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(Circulation. 1998;97:75-81.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Inhibition of ₄ Integrin and ICAM-1 Markedly Attenuate Macrophage Homing to Atherosclerotic Plaques in ApoE-Deficient Mice

Shilpesh S. Patel, MD; Ram Thiagarajan, MD; James T. Willerson, MD; ; Edward T. H. Yeh, MD

From the Department of Internal Medicine (S.S.P., R.T., J.T.W., E.T.H.Y.) and the Institute of Molecular Medicine for the Prevention of Human Diseases (E.T.H.Y.), University of Texas Health Sciences Center, Houston, Tex; and the Texas Heart Institute (J.T.W., E.T.H.Y.), St Luke's Episcopal Hospital, Houston, Tex.

Correspondence to E.T.H. Yeh, Department of Internal Medicine, 6431 Fannin, Suite 4200, UT-Houston HSC, Houston, TX 77030.

	Abstract

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Background—Monocytes/macrophages play a central role in many stages of development of atherosclerotic plaques, including the conversion to an unstable morphology with rupture and fissuring. A better understanding of the mechanism of attachment of monocytes to activated endothelial cells would prove useful in developing strategies aimed at blocking this initial step. Here we describe a novel in vivo model that directly demonstrates homing of macrophages to atherosclerotic plaques.

Methods and Results—Macrophages were loaded with fluorescent microspheres and injected intravenously into 40-week-old apolipoprotein E–deficient mice. After 48 hours, labeled macrophages were observed adhering to all stages of atherosclerotic plaques from the early fatty streak to mature calcified lesion. The mean number of macrophages adherent to atherosclerotic plaques located in the proximal 1 mm of the aortic root was quantitated by counting serial frozen sections and found to be 143±17 macrophages per aortic root. Pretreatment of the apolipoprotein E–deficient mice with monoclonal antibodies directed against the -subunit of the ₄ß₁ integrin and against intracellular cell adhesion molecule (ICAM-1) reduced macrophage homing by 75% and 65%, respectively, as compared with isotype-matched controls (P<.05). Pretreatment with a monoclonal antibody directed against E-selectin did not significantly reduce macrophage homing.

Conclusions—These data demonstrate that ₄ integrin and ICAM-1 play major roles in the recruitment of macrophages to atherosclerotic plaques, whereas E-selectin does not appear to contribute significantly to macrophage recruitment. This model will be useful for studying the mechanism of macrophage recruitment to atherosclerotic plaques and for evaluating the efficacy of inhibitors to adhesion molecules in preventing macrophage recruitment.

Key Words: atherosclerosis • monocytes • macrophages • cells • adhesion • molecules • integrins

	Introduction

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Monocytes/macrophages are involved in many aspects of the development of atherosclerotic plaques.¹ An early microscopic change observed in atherogenesis is monocyte adherence to activated endothelial cells.^{2 3} After transmigrating across the endothelial cell layer, monocytes mature into macrophages, which phagocytose lipids to become foam cells forming the early fatty streak.⁴ Growth factors and cytokines, released from macrophages, then act to transform contractile SMC into secretory SMC, which migrate into the atherosclerotic lesion.^{5 6} Secretory SMC produce extracellular matrix, mostly composed of collagen, laminin, and fibronectin, which contributes significantly to the volume of the plaque resulting in flow-limiting atherosclerotic lesions.⁷ Recent investigations have focused on the central role of macrophages in converting a stable, quiescent plaque to an unstable one with rupture and fissuring.^{8 9 10} Macrophages have been implicated in weakening the fibrous cap of the plaque due to the secretion of matrix-degrading metalloproteinases (MMP).^{11 12} MMP are a family of enzymes that degrade extracellular matrix components, particularly collagen, elastin, and proteoglycans, thereby possibly serving to thin the collagen skeleton of the fibrous cap, leading to plaque rupture and fissuring.¹³ In support of the role of MMP are several observational studies of human coronary atherosclerotic plaques obtained from patients with acute coronary syndromes, presumably due to plaque rupture, which demonstrate significantly increased numbers of macrophages compared with patients with stable coronary syndromes.^{14 15}

An understanding of the initial interaction of monocyte/macrophages with plaques, namely the adherence of monocytes to activated endothelial cells, may prove useful in providing new anti-inflammatory therapeutics in the treatment of atherosclerosis aimed at preventing macrophage entry into plaques. Macrophage adherence occurs through binding of highly regulated cell adhesion molecules expressed on the surface of macrophages and endothelial cells. Evidence for the specific adhesion molecules involved in macrophage adherence comes largely from immunohistochemical and in vitro studies and implicates important roles for selectins, VCAM-1, and ICAM-1.^{16 17 18 19 20 21 22} The expression of VCAM-1²² and ICAM-1²³ has been shown to be upregulated on endothelial cells in regions overlying atheromatous lesions. Monoclonal antibodies against ß₂ integrins, CD14, ICAM-1, and P-selectin have been shown to inhibit monocyte attachment to human atherosclerotic plaques in vitro.²⁴ The regulation of these adhesion molecules is likely in response to locally released cytokines such as interleukin-1ß, tumor necrosis factor-, and interferon-.²⁵

The development of hypercholesterolemic ApoE-deficient mice by the laboratories of Breslow and Madea has provided an excellent animal model for studying many aspects of atherogenesis.^{26 27} By lacking ApoE, these mice develop spontaneous elevations of serum cholesterol to an average of 606 mg/dL on chow diets and lesions of atherosclerosis characteristic in location and histological appearance to those observed in humans. Monocyte adherence has been observed as early as 5 weeks, with fatty streaks and fibrous plaques developing by 10 and 15 weeks, respectively. Furthermore, electron microscopic studies in these animals have demonstrated monocyte attachment to the endothelium continuing through all stages of lesion development from the initial fatty streak to advanced fibrous plaques.²⁸

To more clearly elucidate the mechanism of macrophage adherence to endothelial cells and to provide a model for studying the behavior of macrophages in atherosclerotic lesions, we have developed a novel in vivo model of macrophage homing to atherosclerotic lesions in ApoE-deficient hypercholesterolemic mice. In addition, we have studied the effect of pretreatment with monoclonal antibodies targeted to block the -subunit of the ₄ß₁ integrin, ICAM-1, and E-selectin to provide more direct in vivo evidence for the cell adhesion molecules responsible for macrophage attachment to atherosclerotic plaques.

	Methods

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Mice
Congenic 6- to 8-week-old female C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine) were used to obtain activated peritoneal macrophages. ApoE knockout mice were purchased from Jackson Laboratories as male and female 30- to 40-week-old retired breeders with C57BL/6J background and were fed a normal chow diet. A second colony of ApoE knockout mice with BALB/cJ and C57BL/6J background was generously supplied by J. Breslow (Rockefeller University, New York, NY). The mean serum cholesterol level in the ApoE-deficient mice was 707±177 mg/dL. All experimental groups consisted of 10 or more mice.

Peritoneal Macrophages
Activated peritoneal macrophages were obtained from C57BL/6J mice by peritoneal washings 4 to 5 days after the intraperitoneal injection of 1 mL of 3% aged Brewer's thioglycolate (Difco Laboratories). Peritoneal washings were centrifuged at 600g for 6 minutes and then resuspended in 10 mL of 0.2N normal saline for 60 seconds to hemolyze contaminating erythrocytes followed by 10 mL of 1.8N normal saline to restore isotonicity. Cells were then centrifuged again and resuspended in RPMI 1640 (Gibco Laboratories) with 10% heat-inactivated fetal calf serum. The cell suspensions were then plated on tissue culture plates to allow the macrophages to adhere. Fluorescent microspheres were added immediately to the plated cell suspensions.

Labeling and Purification of Macrophages
Two-micron yellow-green fluorescent latex microspheres (Molecular Probes) were opsonized with 50% normal mouse serum for 30 minutes at 37°C to enhance phagocytosis. Microspheres were then incubated with the peritoneal cell suspensions in a 25:1 ratio of microspheres to cells for 75 minutes at 37°C under 95% oxygen/5% carbon dioxide in standard tissue culture incubators. Macrophages with phagocytized microspheres adhered to the tissue culture plates, allowing free microspheres and other cells to be removed easily by gentle washing with PBS without Ca and Mg (Gibco Laboratories) x2. The purified adhered labeled macrophages were then lifted off the plate by incubation with 10 mmol/L EDTA in PBS without Ca and Mg (without trypsin) for 10 to 15 minutes at 37°C. Macrophages were then washed free of EDTA and resuspended in Hanks' balanced salt solution (HBSS, Gibco Laboratories) before intravenous injection.

Administration of Monoclonal Antibodies and Injection Protocol
R1–2 (anti–-subunit of the ₄ß₁ integrin), 3E2 (anti-mouse ICAM-1), 10E9 (anti-mouse E-selectin), and three isotype-matched antibodies were purchased from Pharmingen and were administered to 30- to 40-week-old ApoE–deficient mice. 100 µg of monoclonal antibody was injected intraperitoneally 6 to 8 hours before the intravenous injection of labeled macrophages to allow sufficient time to obtain adequate serum levels of monoclonal antibody. Labeled macrophages (10x10⁶) were then injected intravenously into the tail vein in a total volume of 0.2 to 0.3 mL of HBSS. All animals were killed 48 hours after the intravenous macrophage injection.

Tissue Preparation and Quantification of Microspheres
Mice were killed by asphyxiation with carbon dioxide. The animals were then perfused with heparinized saline by injection through the apex of the left ventricle. The base of the heart and the ascending aorta were isolated, mounted in TissueTek freezing medium, and frozen in liquid nitrogen. Sections (5 µm) were then cut and stained with hematoxylin with the use of a xylene-free method. Serial sections spanning 1.0 mm of the ascending aorta at the level of the sinus of Valsalva were examined under light and fluorescent microscopy. The number of macrophages containing fluorescent microspheres seen attached to the intimal surface or in atheromatous plaques was quantified for each aorta.

Statistical Analysis
Data are expressed as mean±1 SEM. Comparison of the seven treatment groups was performed using one-way ANOVA followed by Scheffe's test for post hoc pairwise comparisons. All analyses were done using SAS Statistical programs, and P<.05 was considered statistically significant.

	Results

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Distribution of Fluorescent Microsphere-Loaded Macrophages After Intravenous Injection
Mouse peritoneal macrophages plated in tissue culture avidly phagocytosed fluorescent microspheres (Fig 1, a and b). The number of microspheres phagocytosed by macrophages could be adjusted in a dose-dependent manner according to the ratio of microspheres to cells chosen during incubation. Two-micron microspheres were chosen because they were easily detected by low-magnification fluorescent microscopy. Labeled macrophages were harvested from tissue culture plates with EDTA alone (without trypsin) to avoid proteolysis of the macrophage cell surface proteins. Cell suspensions of 10x10⁶ labeled macrophages were injected intravenously into ApoE-deficient hypercholesterolemic mice with no apparent adverse effects. Fluorescence microscopy of various organs of the injected ApoE mice demonstrated numerous labeled macrophages in the highest numbers in the lungs, perhaps because of entrapment by the pulmonary capillary bed, followed by abundant macrophages in the liver and spleen (Fig 1, c through f). In the spleen, macrophages were observed exclusively in the marginal zone and the red pulp, with no observable recruitment to the white pulp, which is the expected distribution for macrophages.²⁹ Only a few macrophages were identified in the kidneys. Thus the injected macrophages appear to behave appropriately by homing to the organs of the reticulo-endothelial system after initial entrapment by the lungs.

View larger version (81K): [in this window] [in a new window]   Figure 1. Distribution of fluorescent microsphere-loaded macrophages after IV injection. Light (a) and fluorescent (b) micrographs of cultured macrophages with internalized fluorescent microspheres. Hematoxylin-stained (c) and fluorescent (d) micrograph of lung section demonstrating the distribution of intravenously injected labeled macrophages. Many macrophages containing more than two microspheres are seen, likely due to entrapment by the pulmonary capillary bed. Combination light and fluorescent micrograph of liver (e) showing abundant macrophages containing mostly single microspheres and of spleen (f) demonstrating the distribution of macrophages exclusively in the red pulp.

Macrophage Homing to Atherosclerotic Lesions
The atherosclerotic plaques developing over the aortic cusps at the level of the sinus of Valsalva have been characterized to be the most advanced lesions in the ApoE-deficient mouse.³⁰ For this reason, 200 consecutive 5-µm frozen histological sections were obtained over the proximal 1 mm of the aortic root (Fig 2). Each section was observed with light and fluorescent microscopy for macrophages labeled with fluorescent microspheres that were adherent to or within the atherosclerotic plaques. Labeled macrophages were readily seen adhering to the endothelial surface overlying atherosclerotic lesions in the sinus of Valsalva and occasionally in the proximal segment of diseased coronary arteries (Fig 3). Although the majority of macrophages were observed adhering to the luminal surface of plaques, fluorescent microspheres were also detected in foam cell regions inside the plaque, suggesting transmigration of the labeled macrophage across the endothelial cell layer. Macrophage homing to atherosclerotic plaques was equally demonstrable in a different strain of ApoE-deficient mice with C57BL/6 and BALB/c background with peritoneal macrophages obtained from normal BALB/c mice.

View larger version (29K): [in this window] [in a new window]   Figure 2. Schematic diagram of the study area depicting a labeled macrophage adhering to the plaque.

View larger version (104K): [in this window] [in a new window]   Figure 3. Macrophages labeled with flourescent microspheres adhere to atherosclerotic plaques. Low-power micrograph of a cross-section of the aortic root (a and b) demonstrating 8 labeled macrophages adhering circumferentially to atherosclerotic plaque. One macrophage containing 10 fluorescent microspheres (c and d) is seen adhering to a fibrous plaque. One aggregation of 4 labeled macrophages (e and f) is seen attached to an advanced plaque. Numerous macrophages (g and h) are seen adhering to this isolated fatty streak, which is mostly comprised of foam cells. A collection of foam cells overlying a fibrous plaque attracts labeled macrophages (i and j). A few microspheres are seen within the area of foam cells, suggesting transmigration of the macrophage across the endothelial cell layer.

Negative control animals, which consisted of aged-matched heterozygous ApoE mice with normal serum cholesterol levels and no detectable atherosclerotic plaques, had minimal adherence of labeled macrophages to the aorta. Furthermore, regions of aorta in the homozygous hypercholesterolemic ApoE mice with no visible plaque burden (ie, ascending aorta and proximal descending aorta) also showed minimal adhesion of labeled macrophages, demonstrating the low nonspecific binding of injected macrophages.

Macrophage adherence was quantified by counting serial frozen sections for the total number of macrophages adhering to the proximal 1 millimeter of the aortic root at the level of the sinus of Valsalva. Fig 4a illustrates the typical distribution of adherent macrophages, with the majority of macrophages seen in the proximal 500 µm of tissue that corresponds to the bulk of the atherosclerotic lesion. The mean number of labeled macrophages seen adhering to the proximal aortic plaque was 143±17 SEM (n=13) in the heterozygous ApoE-deficient mice.

View larger version (23K): [in this window] [in a new window]   Figure 4. a, Distribution of macrophages in the 10-µm section of aorta in a representative experiment; b, inhibition of macrophage recruitment by antibody against 4 or ICAM-1 but not by anti–E-selectin antibody. The group of mice that were not treated with antibody is labeled as positive control. Appropriate isotype-matched antibody was used for each specific antibody. Mean of each treatment group is indicated by a bar. Comparison of the seven treatment groups was performed with one-way ANOVA followed by Scheffé's test for post hoc pairwise comaprisions. All analyses were done with SAS statistical programs; P<.05 was considered statistically significant.

Effect of Antibodies Blocking Cell Adhesion Molecules on Macrophage Homing
ApoE-deficient mice were pretreated with blocking monoclonal antibodies against specific cell adhesion molecules to determine if macrophage adhesion could be attenuated. Animals treated with monoclonal antibody demonstrated no adverse clinical effects. Administration of monoclonal antibody against ₄ß₁ and ICAM-1 significantly reduced the adherence of labeled macrophages to atheromatous regions in the aortic root. The ₄ß₁ MAb reduced macrophage adhesion by 75% compared with isotype-matched IgG2b controls (44±15 SEM macrophages anti-₄ versus 177±25 SEM macrophages isotype control, P<.05). The ICAM-1 MAb reduced macrophage adhesion by 65% compared with isotype-matched IgG2a controls (55±29 SEM macrophages anti-₄ versus 152±9 SEM macrophages isotype control, P<.05). E-selectin MAb had no significant effect on reducing macrophage adhesion (119±20 SEM macrophages anti-E-selectin versus 130±21 SEM macrophages isotype control, P>.05) (Fig 4b). There was no qualitative difference in the distribution of labeled macrophages in the lung, liver, and spleen in the monoclonal antibody–treated animals compared with isotype-matched controls.

	Discussion

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To date, the evidence supporting the role of cell adhesion molecules in the adherence of macrophages to atherosclerotic plaques comes mostly from in vitro experiments using cultured endothelial cells or removed atherosclerotic plaques. These in vitro experiments are complicated by the possibilities that tissue culture conditions (local tissue ischemia, media components, and so forth) may alter the expression and activation of adhesion molecules and that they ignore the influence of blood flow, turbulence, and leukocyte rolling on cell adhesion. Therefore, although useful in raising suspicion about the importance of specific adhesion molecules, in vitro experiments may be less reliable in determining the key molecules for leukocyte adhesion under physiological conditions.

Our data show in a novel in vivo model that labeled macrophages injected intravenously into ApoE-deficient mice home to atherosclerotic plaques. Specifically, the macrophages adhere to the endothelium overlying the plaque and are also observed within foam cell regions, suggesting transmigration of the macrophage across the endothelial cell layer. This process can occur within a period of 48 hours. Furthermore, macrophage adhesion can be significantly blocked by treatment with the monoclonal antibodies directed against the ₄ß₁ integrin or ICAM-1. These results provided strong evidence that the ₄ß₁ integrin and ICAM-1 play major roles in macrophage adhesion to endothelial cells overlying atherosclerotic plaques in vivo.

The ₄ß₁ integrin is one member of the integrin family of surface receptors that are composed of at least 8 different ß-subunits and 15 distinct -subunits. The ₄ß₁ integrin has been shown to be the counterreceptor for VCAM-1 and the CS-1 variant of fibronectin, which are expressed on activated endothelial and SMC and for mucosal addressin cell adhesion molecule (MAdCAM-1) located on Peyer's patches in the intestines. The monoclonal antibody used in our experiments, clone R1–2, has been shown to bind within residues 195 to 268 of the -subunit, which lies within the 108–268 residue binding site for VCAM-1 and fibronectin. Therefore, clone R1–2 is a blocking antibody that prevents binding of ₄ß₁ to VCAM-1 and fibronectin. In other disease models, in vivo studies have shown the R1–2 MAb to reduce leukocyte migration into the epidermis in a contact hypersensitivity model and lymphocyte recruitment and brain swelling in an experimental allergic encephalomyelitis model.

ICAM-1 is an immunoglobulin-like molecule expressed on the surface of endothelial cells, leukocytes, dendritic cells, and fibroblasts.^{31 32 33} The ligands for ICAM-1 are the ß₂ integrins (_Lß₂ and _Mß₂) and CD43.³³ Antibody against ICAM-1 has been demonstrated to inhibit monocyte recruitment to inflamed tissues.^{19 20 21} Our results provide a direct in vivo confirmation of the importance of ICAM-1 in macrophage recruitment. E-selectin is a cytokine-inducible adhesion molecule expressed on the surface of endothelial cells.^{22 31 32} It belongs to a family of adhesion molecules that recognize carbohydrate ligands present on glycoproteins or glycolipids.³⁴ It plays a major role in the initial rolling of leukocytes in circulation.^{31 32} Our observation that anti–E-selectin blockage has minimal effect on macrophage recruitment does not rule out a role for selectin in atherogenesis. It is possible that blockade of both E- and P-selectins are required for the prevention of macrophage recruitment. Furthermore, the inability of the anti-E-selectin MAb to prevent macrophage recruitment clearly demonstrates the specificity of the present model. Thus the effect observed with anti-₄ or anti–ICAM-1 antibody is not due to nonspecific steric hindrance of macrophage attachment to endothelial cells by anti-₄ or anti–ICAM-1 MAb.

The major limitation of this model is the use of labeled macrophages as a surrogate for studying the process of monocyte adhesion. Although macrophages are produced from the differentiation of monocytes, there are clear differences between these two cells. We have not used monocytes because it is difficult to load monocytes with fluorescent microspheres due to their poor phagocytic activity. Nonetheless, labeled macrophages provide a useful surrogate as demonstrated by the specificity of the inhibition studies. We had investigated, in the beginning, the issue of "recycling" of microspheres by injecting free beads into ApoE-deficient mice. Our hypothesis was that injected free microspheres could be engulfed by the ApoE mouse's own macrophages in the liver, spleen, and lungs. These autolabeled macrophages may then enter the circulation and adhere to atherosclerotic plaques. Our data suggest that this does indeed occur but on a much lower scale (15 autolabeled macrophages adherent to the aortic root) as compared with the adherence of injected labeled macrophages (140 labeled macrophages adherent to aortic root).

In conclusion, we describe a novel in vivo quantitative model of macrophage homing to atherosclerotic plaques. Our results implicate an important role for the ₄ integrin and ICAM-1 in the mechanism of macrophage adhesion to endothelial cells overlying atherosclerotic plaques. We believe this model will allow for more detailed in vivo investigations of the roles of macrophages in atherosclerotic plaques, thereby providing direct evidence for the specific adhesion molecules involved in macrophage adherence, and that it will offer a means for testing new small molecule inhibitors of adhesion molecules for their potential use as novel anti-inflammatory therapy in treating atherosclerotic disease.

	Selected Abbreviations and Acronyms

 

Apo = apolipoprotein ICAM-1 = intracellular cell adhesion molecule MAb = monoclonal antibody SMC = smooth muscle cell(s) VCAM-1 = vascular cell adhesion molecule

	Acknowledgments

 
This work was supported in part by grants from the Department of Health and Human Services (T32 HL-07591, R01-HL-50179, and R01-HL-45851), an American Heart Association Established Investigators Award (E.T.H.Y.), and generous support from Texas Biotechnology Corporation. The authors would like to thank Fred Clubb, Bob Bjercke, and Pam Beck for technical advice and Hui-Ming Chang and William Vaughn for statistical analysis.

	Footnotes

 
Guest Editor for this article was Valentin Fuster, MD, Mount Sinai Medical Center, New York.

Received June 26, 1997; revision received September 2, 1997; accepted September 25, 1997.

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