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Circulation Research. 1995;77:1121-1128

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(Circulation Research. 1995;77:1121-1128.)
© 1995 American Heart Association, Inc.

Articles

Mononuclear Leukocytes Invade Rabbit Arterial Intima During Thickening Formation via CD18- and VLA-4–Dependent Mechanisms and Stimulate Smooth Muscle Migration

Dorothee Kling, Jürgen Fingerle, John M. Harlan, Roy R. Lobb, Florian Lang

From the Pharma Division (D.K., J.F.), Preclinical Research, Hoffmann-La Roche Ltd, Basel, Switzerland; the Division of Hematology (J.M.H.), Department of Medicine, Harborview Medical Center, Seattle, Wash; Biogen Inc (R.R.L.), Cambridge, Mass; and the Institute of Physiology (F.L.), University of Tübingen (Germany).

Correspondence to Dr Dorothee Kling, Pharma Division, Preclinical Research, Hoffmann-La Roche Ltd, CH-4002 Basel, Switzerland. E-mail klingd@roche.com.


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
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Abstract The role of mononuclear leukocytes for the migration of smooth muscle cells (SMCs) during intimal thickening was investigated in the rabbit model of electrically stimulated carotid artery. The approach was to inhibit leukocyte entry into the arterial intima with antibodies against the adhesion molecules very late activation antigen-4 (VLA-4) and CD11/CD18. In electrically stimulated control rabbits treated either with saline or a nonspecific antibody, all types of granulocytes, monocytes, and lymphocytes migrated across an intact endothelium into the acellular subendothelial space, followed by the movement of SMCs from the media into the intima within 36 hours of applying electrical current. Treatment of the rabbits with monoclonal antibody (mAb) HP1/2 directed toward the {alpha}4 subunit (CD49d) of VLA-4 inhibited mononuclear leukocyte invasion (consisting of monocytes and lymphocytes) by {approx}70% compared with the IgG-treated control rabbits and completely abolished the minimal influx of basophils and eosinophils after 36 hours. Neutrophil infiltration, however, remained unaffected by anti–VLA-{alpha}4 treatment. Under these conditions, SMC migration across the internal elastic lamina was reduced by 50%. The use of mAb HP1/2 together with mAb 60.3 (directed to the ß2 chain of CD11/CD18) completely abolished the influx of monocytes, lymphocytes, and all types of granulocytes into the arterial intima. This complete blockade of leukocyte infiltration resulted in a 70% reduction of intimal SMC accumulation. Together with our previous findings excluding neutrophils as stimulators of SMC migration, the present results indicate that mononuclear leukocytes promote lesion development by stimulating SMC migration.


Key Words: ß1 and ß2 integrins • arteriosclerosis • monocytes • lymphocytes • granulocytes


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The accumulation of monocytes and lymphocytes is a prominent feature in human and experimental atherosclerosis.1 2 3 They are assumed to contribute to the initiation and progression of atherosclerotic plaques by secreting a variety of inflammatory mediators, cytokines, and growth factors.2 4 5 6 Secretory products of monocytes/macrophages may serve as chemoattractants7 8 9 or mitogens10 11 for vascular SMCs and may hence promote SMC accumulation within the intima during atherogenesis. T lymphocytes, on the other hand, are capable of introducing growth-inhibiting factors for SMCs into the atherosclerotic plaques,2 12 but they may also indirectly stimulate SMC proliferation and/or migration by macrophage activation.2 A few reports exist indicating a link between leukocyte migration into the vascular wall and intimal SMC accumulation during experimental intimal thickening. In particular, intimal lesion formation induced by an endotoxin-soaked thread in rat femoral arteries was prevented by inhibiting leukocyte invasion into the vessel wall after treatment with dexamethasone.13 Likewise, neointimal thickening elicited by balloon catheter injury in rat carotid arteries was reduced after inhibiting the accumulation of CD4-positive mononuclear leukocytes in the intima,14 suggesting a role of CD4-positive mononuclear cells in mediating intimal SMC accumulation.

Our interest was to study the functional significance of mononuclear leukocyte invasion in SMC migration during experimental intimal thickening. We inhibited mononuclear leukocyte infiltration into the nascent thickening by interfering with leukocyte/endothelial interactions, thus gaining additional insight into the recruitment mechanisms of mononuclear leukocytes. Intimal thickening was induced by repeated transmural ES of the rabbit carotid artery. By use of this model, lesions develop in the presence of a continuous yet functionally and structurally altered endothelium.15 16 Granulocytes, monocytes, and lymphocytes invade the acellular intimal space within the first 2 days of application of direct electrical current.16 17 18 Concomitantly, SMCs start to migrate across the internal elastic lamina from the media toward the intima. We previously demonstrated that the initial invasion of neutrophilic granulocytes was completely abolished by treating the rabbits with an antibody directed toward the common ß2 chain (CD18) of the leukocyte adherence glycoprotein complex CD11/CD18, whereas the influx of mononuclear leukocytes was only partially inhibited (by {approx}50%).17 Under these conditions, SMC migration into the intima was not significantly affected, thus excluding neutrophils as initiators of the directed movement of SMCs into the intimal compartment.

The purpose of the present study was (1) to suppress the residual CD18-independent portion of the invasion of mononuclear leukocytes and (2) to investigate the effect of the blocked influx of monocytes and lymphocytes on the migratory behavior of SMCs in the initial phase of lesion development. The blocking agent we used was a monoclonal antibody that binds to the {alpha}4 chain (CD49d) of VLA-4 (CD49d/CD29). VLA-4 is a member of the ß1-integrin subfamily and is expressed on resting monocytes and lymphocytes19 20 and on basophils and eosinophils21 22 23 but not on neutrophils.21 It participates not only in cell-matrix interaction but also in the recruitment of these cells from the bloodstream to areas of infection and inflammation.19 20 23 24 Its counterreceptor on endothelial cells is the cytokine-induced ligand VCAM-1.25

Our adhesion-blocking experiments provide evidence that the initial recruitment of monocytes and lymphocytes during the development of fibromuscular intimal thickening is mediated by VLA-4–dependent as well as CD18-dependent pathways. Moreover, the blockade of mononuclear leukocyte accumulation in the nascent thickening was shown to inhibit SMC migration from the media into the intima, indicating that mononuclear leukocytes promote the formation of intimal thickening and possibly atherogenesis.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
 down arrowReferences
 
Antibodies
The mAb HP1/2 is a murine IgG1 that binds to a functional epitope (characterized as epitope B) on the {alpha}4 subunit (CD49d) of the human integrin VLA-4.26 It cross-reacts with the respective rabbit homologue on mononuclear leukocytes but does not bind to rabbit neutrophils as evaluated by fluorescence-activated cell sorting.27 HP1/2 was purified by protein A and gel filtration chromatography under endotoxin-free conditions23 24 and used as a 6.5 mg/mL stock solution in sterile PBS.

Murine mAb 60.3 is of the IgG2a subclass and recognizes the common ß2 chain of the membrane glycoprotein complex CD11/CD18 expressed on human leukocytes (B and T lymphocytes, monocytes, and the three subclasses of granulocytes but not red blood cells or ECs28 29 ). It has the advantage of cross-reactivity with the respective adhesion molecules on rabbit leukocytes.27 mAb 60.3 was prepared according to Beatty et al,28 and the concentration of the purified antibody protein in the stock solutions was 8.5 mg/mL in sterile saline.

In order to control the nonspecific effects of repeated treatment with foreign antibody proteins, we used the mouse anti-human antibody 17-1A produced by the hybridoma clone 1083-17-1A.30 31 The hybrid clone secreted IgG1 and IgG2a at an early stage, but repeated cloning led to a loss of the myeloma {gamma}-heavy chain. 17-1A binds with a high level of specificity to a surface antigen on human colorectal carcinoma. All mAb solutions contained <0.01 endotoxin unit per milligram.

Animals
Male New Zealand White rabbits obtained from Thomae GmbH (Biberach, FRG) were used for all experiments. They were fed standard rabbit pellets without lipid supplementation (Altromin GmbH) and were kept in the animal house for at least 1 week before the experiments were started. The animals entered the study at a body weight of 1.7 to 2.0 kg. All rabbits were exposed to the same method of transmural ES known to induce fibromuscular intimal thickening under normocholesterolemia,15 16 18 but they were treated with the CD49d mAb HP1/2 (n=5), the combination of HP1/2 and the CD18 mAb 60.3 (n=5), or the nonspecific control antibody (n=3). For additional controls, eight rabbits received sterile saline instead of antibody.

Induction of Early Intimal Lesions and mAb Treatment
Early intimal lesions representing initial stages of fibromuscular thickening were induced in rabbit carotid arteries by applying direct-current impulses to the artery wall, as described previously.17 Briefly, with the rabbits under anesthesia (15 mg metomidate hydrochloride and 0.1 mg IM fentanyl base per kilogram body weight), two graphite-coated gold electrodes (each measuring 1 mm x 5 mm) were attached to the adventitia of the right carotid artery and held in diametrical position by a flexible polytetrafluoroethylene (Teflon) cuff. The cuff was placed loosely around the outer surface of the arterial wall, so as not to interfere with the pulsation and compressibility of the vessel. The electrodes were connected via subcutaneous leads and a microplug fixed in the skull to an extracorporeal stimulation unit generating DC impulses (0.1 mA, 15 milliseconds, 10 Hz). These were applied to the artery wall of the freely moving rabbit in two sessions, starting 14 and 26 hours after surgery and lasting for 30 and 15 minutes, respectively. The left carotid served as an intraindividual control: Those rabbits treated with the combination of HP1/2 and mAb 60.3 or the nonspecific IgG received a so-called "silent" cuff where no current was applied; in case of the other treatments, the artery remained unmanipulated.

The protocol for administration of the different antibodies was identical to the one used in earlier experiments.17 The antibodies were injected via the marginal ear vein three times at an interval of 12 hours, each at a dose of 2 mg/kg body wt. The first injection was given immediately before implantation of the electrodes; the second and third occurred 2 hours before the respective period of ES. This timing guaranteed that mAb administration interfered with the climax of leukocyte invasion into the arterial intima.18 The saline control rabbits were subjected to the same schedule, but with corresponding injections of sterile saline. All antibody or saline treatments were performed with the animals under anesthesia (see above). Blood samples were drawn before each mAb or saline administration as well as at the end of the experiments for total and differential white blood cell counts. The experiments were terminated 36 hours after surgery in order to study the migration of SMCs as they began to cross the internal elastic lamina into the intimal compartment within the first 2 days of applying direct electrical current, as previously described.16 17 18 The animals were anesthetized, and the carotids were perfused through the left ventricle with 1.25% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4, at 80 to 100 mm Hg. After excision, the arteries were immersion-fixed in the same solution for at least 24 hours.

The experimental protocol was reviewed and approved by the animal care committee of the University of Tübingen.

Electron Microscopic Analysis
After we removed the cuff and ear-marked the anodal side of the artery wall where the eccentric intimal thickening develops,15 16 the tissue was processed for transmission electron microscopy. Both the stimulated and the control carotid arteries were subdivided into rings {approx}3 mm in length. All samples were postfixed in 1% buffered OsO4 in 0.1 mol/L sodium cacodylate, dehydrated through graded alcohols, stained en bloc in alcoholic uranyl acetate, embedded in araldite, and transversely sectioned on an LKB ultramicrotome. Semithin sections were stained with toluidine blue. Ultrathin sections were taken from the anodal area of the block face as revealed by light electron microscopy, collected on 75-mesh copper grids, stained with lead citrate, and examined with a Zeiss EM 10.

The different cell types within the intimal cell population, ie, monocytes, lymphocytes, the subclasses of granulocytes, and SMCs, were identified on the basis of their ultrastructural features, as previously reported.17 The subtypes of granulocytes, ie, neutrophils, basophils, and eosinophils, were clearly identifiable and distinguishable from monocytes and lymphocytes by their specific granules.32 A sharp distinction between monocytes and lymphocytes was not always possible, depending on the cutting level. For quantification, they were therefore grouped as mononuclear leukocytes. However, the proportion of lymphocytes unambiguously identifiable by their fine structural characteristics (such as an oval nucleus, narrow rim of cytoplasm, tiny Golgi area, small number of lysosomes, and few large mitochondria) was determined, possibly underestimating their true quantities. Intimal SMCs were easily distinguished from leukocytes by the presence of a thin external lamina (sometimes only present in fragments), abundant micropinocytotic vesicles, myofilaments, and dense attachments on the cytoplasmic aspect of the membrane. Immunohistochemical analysis of paraformaldehyde-fixed paraffin-embedded tissue did not prove to be useful for cell typing in the early lesions under investigation, since the usual markers for identifying SMCs and macrophages could not be detected within the nascent thickening during the first 36 hours after electrode/cuff positioning. {alpha}-Actin, which is known to modulate its expression with the differentiation state of the cell,33 was first detectable in intimal cells 8 days after the onset of ES, and the cell type–specific antigen of rabbit macrophages recognized by RAM-11 was expressed by subendothelial cells at the earliest 2 days after ES was started (data not shown).

For quantitative analysis, ultrathin cross sections through the midregion of the cuff-bearing artery segment were selected from a minimum of four different planes separated by at least 0.1 mm. The cellular composition of the entire cross-sectional area of the intimal lesion developing next to the anode and covered by {approx}80 to 110 ECs was analyzed in a blind fashion. The numbers of intimal cells arranged in one or two cell layers beneath the endothelium were determined for each cell type and expressed in relation to the number of ECs overlying the lesion. The total count of ECs ranged from 454 to 677 per animal. The numbers of subendothelial cell profiles (with and without a sectioned part of the nucleus) belonging to a particular cell type were calculated per 100 ECs for each animal. The relative cellular composition was additionally determined by calculating the percentages of the different cell types within the intimal cell population.

Statistics
Data are presented as mean±SEM. Global effects of the treatment with specific antibodies on the composition of the intimal cell population were evaluated by one-way ANOVA after the data were normalized with logarithmic transformation. In the case of overall significance (attributed to P<.05), contrasts of selected pairs of group means were computed and adjusted by Holm-Bonferroni corrections.34 35 The computer program SUPERANOVA was used in these computations.

To test for the effect of the antibody treatment on leukocyte counts in peripheral blood, the area under the curve (each curve representing leukocyte counts over time) was computed for each animal in the different treatment and control groups. The data were subjected to one-way ANOVA by using the logarithm of the area under the curve as the target variate.


*    Results
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 up arrowMaterials and Methods
 *Results
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Early Intimal Lesions of Control Arteries
The nonmanipulated left carotid arteries of the control rabbits treated with saline had a normal appearance 36 hours after surgery. A morphologically intact endothelium covered the cell-free subendothelial space. No leukocytes were observed in association with the endothelial lining. In contrast, in the right carotid arteries exposed to two sessions of ES, leukocytes had populated the widened subendothelial space, forming lesions similar to those found in the stimulated carotid arteries of the rabbits injected with the nonspecific control antibody 17-1A. The leukocytes were arranged in one or two cell layers beneath a continuous endothelium with normal-appearing junctional complexes (Fig 1ADown). Additionally, they were seen adhering to, as well as trafficking through, the endothelium at this time (Fig 1BDown). When crossing the carotid endothelium, the leukocytes preferentially used the intercellular pathway through junctional complexes. PMNs and monocytes were the predominant cell types in the population of leukocytes invading the arterial intima, whereas lymphocytes, basophils, and eosinophils were only occasionally seen. The number of intimal PMNs and mononuclear leukocytes, including monocytes and lymphocytes, did not significantly differ between the 17-1A–treated control and the saline-treated rabbits (P>.35, Fig 2Down). The proportion of unambiguously identified lymphocytes within the mononuclear leukocyte population was also similar in the two control groups and amounted to 8.3±3.5% in the 17-1A group and 7.9±2.5% in the saline group, respectively. Basophils and eosinophils were present within the stimulated intima in percentages <1%, without significant differences between the two control groups.



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  		Figure 1. Electron micrographs showing characteristic features of intimal lesions induced by ES within 36 hours in carotid arteries from IgG-injected rabbits. A, Accumulation of PMNs and mononuclear leukocytes (M) between the intact endothelium (E) and the internal elastic lamina (IEL). B, Transendothelial migration of a monocyte (M). C, SMC in the process of migration from the media into the intima, as judged by its protrusion (arrowhead) through a pore of the IEL. Bars=2 µm.



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  		Figure 2. Bar graph showing accumulation of PMNs, mononuclear leukocytes (MLs), and SMCs within the intimal lesion induced by ES of rabbits treated with saline (n=8), nonspecific IgG (n=3), and anti-CD49d alone (n=5) or together with anti-CD18 (n=5). Antibodies and saline were injected intravenously at 0, 12, and 24 hours. The number of intimal cells is expressed in relation to 100 overlying ECs. For further experimental details, see "Materials and Methods." Data represent mean±SEM. *Significant difference from either control group treated with saline or nonspecific IgG (P<.05 by ANOVA and Holm-Bonferroni–adjusted contrasts).

Not only did leukocytes accumulate in the stimulated intima of the control groups within 36 hours, but SMCs also started to migrate from the underlying media into the intimal compartment, which normally was void of SMCs. They were predominantly observed squeezing their way through pores in the internal elastic lamina and spreading out in the intima (Fig 1CUp). The number of SMCs that had already reached the intima was similar in the two control groups (17-1A control group, 14.2±2.5 per 100 ECs; saline-treated control group, 13.1±1.1 per 100 ECs; P=.89; Fig 2Up). SMC migration was the dominant factor contributing to the accumulation of SMCs in the intima at 36 hours, since proliferation of intimal SMCs was first detectable after 2 days of ES, as shown in former studies using bromdeoxyuridine labeling.36

In the left carotid arteries of the 17-1A control rabbits surrounded by silent cuffs, mononuclear leukocytes also migrated across the endothelium and populated the normally acellular intimal compartment, yet at a clearly reduced number compared with the electrically stimulated right carotid arteries (5.6±1.7 versus 12.6±2.5 per 100 ECs, P=.017). Likewise, SMCs were present in the intima under silent cuffs; their number was also significantly lower than in the corresponding segments exposed to ES (2.3±0.9 versus 14.2±2.5 per 100 ECs, P=.04).

Early Intimal Lesions After Treatment With Integrin-Recognizing mAbs
The systemic application of the anti–VLA-{alpha}4 antibody HP1/2 did not affect PMN invasion into the electrically stimulated intima. PMNs were found to be adherent to, trafficking through, and lying beneath the endothelium. Their number within the nascent thickening was similar to that determined in the control arteries treated either with the nonspecific IgG (P=.21) or saline (P=.08), as indicated in Fig 2Up. Basophils and eosinophils, however, which constituted <1% of the intimal leukocyte population in each control group, were fully blocked in their invasion into the intima by HP1/2. Monocytes and lymphocytes still invaded the stimulated arterial intima after HP1/2 treatment, but their number was reduced to {approx}30% of the IgG control value (Fig 2Up, P=.0003). Under these conditions, the number of intimal SMCs was significantly diminished by {approx}50% compared with the control arteries (Fig 2Up; P=.02 versus saline, P=.04 versus IgG).

When anti–VLA-{alpha}4 was given in combination with the anti-CD18 mAb 60.3, the invasion of monocytes, lymphocytes, and granulocytes (all subsets) into the intima of the stimulated right carotid artery was completely abolished after 36 hours (Figs 2Up and 3ADown). Now, the number of SMCs that had migrated into the intima in response to ES was reduced to a greater extent than after anti–VLA-{alpha}4 treatment alone, reaching {approx}30% of the IgG control value (Fig 2Up, P=.006). As shown in previous experiments performed under identical conditions with the anti-CD18 mAb alone,17 total blockade of neutrophil invasion did not affect SMC migration. Thus, we can conclude that the reduction of mononuclear leukocyte infiltration by >70%, achieved in the present experiments, accounts for the inhibition of SMC movement from the media into the intimal compartment.




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  		Figure 3. Electron micrographs showing characteristic features of intimal lesions induced by ES after treatment with anti-CD49d and anti-CD18. A, Representative part of a transverse section through the stimulated carotid artery 36 hours after electrode/cuff positioning and combined antibody treatment. The subendothelium is void of leukocytes. B, SMC migration into the intima despite complete abolishment of leukocyte invasion after administration of both antibodies. Migration occurs beneath interendothelial contacts (arrow) and in proximity to basal protrusions of ECs (arrowhead). IEL indicates internal elastic lamina; E, endothelium. Bars=2 µm.

The number of intimal SMCs observed in the ES arteries after the combined antibody treatment (4.2±1.5 SMCs per 100 ECs) was similar to that found in the contralateral arteries surrounded by cuffs alone (3.7±1.5 SMCs per 100 ECs, P=.54). This minimal migratory response of SMCs was apparently unaffected by leukocytes, since in both the cuffed and ES arteries of the combined antibody treatment group leukocytes were absent. Interestingly, in those rabbits receiving IgG the cuffed but otherwise nonstimulated carotid arteries showed similar numbers of intimal SMCs (2.3±0.9 SMCs per 100 ECs, P=.41) as after combined antibody treatment.

Circulating Leukocytes
In the two control groups, total leukocyte counts remained on baseline levels within the first 24 hours after implantation of the electrodes without significant difference between the saline- and IgG-treated animals (Fig 4ADown, P=.12). In contrast, peripheral leukocyte counts of the other groups receiving either anti–VLA-{alpha}4 alone or in combination with anti-CD18 increased dramatically within 24 hours (Fig 4ADown, overall significance at P=.0001 evaluated by one-way ANOVA). The combined use of anti–VLA-{alpha}4 and anti-CD18 resulted in maximal leukocyte numbers of 51.7±5.7x103/µL, which was about eightfold the value found in the saline control arteries, whereas in the anti–VLA-{alpha}4 group, leukocytes reached values of 16.1±1.7x103/µL.



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  		Figure 4. Line plot of number of circulating leukocytes in peripheral blood of rabbits treated with saline (n=8), nonspecific IgG (n=3), and anti-CD49d alone (n=5) or together with anti-CD18 (n=5) (antibody and saline administration similar to that described in Fig 2Up). A, Time dependence of total leukocyte counts after the different treatments. Data are expressed as mean±SEM. Leukocyte counts of the saline and IgG control group remained on baseline levels, whereas anti-CD49d alone and together with anti-CD18 increased leukocyte numbers (P=.0001 by ANOVA after determination of the area under the curve). B, Numbers of circulating lymphocytes (lympho), PMNs, and monocytes (mono) at 0 and 24 hours (note the different scale for monocyte counts).

Leukocytosis evoked by anti–VLA-{alpha}4 was characterized by a marked increase in the number of lymphocytes within the experimental period from 0 to 24 hours (Fig 4BUp). PMN levels, however, remained within the range of the control levels during this time. When anti–VLA-{alpha}4 and anti-CD18 were given together, both lymphocytes and PMN numbers increased, reaching significantly higher levels than those found in the animals treated with anti–VLA-{alpha}4 alone (Fig 4BUp). Neutrophilia was based on a dramatic rise in juvenile PMNs from 1.0±0.03x103 at 0 hours to 10.6±0.9x103/µL at 24 hours. Only in this group of combined antibody treatment did monocyte numbers clearly exceed control values at 24 hours (Fig 4BUp; P=.0002 versus IgG, P=.0009 versus saline).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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The central cellular feature of atherosclerotic lesions is the accumulation of SMCs, together with monocytes/macrophages and lymphocytes, in the arterial intima. The SMCs are either derived from existing intimal cells by proliferation or from cells that migrate in from the underlying media. The present study provides insight into the mechanisms underlying the recruitment of monocytes and lymphocytes during experimental intimal thickening and the functional significance of these leukocytes for SMC migration from the media into the intima. The principal findings were that (1) monocytes and lymphocytes that initially invade the intimal thickening through the endothelium use pathways dependent on both the leukocytic integrins CD11/CD18 and VLA-4, and (2) the mononuclear leukocytes promote the directed movement of medial SMCs into the intima.

Contribution of CD18 and VLA-4 to Leukocyte Infiltration During Arterial Intimal Thickening
Using the model of electrically stimulated rabbit carotid artery, we recently demonstrated that the invasion of monocytes and lymphocytes into the arterial intima within the first 36 hours of the formation of fibromuscular thickening was in part CD18 dependent.17 The present experiments extend this finding by showing that VLA-4 accounts for the CD18-independent portion of mononuclear leukocyte invasion during intimal thickening, since (1) the antibody HP1/2 recognizing the {alpha}4 subunit of the ß1 integrin VLA-4 inhibited the stimulated influx of mononuclear leukocytes into the intima by {approx}70% compared with the nonspecific IgG controls, and (2) the combined use of the anti–VLA-{alpha}4 and anti-CD18 mAb completely blocked mononuclear leukocyte emigration. Both monocytes and lymphocytes are known to use multiple pathways for adhering to ECs under both in vitro and in vivo conditions.20 37 38 39 40 These include interactions between (1) the leukocytic receptors CD11a/CD18 or CD11b/CD18 and endothelial ICAM-1 and/or ICAM-2,39 40 41 42 43 44 (2) VLA-4 and the inducible VCAM-1,19 25 45 46 (3) the endothelial leukocyte adhesion molecule-1 (also designated E-selectin) and its leukocytic ligand(s),37 47 48 49 and possibly (4) additional adhesion pathways that are as yet poorly characterized.40 50 The dominance or hierarchy of the utilized pathways varies with the activation and differentiation state of the interacting cells51 as well as with time.27 In our experiments, the CD18-dependent and VLA-4–dependent interactions dominated and appeared to be sufficient for the recruitment of mononuclear leukocytes within the first 36 hours of experimental intimal thickening. Interestingly, despite blockade of both CD49d and CD18, some mononuclear leukocytes were observed adhering to the endothelium, suggesting a potential involvement of additional receptor/ligand pairs in the adhesion process. However, these adhesive interactions seemed to be "abortive" under the applied conditions, since they did not result in final accumulation in the subendothelial space of the carotid intima. The present study was not intended to distinguish whether the adhesion molecules are preferentially involved in adherence or transendothelial migration. As suggested from a substantial body of in vitro studies, the adhesion of monocytes,52 lymphocytes,53 and PMNs52 54 to the endothelium is predominantly mediated by CD18-independent pathways, whereas the migration across the endothelium is dominated by CD18-dependent mechanisms. However, a significant VLA-4–dependent component in the chemotactic factor–induced migration of monocytes across activated endothelium has also been reported.55 Future experiments are required to determine whether differences in the utilization of CD18 and VLA-4 during the adhesion and transendothelial migration also exist in our model.

The dependence of mononuclear leukocyte recruitment on VLA-4 and CD11/CD18 during fibromuscular thickening provides indirect evidence for the expression of the appropriate counterreceptors on the endothelial surface. VCAM-1 has been reported to be expressed by endothelial cells and by intimal subpopulations of SMCs and macrophages in both diet-induced56 57 58 and human atherosclerotic lesions.59 60 Additionally, increased immunoreactivity for ICAM-1 has been demonstrated at the arterial luminal surface of human atherosclerotic plaques.59 61 62 On the basis of our findings, it may be speculated that both of these inducible endothelial adhesion molecules are expressed by the carotid endothelium during fibromuscular thickening in response to ES.

As far as granulocytes are concerned, the present work also provides information on the pathways underlying the immigration of basophils and eosinophils into the developing lesion. The occurrence of basophils and eosinophils in arterial thickening, although minimal compared with neutrophil invasion, is not a curiosity of our model. All subclasses of granulocytes have been described to be present in other models of experimental arteriosclerosis.13 63 64 65 On their way into the electrically induced thickening, basophils and eosinophils used the VLA-4–dependent pathway, as judged by the complete abolishment of their influx by the anti–VLA-{alpha}4 mAb. Neutrophil involvement, however, remained unaffected by this antibody. These results extend our previous observations that the anti-CD18 mAb 60.3 had no detectable effect on the accumulation of basophils and eosinophils in the electrically stimulated arterial intima.17 Additionally, our data are in line with in vitro studies demonstrating that the adherence of basophils and eosinophils to cytokine-activated cultured ECs is dominated by the VLA-4 pathway and scarcely affected by CD18 antibodies,21 22 23 whereas neutrophil binding was shown to be VLA-4 independent.21 22 52

Effect of Mononuclear Leukocytes on SMC Migration
The time frame in the present study (ie, termination of all experiments 36 hours after electrode implantation) was designed to examine the initial migration of SMCs as they start to move across the internal elastic lamina from the underlying media into the intimal compartment within the first 2 days of ES.16 17 18 At this time, an increase in the replication rate of SMCs was not yet detectable in the intima,36 suggesting that the SMCs identified at 36 hours within the ES intima were derived from cell migration but not cell proliferation.

From previous experiments, we can exclude PMNs as cellular mediators in SMC migration.17 The present experiments indicate that the mononuclear leukocytes that accumulate within the nascent thickening promote SMC movement into the intima, since inhibition of mononuclear leukocyte infiltration by {approx}70% achieved with anti–VLA-{alpha}4 treatment resulted in reduced SMC migration. Moreover, the complete abolishment of mononuclear leukocyte influx by anti–VLA-{alpha}4 plus anti-CD18 led to an even more pronounced reduction in the number of intimal SMCs. Monocytes/macrophages, which prevail in the population of infiltrating mononuclear leukocytes (at {approx}90%), are good candidates for promoting SMC migration. They have the capacity to secrete not only proteolytic enzymes, which are capable of degrading the cage of extracellular matrix surrounding SMCs,66 67 68 but also chemotactic substances for SMCs, eg, PDGF.11 In fact, a rapid increase in PDGF-B chain expression was detected in rat carotid arteries exposed to the same method of ES as used in the present study69 ; however, without determining the cellular association. It is intriguing to raise the hypothesis that PDGF-B is indeed expressed in monocytes/macrophages in our model, as determined in human and nonhuman primate atherosclerosis7 and that PDGF is one of the mediators of the migration-promoting effect on SMCs, as shown in other in vivo models of intimal thickening.8 9 70 Whether the lymphocytes, which are also members of the mononuclear leukocyte population, also affect SMC migration, eg, by activating macrophages or by altering the responsiveness of SMCs to chemotactic factors, remains to be determined. It is less likely that the anti-CD18 or anti–VLA-{alpha}4 antibody may exert a direct suppressive effect on SMC migration, since the CD18 integrins are exclusively expressed on leukocytes20 and VLA-4 has so far not been demonstrated on adult vascular SMCs,58 71 although {alpha}4 expression has been detected on fetal SMCs.72 However, it cannot be excluded that the antibodies indirectly affect SMC functions by interfering with SMC/leukocyte interactions mediated by ICAM-1 or VCAM-1, which both were shown to be expressed by SMCs in atherosclerotic lesions.58 60 61 62 In the case of the combined treatment with anti–VLA-{alpha}4 and anti-CD18, however, SMC/leukocyte interactions seem unlikely to occur, because leukocyte entry into the intima was completely blocked.

In addition to the leukocyte-dependent portion of SMC migration, our results also suggest that at least one third of the SMCs present in the early intimal lesion migrated independently of mononuclear leukocyte infiltration. This suggests that mononuclear leukocytes contribute only partially to the control of SMC migration and that additional systems are involved in regulating the migratory behavior of SMCs. ECs, for example, may be an additional regulatory component because of their ability to synthesize and release chemoattractants for SMCs73 74 and to generate plasminogen activators that can induce and amplify the degradation of the matrix surrounding stimulated SMCs.75 It is interesting to note that SMCs that migrated in the absence of mononuclear leukocytes into the intima were often observed closely interconnected with basal protrusions of ECs or beneath interendothelial contacts. It cannot be excluded that substances derived from the plasma that gain access to the arterial intima (eg, by enhanced endothelial permeability as observed after brief ES16 ) may also play a role in promoting SMC migration.

In conclusion, our findings show that in the rabbit model of electrically induced intimal thickening, the abolishment of mononuclear leukocyte invasion into the arterial intima reduced SMC migration, indicating a role for these leukocytes in stimulating lesion development and, possibly, in atherogenesis. The entry of mononuclear leukocytes was inhibited by blocking VLA-4–dependent and CD18-dependent adhesion mechanisms, suggesting that the expression of the respective endothelial counterreceptors is indirectly involved in mediating SMC migration.


*    Selected Abbreviations and Acronyms
 
EC = endothelial cell
ES = electrical stimulation (electrically stimulated)
ICAM = intercellular adhesion molecule
mAb = monoclonal antibody
PDGF = platelet-derived growth factor
PMN = polymorphonuclear neutrophil
SMC = smooth muscle cell
VCAM = vascular cell adhesion molecule
VLA = very late activation antigen


*    Acknowledgments
 
We wish to thank Rosemarie Weidler for the implantation of the electrodes and Antje Rummel for her expert assistance in electron microscopy.

Received July 10, 1995; accepted August 11, 1995.


*    References
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 *References
 

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