthermore, plasma levels of myeloperoxidase, the neutrophils’ most abundant enzyme stored in the azurophilic granules, are associated with the presence of coronary artery disease in humans.9 In addition, MPO levels are elevated in patients with chest pain and predict the risk of a major cardiac adverse event.10 Besides, a correlation has been found between neutrophil counts with the occurrence and severity of carotid atherosclerosis in patients.11 However, not all studies have found that plasma levels of myeloperoxidase were related to coronary artery disease (CAD).12 Overall, however, the role of neutrophils in atherogenesis remains unclear. As a first step to address this issue, the present study was designed to investigate the presence of neutrophils and myeloperoxidase during the course of atherosclerotic lesion development in atherosclerosis-prone low-density lipoprotein receptor–deficient mice (LDLR–/– mice).
Methods
Female LDLR–/– and apoE–/– mice on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, Me). Three experimental groups of 7 and 1 control group of 6 LDLR–/– mice aged 14 to 16 weeks were fed a high-fat diet (Hope Farms) or chow. After 6, 10, and 16 weeks of diet or 16 weeks of normal chow, mice were euthanized after overnight fasting. Furthermore, a first comparison was made between LDLR–/– mice and apoE –/– mice (another mouse model for atherosclerosis). Plasma cholesterol (Sigma-Aldrich) and plasma myeloperoxidase levels (Hycult Biotechnology) were determined using commercial kits. All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc) and SPSS 12.0 for Windows (see Supplemental File I, available online at http://atvb.ahajournals.org).
Tissue Processing and Atherosclerotic Lesion Analysis
Mice were anesthetized and hearts and aortic arches were removed from the body and bisected perpendicular to the heart axis just below the atrial tips. Sections of 7 µm were collected starting from where the atrioventricular valves were visible. Aortic lesion areas were quantified using serial cross-sections obtained every 42 µmol/L beginning at the start of the atrioventricular valves and spanning 250 µmol/L. Lesion severity was classified by staining with Sirius red for collagen content and categorized as described previously.13 Both severity and necrosis of the atherosclerotic lesion were determined by an experimental pathologist (M.G.; see Supplemental File I).
Immunohistochemistry
All antibodies used for immunohistochemistry were initially titrated and used at saturated concentrations. Isotype controls were used at the same concentrations as the respective primary antibodies. All isotype control stainings showed absence of staining (data not shown). To quantify neutrophils, monoclonal antibody Ly-6G (clone 1A8; BD Biosciences Pharmingen, 2.5 µg/mL), a specific neutrophil marker14,15 was used. To detect myeloperoxidase, fluorescein isothiocyanate (FITC)-labeled mouse anti-mouse MPO monoclonal antibody 8F4 (20 µg/mL)16 that had been generated at our department (Supplemental data including Figure IIA and IIB) was used. Adhesion molecule E-selectin was identified by monoclonal antibody clone MES-1 and macrophages by monoclonal antibody CD68 (clone FA-11), using hybridoma culture supernatants. Seven-µm-thick frozen sections were acetone-fixed at room temperature and air-dried. For detailed protocols concerning immunohistochemistry, please see supplemental data (see Supplemental File I). Immunofluorescence pictures were digitally merged with Adobe Photoshop, version 7.0, confocal microscopy pictures were merged by ImageJ version 1.37. Neutrophils in the lesions and in the adventitial layer were counted and scored in a blinded fashion by 1 observer. Neutrophils in the lesions were expressed relative to the lesion area per mm2. The neutrophils in the adventitial layer, neighboring the lesion, were graded according to the following severity scale: 0=no staining (none), 1=1 to 10 counted neutrophils (mild staining), 2=11 to 25 counted neutrophils (moderate staining), and 3=more than 25 counted neutrophils (abundant staining).
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Results
Atherosclerosis Characterization
To study the presence of neutrophils in atherosclerosis, LDLR–/– mice were fed a high-fat diet and euthanized after 6, 10, and 16 weeks to study initiation and progression of atherosclerosis. As control 1 group of mice was kept on chow for 16 weeks. Body weight and cholesterol levels were measured before starting the high-fat diet. The mean weight and cholesterol levels for the 3 experimental groups and the control group are presented in supplemental Table I (see Supplemental File III). There were no significant differences between groups in body weight and cholesterol levels before the start of the experiment. During the experiment, body weight and cholesterol levels increased in the experimental groups whereas the control group only developed an increase in body weight. The mean atherosclerotic lesion area in the experimental groups showed a significant increase over time.
Lesion Severity in LDLR–/– Mice
After 6, 10, and 16 weeks of high-fat diet, atherosclerotic lesions in the aortic valve area (AVA) were characterized for severity. At week 6 mixed early, intermediate, and advanced lesions were observed. After 10 weeks, lesions of the intermediate and advanced stage predominated whereas after 16 weeks, the majority of the lesions were characterized as advanced (see Supplemental File IV).
Accumulation of Neutrophils During Atherosclerosis Development
During atherosclerosis progression, macrophages are abundantly present in lesions as shown (Supplemental file V, Figure VA, VC, and VE). To identify neutrophils in the atherosclerotic lesions, immunohistochemistry was performed with the neutrophil specific marker Ly-6G (supplemental Figure VB, VD, and VF). On classification of the lesions and correlation with diet-period we found that Ly-6G positive-neutrophils were present in the lesions at all 3 time points. Neutrophils were not present in early lesions, which were only observed in the group euthanized at 6 weeks, and consisted mainly of macrophages (Figure 1A through 1C). On the other hand, in the 6 week group, many infiltrating neutrophils were seen in intermediate and advanced lesions, resulting in a high average neutrophil influx score at this time point (Figure 3). The time points 10 and 16 weeks also showed neutrophil accumulation in the intermediate and advanced lesions (Figure 1H and 1K). In the lesions, neutrophils could be detected at two different locations: in the atherosclerotic lesion itself and attached to the cap (Figure 1M). In addition, at the site of atherosclerotic lesions neutrophils were found in the lamina adventitia of the aorta (Figure 1N). The majority of the lesions showed neutrophils present in both the lesional cap and the adventitia, whereas they were rarely observed in the core of the lesions that consisted of macrophage-derived foam cells. A typical example of this neutrophil distribution pattern is shown in Figure 1P. Figure 1O shows a high magnification of a Ly-6G–positive neutrophil with a multilobed nucleus and a Ly-6G–negative monocyte with a bean-shaped nucleus. To study in more detail the neutrophil distribution in comparison to that of macrophages, a CD68 staining for macrophages was performed on adjacent sections. As shown in Figure 1, (C, F, I, L, and Q) within the atherosclerotic lesion macrophage distribution was different from that of neutrophils. Double staining with CD68 and Ly-6G was analyzed with confocal microscopy and showed absence of colocalization, confirming that Ly-6G was exclusive for neutrophils and did not stain CD68 positive monocytes or macrophages (Figure 2). To investigate whether neutrophil accumulation in atherosclerosis is not just a specific characteristic of the LDLR–/– mouse model, atherosclerotic lesions of apoE–/– mice were also studied for the presence of neutrophils. Within these lesions, the presence of neutrophils was demonstrated as well (Figure 1R), suggesting that neutrophils in general are involved in atherogenic mouse models.
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In the LDLR–/– mouse model, no significant differences between the different time-points were found for the number of neutrophils when adjusted for mean lesion area (Figure 3). Although mean values showed a trend toward smaller numbers of neutrophils in the 16 week diet group in comparison with the 10 week diet group (Mean 10 week HFD: 180 neutrophils per mm2; 16 week HFD: 33 neutrophils per mm2). The absolute number of neutrophils in the lesions correlated strongly with the severity score of the neutrophils in the adventitial layer (0.83, 95% CI 0.71 to 0.89, P<0.0001; Supplemental File VI). This indicates that these different sites are similarly involved in neutrophil recruitment in the atherosclerotic lesion. Because in inflammatory conditions the attraction and infiltration of neutrophils often coincides with necrosis we compared necrotic areas in the lesions with the presence of neutrophils. The area of necrosis did not correlate with neutrophil accumulation (data not shown). Interestingly however, almost no neutrophils were found in close proximity of the necrotic cores in atherosclerotic lesions.
To study factors involved in the extravasation of neutrophils into the atherosclerotic lesion, expression of the adhesion molecule E-selectin was investigated. E-selectin staining could be occasionally observed in endothelial cells lining the lesion (Supplemental File VII). By using immunostaining of consecutive sections we noticed that E-selectin staining of the endothelial lining always coincided with neutrophil presence.
Abundant Expression of MPO-Containing Neutrophils in the Lesion
Myeloperoxidase (MPO) is an enzyme present in human atherosclerotic lesions17 and known to be the most abundant enzyme of neutrophils. We studied for the distribution of MPO in murine atherosclerotic lesions using a monoclonal antibody 8F4 that was recently generated in our laboratory.16
In murine spleen sections, immunofluorescent double-staining of MPO and Ly-6G showed that neutrophils are the predominant MPO-containing cell type in mice whereas weak MPO expression was observed in other cells, most likely monocytes (Figure 4, Section 1, A through C). Double staining for MPO and neutrophils in atherosclerotic lesions at 6, 10, and 16 weeks after a high-fat diet showed that MPO was present in lesions and colocalized strongly with neutrophils (Figure 4, Section 1, D through G). Additionally, we used a polyclonal antibody to MPO,18 which showed a similar staining pattern as the monoclonal antibody 8F4 on atherosclerotic lesions (Supplemental Figure IIC).
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There was no colocalization of MPO with macrophages, which is in accordance with previous findings that macrophages in murine atherosclerotic lesions do not express MPO.19,20 That indeed MPO was predominantly expressed by neutrophils was also shown by confocal microscopy analysis of murine spleen sections. A cytoplasmic granular MPO staining pattern was observed in cells with a distinct multilobed nucleus, representative for neutrophils (Figure 4, Section 2, A through C). Also in atherosclerotic lesions this characteristic staining pattern was observed (Figure 4, Section 2, D through N).
Circulating Plasma Levels of MPO Are Increased During Atherosclerosis Development
Because MPO is also secreted into the circulation, we measured plasma levels of MPO during the development of atherosclerosis. When comparing the control group with the experimental groups, the groups fed a high-fat diet showed strongly increased levels of circulating MPO (Figure 5). Six weeks after introducing the high-fat diet, significantly higher levels of circulating MPO were observed as compared with baseline MPO levels. After 10 weeks of diet, MPO plasma levels decreased, however mean values were still increased (although not significantly) from baseline levels (Mean baseline: 39 ng/mL; Mean 10 weeks HFD group: 86 ng/mL). At 16 weeks of diet circulating MPO levels dropped further, although they remained slightly elevated as compared with plasma samples obtained from animals before starting the diet or animals that had been kept on normal chow.
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Discussion
In the present study, we investigated the presence and distribution of neutrophils and the enzyme MPO during the development and progression of atherosclerosis in LDLR–/– mice. Neutrophils were found to be present in the various intermediate and advanced stages of atherosclerosis. No neutrophils could be identified in the early lesions that only consisted of macrophage-derived foam cells. Neutrophils were observed in the atherosclerotic lesions, attached to the cap and in the adventitial layer. A strong correlation was found between the number of neutrophils in the lesion and the severity score of neutrophils in the surrounding adventitia. Occasionally, neutrophil infiltration colocalizes with E-selectin staining on the endothelial lining. To our knowledge, this study is the first detailed description of neutrophil involvement and localization during atherogenesis in LDLR–/– mice, one of the most widely used animal models for atherosclerosis.
Neutrophils have received little attention in the atherosclerosis field so far. However, already in 1982 Trillo21 described the presence of neutrophils in atherosclerotic lesions in monkeys based on identification of nuclear morphology. In addition, there is indirect evidence for a role of neutrophils in atherosclerosis. For example, mice deficient in P-selectin, E-selectin, or both5,6 showed delayed formation of atherosclerotic lesions. This diminished lesion development was explained to result mainly from a decreased influx of monocytes attributable to the absence of the selectins. However, although not addressed in these studies, decreased lesion development may also have been the result of impaired neutrophil infiltration, because selectins are key regulators in neutrophil infiltration in inflammatory sites. Studies by Eriksson et al22 have demonstrated that the leukocytes interacting in vivo with atherosclerotic endothelium are predominantly neutrophils. Hence, they suggested an important contributing role for neutrophils in atherogenesis. Also, Chen et al23 raised evidence for a potential role of neutrophils in atherosclerotic lesions by demonstrating in in vitro studies that under complex flow environments, as found in atherosclerotic lesions, neutrophils attached to the endothelial layer. An additional indication for neutrophil infiltration and involvement in lesion development comes from studies describing the presence of the chemoattractant interleukin (IL)-8 in atherosclerotic lesions.24 IL-8 is a chemokine important for neutrophil recruitment to sites of inflammation. In mice, the chemokines MIP-2 and KC, functional homologues of human IL-8, have been shown to be related to the extent of atherosclerosis.24,25 The above mentioned data together with our observations showing the presence of neutrophils in atherosclerotic lesions, suggests a role for neutrophils in the progression of atherogenesis. In this respect, monocytes and macrophages which are the first inflammatory cells observed in early lesions are putative candidates for initiating neutrophil recruitment.26
The precise role of neutrophils in atherosclerosis, however, remains unclear. Neutrophils may contribute to atherogenesis through the secretion of several inflammatory mediators. Two important classes of products that are released by activated neutrophils are reactive oxygen intermediates (ROI) and neutrophil-derived proteases.3,26 First, activation of the respiratory burst and MPO-derived oxidants such as hypochlorous acid, can contribute to tissue damage by promoting lipid peroxidation.27 Our demonstration of MPO-positive neutrophils in the lesions and the significantly higher plasma MPO levels as compared with control nonatherogenic mice may indicate an important role for MPO in murine atherosclerosis. Multiple studies in humans have already shown an association between MPO levels in the circulation and risk for cardiovascular disease.9,10,28 In our study, we found a relationship between plasma MPO levels and the extent of atherosclerosis. Particularly in the initiation of atherosclerosis (week 6), high levels of circulating MPO were measured, which subsequently dampened after 10 and 16 weeks but mean levels were still increased. It cannot be excluded that these increased plasma MPO levels at week 6 were initiated by introduction of a high-fat diet. In transgenic hypercholesterolemic mice overexpressing human MPO in macrophages, atherosclerosis was increased indicating an aggravating role of MPO.20 In contrast, a protective effect of the enzyme MPO was found when MPO-deficient animals were studied in atherosclerosis.19 As such, the precise role of MPO in atherosclerosis is still inconclusive. In addition to the respiratory burst, degranulation of activated neutrophils causes the release of various proteases. For example, matrix metalloproteinase (MMP)-8 and MMP-9 are responsible for degradation of matrix components such as laminin, collagen and proteoglycans.26 The products mentioned above have an antimicrobial function and are secreted to protect the host against pathogens. However, when released in the context of an inflammatory response such as in atherosclerosis these factors can have a proatherogenic effect29 and might influence the development of atherosclerotic lesions by causing rupture. Several studies7,8 already showed infiltration of neutrophils at sites close to thrombi suggesting a potential role for neutrophils in destabilizing the lesion. Recently, the work of Leclercq et al30 provides substantial evidence for the involvement of neutrophils in intraplaque hemorrhage in human atherosclerosis.
Neutrophils may also, by releasing their lysosomal constituents, contribute to the development of necrosis. On the other hand, necrosis may be an initiating factor for recruitment of neutrophils. Here, we did not find a correlation between necrosis and presence of neutrophils suggesting that other factors than necrosis mediates the recruitment of neutrophils.
In our studies, we have used the marker Ly-6G to uniquely identify neutrophils in atherosclerotic lesions. Henderson et al15 described that markers such as NIMP-R14 and GR-1, widely used to define the neutrophil lineage, not only detect neutrophils but also subsets of macrophages and even lymphocytes. Our study shows that Ly-6G, reacting only with neutrophils, is a very useful marker to detect specifically cells of the neutrophil lineage,14 which was also recently described in a study by Tsou et al.31
In addition to the presence of neutrophils in the atherosclerotic lesion, likely infiltrated through the E-selectin–expressing endothelium lining on the lesion, neutrophils were also found in the adventitial layer and in between the media fibers surrounding the lesion. This may indicate two alternative ways of entrance of neutrophils into the lesion. Recently, Moos et al31 found adventitial lymphocyte infiltration to be important during atherogenesis in apoE–/– mice. Our results suggest that adventitial infiltration of neutrophils may also play a role in atherogenesis.
In summary, our study shows, for the first time, the presence and distribution of MPO-positive neutrophils in murine intermediate and advanced atherosclerotic lesions. These data point to a significant but as yet undefined role for neutrophils in the progression of atherosclerosis. This urges for more investigations into the pathophysiological role of neutrophils in the pathology of atherosclerosis in mice and humans.
Acknowledgments
We thank E. Lutgens for sections of the apoE–/– mice, J. Jaspers for technical assistance, D. Huugen for providing the anti-MPO 8F4 antibody, I. Ferreira for statistical advice, and B. Schutte for his assistance on the confocal microscopy.
Sources of Funding
This work was supported by grants from the Netherlands Organization for Scientific Research (VIDI 917.66.329 (M.P.J. de Winther) and VIDI 917.66.341 (P. Heeringa) and the European Vascular Genomics Network (EVGN).
Disclosures
None.
Footnotes
Original received February 22, 2007; final version accepted October 31, 2007.
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