Macrophages - NCBI

Arterial Foam Cells With Distinctive Immunomorphologic and Histochemical Features of Macrophages Thomas Schaffner, MD, Katherine Taylor, MS, Eugene J. Bartucci, BA, Katti Fischer-Dzoga, MD, James H. Beeson, PhD, MD, Seymour Glagov, MD, and Robert W. Wissler, PhD, MD

A variable population of fat-filled "foam" cells in diet-induced experimental arterial intimal plaques of rabbits and monkeys were analyzed for several features characteristic of macrophages. These included: l)surface binding and phagocytosis of antibody-coated or complement-coated erythrocytes to detect specific surface receptors; 2) cytochemical tests and ultrastructural features to evaluate cell function and structure; and 3) rapid adherence to glass, a feature of macrophage activity, to isolate and identify a homogeneous population of fat-filled foam cells from excised and disrupted arterial lesions. Mixed populations of cells grown in culture from explants of lesions were also analyzed and lipidfilled cells were studied in histologic sections of adjacent lesions. Eighty to ninety percent of the easily dislodged glass-adherent cells from lesions had surface receptors for the Fc portion of immunoglobulin G and for the third component of complement. Coated red blood cells were readily phagocytized, but noncoated cells were not. Acid lipase activity was demonstrated in the Fc-receptor-positive cells. These cells were also devoid of ultrastructural features of smooth muscle. Among the cells growing or migrating out of explants, a population of large round foam cells possessed all of the macrophage features found in the glass-adherent cells from lesions and lacked ultrastructural characteristics of smooth muscle. Fusiform lipid vacuolated cells also grew out of the explants but did not exhibit surface receptors, failed to phagocytize coated or noncoated erythrocytes and did not stain for acid lipase activity; these cells showed distinctive morphologic features of smooth muscle. In histologic sections of nearby lesions foam cells that showed macrophage characteristics, ie, acid lipase activity and the presence of lysozymelike antigen, lacked ultrastructural smooth muscle features. Smooth muscle cells in lesion sections often contained lipid but demonstrated no lysozyme or acid lipase activity. The occurrence of a population of cells with several functional and structural features of macrophages among the lipid-laden cells of experimental diet-induced arterial lesions suggests that some foam cells may not be of smooth muscle origin but may be derived from monocytes. An alternative explanation, that metabolically altered autochthonous arterial wall cells assume one or more characteristics of mononuclear phagocytes is less likely, since some of the markers used in these experiments are unrelated. Both explanations deserve further careful study. (Am J Pathol 1980, 100:57-80)

IT IS CLEAR from many studies 1-4 that smooth muscle cells are prominent in atherosclerotic lesions in many species. The accumulation of From the Departments of Pathology and Medicine, University of Chicago, and The Specialized Centers of Research in Atherosclerosis and Ischemic Heart Disease. Supported by USPHS Grants HL-15062-08 and HL-17648-05, by the Block Fund at the University of Chicago, and by the Heart Research Foundation, Inc. Accepted for publication Febnrary 6, 1980. Address reprint requests to Dr. T. Schaffner, Pathologisches Institut des Universitat, Bern Freiburgstrasse 30, CH 3000 Bern, Switzerland. 57 0002-9440/80/0709-0057$01.00 © American Association of Pathologists

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American Journal of Pathology

lipids, including cholesterol, within and about the cells that populate atherosclerotic lesions is also a characteristic feature. Nevertheless, it does not follow from these findings that all of the lipid-containing cells in arterial intimal plaques are of smooth muscle origin.3 In previous reports from our laboratory, it was noted that although smooth muscle cells predominate in progressive human and rhesus monkey plaques, many cells in lesions of other species appear to be of a different type.>7 Other investigators have maintained that at least some of the large, round, markedly vacuolated elements known as foam cells, often numerous in relatively flat, fatty human lesions 8 and in diet-induced intimal fatty streaks in several experimental models,9-12 are actually mononuclear phagocytes, ie, macrophages of monocytic origin.8"0"2-8 In a previous report, one of us' sum marized the evidence that a predominance of fat-filled cells of monocyte origin could help explain some of the peculiarities of experimental atheromatous disease in the rabbit. More direct evidence for the presence of more than one cell type in intimal plaques in the rabbit had been given recently by Haley et al " who described two separate cell populations in diet-induced lesions on the basis of differences in cell density. Foam cells in experimental lesions have actually been shown to have cytochemical properties characteristic of macrophages,13"14"16'8 to resemble macrophages by adhering rapidly to glass,9 and to lack ultrastructural features characteristic of smooth muscle.3""0"5 Although none of these observations constitute proof that any of the lipid-laden cells in atherosclerotic lesions are necessarily mononuclear phagocytes distinct in origin from smooth muscle cells, they do suggest that more detailed characterizations of foam cells could reveal that some are indeed derived from more than one source and/or that they reflect an altered functional state of arterial wall cells. In addition to providing data concerning the regulation of cell function, such findings could also provide new insights into the pathogenesis of early atheromatous lesions and into the nature of cell changes that characterize progression and regression of experimental plaques. We and others 19-22 have described techniques for the identification of macrophage markers in cells derived from diet-induced arterial lesions. In preliminary reports, we submitted evidence that foam cells dislodged mechanically from experimental arterial plaques exhibited receptors for the Fc portion of immunoglobulin G (IgG-Fc) in vitro.Y,21 In addition, we were able to distinguish the receptor-positive, large, round, fat-filled foam cells from fusiform, lipid-laden smooth muscle cells growing out of explanted lesions in tissue culture.19-21 In the present report we further validate the results of these studies by means of suitable controls and provide additional new supportive data for macrophage-specific functions 3 of

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foam cells. We will present new immunomorphologic and histochemical findings, using additional unrelated macrophage markers and compare in vitro findings with findings in tissue sections. In addition to acid lipase activity and receptors for IgG-Fc,24 we will demonstrate receptors for the third component of complement 5 and the presence of intracellular lysozyme-like antigen. Since we use a morphologic assay system to show that specific antibody-coated erythrocytes form immune-adherence rosettes with arterial foam cells, we will also demonstrate phagocytosis of the coated erythrocytes, an additional characteristic of mononuclear macrophages. Materials and Methods Atheromatous arterial disease was induced in adult male New Zealand white rabbits and male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys. The rabbits were fed rabbit chow (Purina) mixed with 0.4% (wt/wt) cholesterol dissolved in 4% corn oil, for 4 months. Unless otherwise indicated, the monkeys ate a special monkey chow (Purina) mixed with 2% cholesterol and 25% peanut oil, for 3 or 6 months. At the time of autopsy, animals were anesthetized with sodium pentabarbital (50 mg/kg body weight) and exsanguinated by way of a needle puncture at the aortic bifurcation. Isolation and Culture of Cells Glass-Adherent Cells

Intimal lesions were dissected from the aorta of 6 rabbits and from the carotid and femoral arteries of 7 cynomolgus and 4 rhesus monkeys. The plaques were then stripped from the media with forceps, and cell suspensions were prepared by disrupting individual lesions in 2 ml of Hanks' balanced salt solution (HBSS). The plaques were disrupted by means of multiple strokes with a sharp dissecting needle. Tissue debris was allowed to settle for 1 minute, after which the fluid was incubated in Lab-Tek slide chambers for 30 minutes in HBSS at 37 C. The HBSS, along with remaining tissue debris and unattached cells, was then decanted. The remaining adherent cells were tested for the presence of immune adherence receptors within 1 hour of the death of the animals. Culture of Explants

Small pieces of lesions were cultured as described elsewhere.'- In brief, aseptically stripped intima-media preparations of lesions from rabbit aortas and from femoral and carotid arteries of monkeys were cut into small pieces approximately 1 mm in diameter. The fragments were then attached to vertical 25-ml polystyrene tissue culture flasks by drying them onto the bottom for approximately 30 minutes. The fragments were subsequently carefully flooded with Eagle's medium containing 10%o fetal calf serum by bringing the flask into a horizontal position. Incubation was carried out in air with 5% CO2 at 37 C for extended periods of up to 60 days. Cultures were assayed for receptors after one brief rinse with HBSS. Receptor Assays IgG-coated erythrocytes (EAIgG) were obtained by incubating sheep or human (type A, Rh-positive) erythrocytes in one-half the least agglutinating concentration of antierythrocyte IgG (Cappel Laboratories) or antierythrocyte F (ab')2 27 dissolved in HBSS (1%

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vol/vol, erythrocytes to medium) at 37 C for 30 minutes. Washed EAIgG was adjusted to 1% (vol/vol) for use in the receptor assays. IgM-coated erythrocytes (EAIgM) were prepared by incubation of sheep erythrocytes (2.5% vol/vol) in a 1: 500 dilution of hemolysin (Cappel Laboratories) at 37 C for 30 minutes. Erythrocytes coated with the third component of complement (EAIgMC) were obtained by incubation of the EAIgM (2.5% vol/ vol) in nonlytic, C5 deficient, AKR-mouse serum, diluted 1: 20 in HBSS at 37 C for 30 minutes after one washing in HBSS. After an additional washing after incubation and storage on ice for 1 hour, 0.5% suspensions (vol/vol) of EAIgM and EAIgMC were used in complement receptor assays. Erythrocytes not coated with either of the immunoglobulins were prepared by incubation in HBSS or dilute mouse serum alone. All erythrocytes used in these experiments were incubated in their respective solutions within 48 hours of venipuncture and after 5 preliminary washings in HBSS. In control experiments we used erythrocytes coated with F(ab')2 fragments of IgG, prepared as described previously,25 or erythrocytes incubated with AKR-mouse serum alone. Other controls included the addition of rabbit antiferritin IgG or antiferritin F(ab')2 to the coated erythrocyte suspensions. Glass-adherent cells isolated directly from lesions or cells growing or migrating out of cultured lesion fragments were overlaid for 30 minutes with 1% suspensions of antibodycoated erythrocytes at 25 C and 0.5% suspensions of complement-coated erythrocytes at 37 C. The nonbound erythrocytes were then removed by four rinses with HBSS. After the removal of nonbound erythrocytes from the cultures, the cultures were fixed by flooding with 2.5% phosphate-buffered glutaraldehyde. Some preparations were then processed for scanning or transmission electron microscopy as described in detail elsewhere.2' Other preparations were stained with hematoxylin and eosin and/or oil red 0 and scanned at a magnification of 500 to determine the relative number of receptor-positive cells with lipid vacuolization. Two hundred fifty cells were counted for each determination. Enzyme and Immunohistochemical Procedures Acid Lipase

Samples obtained from cultures and from cell suspensions were fixed for 20 minutes at 4 C in 2.5% purified glutaraldehyde (Sigma, St. Louis, Mo) in 0.15 M sodium phosphate buffer, pH 7.4. After one rinse in refrigerated 0.2 M tris-HCI, pH 7.2, for five minutes, acid lipase activity was visualized as described elsewhere.'8 In brief, 40 mg naphthylpalmitate, 650 mg Triton X-100, and 5 ml 0.1 sodium acetate buffer, pH 5.2, were mixed in a test tube for 3 minutes and then heated in a waterbath to 70 C. The test tube was then capped and shaken vigorously until the solution became clear. The naphthylpalmitate micelle preparation was then added to 50 ml 0.1 M sodium acetate buffer containing 2 ml hexazotized pararosaniline (2% wt/vol) and adjusted to pH 5.2. Incubation of cultures in this mixture was carried out for 6 hours at 25 C. The cultures were then washed and examined by light microscopy. The same incubation medium was used to detect acid lipase activity in intact lesions, fixed and sectioned as described previously.'8 Macrophages in all tissues which have been examined thus far stain intensely by this technique. Lysozyme Lysozyme was visualized by indirect immunofluorescence, with the use of a modification of the peroxidase procedure of Spicer et al.' Tissue samples were fixed for 6 hours at 4 C by successive changes of 20 minutes each in 50%/o, 70%, 95%, and 100% ethanol. After the tissue had been warmed to room temperature, one more change of 100%o ethanol and two changes of toluene, 30 minutes each, were used to assure maximum dehydration. The tissue was then infiltrated with paraffin at 60 C for 1 hour, embedded, and kept in the freezer overnight. Sections cut at 6 ,u were floated on water at 52 C, taken up and dried onto slides, and kept at -18 C until used. All sections were either used within 1 week after cutting or discarded. For immunofluorescence staining, the sections were placed in a 56 C

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oven for 10 minutes, cleared of paraffin in xylene, and rehydrated through 100%7o, 95%, and 70% ethanol to 0.5 M tris-HCl at pH 7.4. Preincubation for 15 minutes with normal rabbit serum was used to prevent nonspecific staining due to hydrophobic binding. Goat IgG antihuman lysozyme (Miles Laboratories) diluted 1:40 with phosphate-buffered saline (PBS), normal goat serum (1: 40), or goat IgG antihuman Apo B (1: 40) were then applied to the sections for 30 minutes in a humidified chamber at room temperature and followed by three washes in PBS of 5 minutes each. Antibody binding was visualized by incubating the sections in fluorescein-conjugated rabbit IgG antigoat IgG (Cappel Laboratories, 1: 20) for 15 minutes. Sections were then washed twice in PBS, mounted in phosphate-buffered saline glycerol (1: 2), and viewed with an American Optical mercury-arc epifluorescence microscope with built in fluorescein excitation and barrier filters. Photographs of antilysozyme-incubated and control sections were taken at identical time exposures in order to permit comparison of staining intensity. In blocking experiments, 0.5 mg/ml of a human lysozyme preparation obtained from human spleen was added to the diluted serum containing antilysozyme. The partially purified human lysozyme was prepared from human spleen obtained at autopsy according to the method described by Jolles.' In brief, 300 g of spleen was homogenized in 1 1 deionized water in a Waring blender. After centrifugation for 20 minutes at 20,000g, the homogenate was acidified to pH 5.5 with acetic acid and then heated and kept at 100 C for 5 minutes in an autoclave. The precipitated, denatured protein was then cleared by centrifugation, and the lysozyme remaining in the supernatant was absorbed for 1 hour onto Biorex AG-70 ion-exchange resin, which had been equilibrated with 0.1 M sodium phosphate, pH 6.55. The lysozyme-containing fraction was then eluted with 10 volumes of 1 M sodium phosphate, pH 6.55. After dialysis for 48 hours at 4 C against distilled water, the preparation was filtered, freeze-dried, resuspended in 4 ml PBS (approximately 4 mg protein per ml), and frozen in 0.5-ml units. When tested on an Ouchterlony immunodiffusion plate, this lysozyme preparation formed a line of precipitation with undiluted goat antilysozyme (0.5 mg specific antibody/ml) halfway between the respective walls and did not react with any of the other control reagents. A 1: 10 dilution of this preparation cleared a 0.01% suspension of Micrococcus lysodeikticus. Staining controls included omission of the primary goat antibody or the secondary fluorescent rabbit antibody.

Results

The lesions that form the basis of this report were slightly raised, yellow fatty streaks and plaques resembling those described in our preliminary accounts.21'30'3' The femoral and carotid artery lesions of the monkeys consisted of clusters of large foam cells in several layers beneath the lumenal surface as well as lipid-laden smooth muscle cells at the base of the lesions. In general, the rhesus monkey lesions contained a higher proportion of smooth muscle cells. There was little involvement of the media in the rhesus, but the cynomolgus monkeys showed occasional large medial clusters of lipid-laden cells. The aortic lesions of the rabbits were more heterogeneous. Some were yellow fatty streaks, laden with foam cells, quite similar in all respects to those found in the cynomolgus monkeys, while others were raised, pale lesions with varying degrees of cellularity, and foam cells in clusters at the edges, near the endothelial surface or interspersed with layers of smooth muscle cells near the media. Typical rabbit and cynomolgus lesions are illustrated in Figures 1 and 2.

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Foam Cells Isolated From Suspensions by Substrate Adherence

Cells dislodged from mechanically disrupted lesions and attached to glass varied greatly in size, ranging from 7 IL to 50 IL in diameter. In preparations stained with hematoxylin and eosin most of these cells had the usual foam cell appearance. Up to 0.5% of the cells in preparations derived from the rabbit lesions were multinucleate. Nuclear pyknosis and rhexis were seen frequently. Nearly all of the glass-attached cells stained intensely with oil red 0, indicating the presence of neutral lipid. In none of our preparations were fewer than 90% of the cells stained. In unstained, fixed preparations viewed with polarized light, intracellular lipid droplets showed birefringence with frequent Maltese crosses, characteristic of the liquid crystalline and smectic states of cholesterol esters. Ninety percent or more of the glass-attached cells stained for acid lipase activity. The functional and morphologic features of these cells are illustrated in Figures 3-7. Eighty to ninety percent of the glass-adherent cells isolated from lesions of all three species reacted strongly with the antibody-coated erythrocytes (Table 1). Erythrocytes coated with antierythrocyte IgG formed rosettes (EAIgG rosettes), indicating the presence of surface receptors for IgG. Erythrocytes coated with the third component of complement by activation of the complement system of nonlytic mouse serum by IgM also formed rosettes (EAIgMC rosettes) with the foam cells, indicating the presence of surface receptors for the third component of complement. The proportion of rabbit aortic foam cells positive for complement receptor dropped to less than 50% if the attached cells were kept in vitro for longer than an hour before the receptor assays, while the reactivity of primate artery cells was maintained for up to 4 hours. The controls indicated that surface binding of erythrocytes to the foam cells was specific and due to surface Fc receptors (Table 1). Removal of Fc portions of the antibody by peptic digestion (F[ab']2 fragments) prevented the formation of rosettes; intact antiferritin IgG inhibited EAIgG rosette formation, whereas antiferritin IgG rendered Fc-deficient had little inhibitory effect. The ultrastructural appearance of the receptor-positive foam cells is shown in Figures 5 and 7. The cells lacked basal lamina, showed abundant microvilli intricately associated with the attached erythrocytes, and contained numerous lipid inclusions, many of which were surrounded by a thick single membrane. Intracellular erythrocytes were prominent. Dense granules, resembling lysosomes, were seen throughout the cytoplasm but most notably at the periphery of large Golgi areas. Thin filaments were noted near the cell surface and about the Golgi regions. There were minor distinctions between cells derived from rabbit aortas and those derived

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Table 1-Formation of Rosettes of Antibody-Coated Erythrocytes on Glass-Adherent Foam Cells Dislodged From Rabbit Aortic and Monkey Arterial Fatty Streaks Percentage of cells forming Displacing rosettest reagent Source of foam cells* Coating of test erythrocytes

Rabbit aortas 1

IgG Uncoated

None None

97.7 0.0

IgG F(ab')2

None None

86.2 0.0

IgG

None None IgG 11

84.3 2.8 5.8 58.0

None None

IgG * *

89.0 0.3 82.4

IgM and Complement

None None IgG* *

86.2 1.7 90-.8

1

II gG F(ab')2 Uncoated

None None None

77.6 1.4 1.7

2i

IgG Uncoated

None None

89.8 3.6

3f

IgM and Complement

None None

86.6 0.5

4§

IgM and Complement IgG F(ab')2

IgG* * None None

77.4 76.6 1.5

5§

IgG IgM and Complement

None None None

88.0 79.7 4.2

2

3

F(ab')2 IgG IgG 4

IgM and Complement

IgM IgM and Complement 5

IgM and Complement

IgM

F(ab')21

Primate arteries

IgM

IgM

Summary of 10 typical experiments. Erythrocytes coated with complete IgG antibody formed EAIgG rosettes, whereas Fc-region-deficient antibody-coated erythrocytes used at the same titer were not able to form rosettes. EAIgG rosette formation was markedly inhibited by simultaneous exposure to soluble, complete IgG but was only slightly inhibited by partially purified

F(ab9)2 fragments deficient in Fc regions. lgM antibody coating alone did not produce rosettes

but C3 complement fixed by this antibody to erythrocytes did produce EAIgMC rosettes. In addition to the results tabulated here, erythrocytes incubated with mouse serum at a dilution of 1: 10 did not result in rosette formation. * Each experiment performed on different animal. t 250 cells counted for each determination. t Cynomolgus carotid. § Rhesus femoral. 0.5 mg. 2.0 mg. * * Rabbit antiferritin 1.0 mg/mi.

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from monkey arteries. The latter were usually smaller than the former, rarely exceeding 20 IL in diameter. The foam cells of primate arteries also spread and flattened less on the substrate, contained fewer lipid vacuoles, and were better preserved than the rabbit aorta cells. In summary, the cell population obtained from mechanically disrupted lesions by their ability to attach rapidly to glass, exhibited several macrophage properties, ie, two distinct immune adherence receptors, macrophagocytosis, and histochemically detectable acid lipase activity, in addition to their neutral lipid and cholesterol ester content and the absence of distinctive morphologic features of smooth muscle. Cells Growing or Migrating From Explanted Lesion Fragments

At 48 hours, two distinct cell types were discernible. Large, flat, polygonal cells, as well as smaller, rounded cells, appeared about the explants. When the preparations were exposed to oil red 0, nearly all of the rounded cells were observed to be completely stained, ie, filled with neutral lipid, whereas the majority of the flat polygonal cells contained only discrete oil-red-0-positive lipid droplets. Nearly all of the round cells formed EAIgG and EAIgMC rosettes, indicating the presence of surface receptors for IgG and complement. These cells also stained intensely for acid lipase activity. The flat, polygonal cells showed no evidence of rosette formation with either immune receptor system and did not stain for acid lipase activity even after prolonged incubations up to 24 hours (Figure 13). In cultures followed for up to 60 days, the rounded cells retained immunoreactivity and acid lipase staining, while the polygonal cells remained unreactive (Figures 8-10). The only distinctive change in the receptor-positive population after prolonged culture was a reduction in cell size and a gradual reduction in intensity of oil red 0 staining for neutral lipid, beginning after 4-7 days in culture (Figure 9). After 2 weeks, the receptor-negative cells began to assume the characteristic growth pattern of smooth muscle, forming numerous spindle-shaped elements that formed confluent masses with the typical surface configuration of hills and valleys (Figure 8). Some of the explants showed, in addition, flat polygonal cells that tended to layer on top of the spindle-shaped cells in the immediate vicinity of the explant (Figure 11). These surface cells became visible by phase contrast microscopy at 2 weeks and were tentatively identified as endothelial cells by the strong staining of their regular and prominent intercellular junctions with 0.1% silver nitrate prior to fixation. These elements did not react with IgG-coated erythrocytes and showed no acid lipase activity. Except for the gradual diminution of their lipid content after 4-7 days in culture, the rounded receptor-positive

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foam cells appearing in outgrowths from explants (Figure 9) differed little in ultrastructural appearance from the glass-adherent foam cells isolated directly from the disrupted lesions. They remained actively phagocytic of coated erythrocytes throughout. The flat, polygonal receptor-negative cells (Figure 10) resembled cultured smooth muscle cells described by others 32 and were closely associated with characteristic extracellular matrix materials such as basal lamina, collagen, and elastin. Thus, two distinct cell populations either grew or migrated from the cultured lesion explants. One type had characteristics identical to the macrophagelike foam cells that adhered to glass after lesion disruption. and the other type was typical of cultured smooth muscle, containing discrete lipid vacuoles and showing no evidence of immunoreactivity or acid lipase content. A third cell type was noted after prolonged culture. These cells were probably endothelium and were neither immunoreactive nor lipid-containing. Sections of Intact Lesions

Frozen sections of lesions sampled adjacent to those used for the in vitro studies revealed large, rounded lipid-positive foam cells that stained intensely for acid lipase activity, and smaller spindle-shaped lipid-containing cells, endothelial cells, and medial smooth muscle cells, all of which were negative for acid lipase activity. The location of the acid-lipase-positive cells within the lesions corresponded to the location of the large foam cells identified by light microscopy of thick sections of Epon-embedded lesions (Figures 1, 2, 14-16). In size, intensity of oil red 0 staining, presence of cholesterol esters by polarizing microscopy, and ultrastructural appearance, these cells were similar in every respect to the glass-adherent foam cells dislodged from lesions and to those growing from lesion explants. Lysozyme was detected exclusively in the large foam cells of the primate lesions. The specificity of lysozyme antibody binding to the foam cells was supported by the controls employed. Antibody binding was blocked by exposure to the human lysozyme preparation, and there was no nonspecific staining of foam cells with an unrelated antibody and normal goat serum (Figure 12). Foam cells in rabbit lesions. stained only weakly at an antilysozyme antibody dilution of 1: 20. These observations on foam cells in sections of monkey arterial lesions suggest that the cells that showed macrophage properties, ie, acid lipase activity and the presence of lysozyme, are representatives of the same population as the macrophagelike foam cells dislodged from lesions or those growing from lesion explants.

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Discussion

Our findings provide further evidence that a population of foam cells obtained from experimental diet-induced arterial lesions can be characterized as macrophages on the basis of several distinct markers and functional properties.23 Foam cells dislodged from lesions by mechanical disruption adhered to glass, had surface receptors for the Fc portion of IgG and for the third component of complement, phagocytized antibodycoated erythrocytes, and were strongly positive for acid lipase activity; these cells had no morphologic features that distinguished them as smooth muscle. Of the cell types which grew or migrated from cultured lesion fragments, lipid-filled large round cells had surface receptors for IgG and the third component of complement, were strongly positive for acid lipase, and lacked specific morphologic features of smooth muscle. Flat or fusiform cells that originated from the same explants usually contained more dispersed lipid droplets and had ultrastructural morphologic features of smooth muscle. These cells, however, had no demonstrable immune-adherence surface receptors and no stainable acid lipase activity. Many of the large, round foam cells in histologic sections of lesions were positive for acid lipase and lysozyme and lacked morphologic features of smooth muscle. Smooth muscle cells in the same tissue sections, many of which contained discrete lipid vacuoles, were not reactive for acid lipase or lysozyme. If we assume, on the basis of functional and morphologic similarity, that the foam cells that adhered to glass and the round foam cells that grew out of the explants were representative of the acid-lipase-positive, lysozyme-containing foam cell population noted in the intact lesions, we must infer that cells functioning principally as phagocytes are abundant in early atherosclerotic fatty streaks of rabbits and some monkeys. Such a finding does not necessarily represent either a refutation or a challenge to the well-documented concept that lipid-containing smooth muscle cells are a characteristic feature of progressive and advanced atherosclerotic lesions. It is rather the origin, relative abundance, turnover, and significance of the macrophagelike cells that require further investigation. Although macrophagelike cells in lesions are probably derived from circulating monocytes, one cannot rule out the possibility that some come from uncommitted mesenchymal cells already present in the arterial wall or from arterial multifunctional medial smooth muscle cells that have been markedly altered by their participation in the atherosclerotic process. If the phagocytic foam cells that form the basis of this report were derived from smooth muscle, the basal lamina, a consistent morphologic fea-

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ture for positive identification of the smooth muscle cells,32 appeared to have been lost in the process. Attempts have been made with some reported success to characterize transitional forms between smooth muscle and large foam cells.333' The main criterion for smooth muscle origin in these predominantly ultrastructural studies was the presence of myofilaments in the foam cells. The presence of myofilaments is, however, probably not sufficient for establishing smooth muscle origin, for thin filaments have been identified in otherwise typical macrophages from other tissues,, and the major contractile proteins have been identified and characterized in extracts of alveolar macrophages.6 In any event, the glass-adherent cells, the large, rounded foam cells growing from explants, and the enzyme staining foam cells present in intact lesions all failed to show either basal lamina or typical dense arrays of myofilaments characteristic of smooth muscle, while the easily distinguishable fusiform cells in culture and in lesions showed both these cytoplasmic structures. Some properties of macrophage activity have been detected in cell types that are not usually regarded as mononuclear phagocytes, 3 and one marker, the Fc receptor, may be acquired by cells that are transformed or infected by virus.37 Polymorphonuclear granulocytes, for example, have many features of macrophages, except that they contain no histochemically demonstrable acid lipase activity.'8'23 Their distinct nuclear morphology and short survival in culture would tend to exclude these cells as precursors of arterial foam cells. Lymphocytes possess complement receptors as well as Fc receptors but do not usually phagocytize large particles or produce lysozyme 23 and are probably acid-lipase-negative.'823 Lymphocytes are therefore unlikely to be progenitors of foam cells. Nontransformed, nonmalignant fibroblasts do not exhibit IgG or complement receptors in vitro.' The lipid-laden morphologically distinctive fusiform smooth muscle cells that grew from lesion explants in our material also never exhibited immune-adherent receptors. While one could speculate that Fc receptors could occur also on transformed smooth muscle cells, it would be difficult to explain the simultaneous occurrence of both complement receptors and other macrophage characteristics on such transformed cells. It should be noted in this regard that complement receptors have not been demonstrated on transformed or malignant cells other than leukocytes. Recent evidence appears to suggest that endothelial cells derived from human umbilical cord exhibit Fc receptors and a putative complement Clq receptor.38 These findings are based on a fluorescence method. Rosette formation with either IgG-coated or complement(C3)coated erythrocytes was not demonstrated in these studies, and it remains to be established that detection of specific markers on endothelium by flu-

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oresceinated reagents is actually due to surface binding by discrete receptors. We could demonstrate neither lysozyme nor acid lipase activity in endothelium in tissue sections of the lesions we studied, nor did the cells resembling endothelium that grew out of explants show Fc surface receptor activity or acid lipase staining. Thus, although none of the functional features that we or others have used for identification of macrophage properties is by itself unique to the mononuclear macrophage, we base our proposal that the cells we have studied in vitro are probably macrophages of blood cell origin on the fact that these cells exhibited a spectrum of unrelated functional and morphologic characteristics. Of the criteria we have chosen to characterize macrophages in culture, the histochemical detection of acid lipase activity requires critical evaluation. Fowler et al 3 have demonstrated that lysosomal enzyme activity, including that of acid lipase and/or acid cholesterol esterase, varies greatly with culture conditions and the number of subculture passages. It is therefore conceivable that we induced the lysosomal enzyme activity that we demonstrated in cultured foam cells and that this activity could also be induced in smooth muscle cells of lesions under suitable conditions." Thus, our finding of acid lipase in foam cells but not in lipid-containing smooth muscle cells does not necessarily negate the smooth muscle origin of the foam cells. Indeed, the fact that typical smooth muscle cells in human and other primate lesions show abundant phosphatase activity (Yang C, Vesselinovitch D, Schaffner T, Wissler RW: Unpublished observations) would appear to indicate that markedly elevated lysosomal enzyme activities may be induced in smooth muscle cells. Acid lipase activity may, however, be an exception to the lysosomal enzyme induction phenomenon, for we were not able to stain for this enzyme activity in subcultures of monkey aortic smooth muscle or human fibroblasts by the method we have developed.'8 If acid lipase was present in these cells, the threshold of enzyme activity necessary for the formation of precipitable dye was not attained. The findings in the present experiments, however, suggest that induction of acid lipase in culture may be minimal. The fusiform smooth muscle cells growing out of explants, although lipid-vacuolated, did not stain for acid lipase activity even after prolonged incubation, while the immune-receptor-positive round foam cells stained strongly after two hours. Since this disparity in staining between smooth muscle and rosette forming macrophagelike cells remained detectable for several weeks in vitro, we must conclude for the time being that longterm exposure of the two cell types to the same culture medium did not interfere with or modify these phenotypic features.2' This observation lends further support to the idea that these cell types are of different origin.

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Fowler et al have recently reported findings similar to ours in studies of foam cells obtained by density gradient centrifugation of enzymatically digested aorta of cholesterol-fed rabbits.'2 These investigators found Fc receptors on the majority of the cells in the low-density cell fraction, but complement receptors were present on only 25% of the cells. The discrepancy between their findings and ours may be a result of cell injury caused by the necessity for prolonged digestion of their tissue and the manner of separation of cells in their experiments. Our observation that prolonged incubation in HBSS led to a reduction in the number of complement-receptor-positive cells, while binding of IgG-coated erythroyctes occurred even when cells had pyknotic nuclei and were quite fragile, supports this explanation. Such findings are not unexpected, since strong C3 receptormediated binding requires energy as well as integrity of the cytoskeleton.404' Furthermore, complement receptors are susceptible to the action of proteolytic enzyme, whereas Fc receptors withstand such treatment.42 Oxygenated cholesterol derivatives or cholesterol itself may also affect membrane requirements for rosette formation.43 Thus, the metabolic and structural state of the cells may be critical for any quantitative comparison of receptor binding by morphologic techniques. We suggest that our method of obtaining foam cells by relatively gentle mechanical disruption of lesions and rapid adhesion to glass not only was selective for a population of foam cells that was loosely bound in the lesion and had macrophage properties but probably also selected for comparatively wellpreserved cells. Since the process of substrate adherence is itself energydependent,42 our method may even have selected for preservation of adhesive capability. It is therefore possible that foam cells that lack the ability to adhere to glass and/or lack other specific macrophage properties were present in the lesions but were not isolated by our method, so that our impression of nearly total foam cell reactivity is not an accurate representation of the proportion of such cells in actual lesions. The observation of Zucker-Franklin 44 that monocytes that become foam cells in vitro lose their complement receptors but not their Fc receptors would tend to support this possibility. Thus, the isolation procedure of Fowler et al ' was less selective than ours and probably yielded a more comprehensive set of cells, but it is likely that the cells they studied were more damaged than ours. Even if our cell yield was relatively small and the cell population we analyzed may have been biased toward a more uniform level of cell function, the evidence derived from the explants and the sections does tend to substantiate the cell suspension findings. The reported recovery of cells in the experiments of Haley et al " was also relatively low, ie, around 20%. A quantitative assessment of a full population of well-preserved aortic cells is therefore desirable but remains to be done.

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We concur with Fowler et al 22 that very little lysozyme is demonstrable in foam cells of rabbit aorta, but we did find abundant lysozyme in monkey foam cells in atheromatous lesions. Although we noted some staining of rabbit cells with antilysozyme antibody diluted 1: 10 and were able to block this staining with added, partially purified human lysozyme, similar staining was noted with control serum used at the same concentration. This observation does not exclude the possibility that rabbit foam cells are macrophages, for the lysozyme content of rabbit macrophages has been reported to be variable, depending on the source of the cells, and lysozyme may be undetectable in some macrophages.4546 The method we have used for the demonstration of lysozyme appears to be acceptable, since macrophage-derived lysozymes of different mammalian species are apparently sufficiently similar in their immunogenic determinants to permit detection with heterologous antisera.232 Spraragen has demonstrated that labeled, circulating mononuclear blood cells participate in atheromatous lesion formation,47 and many investigators have indicated that monocytoid cells may traverse the endothelial barrier and gain access to arterial lesions.'2,15,21,48,49 Phagocytic cells in other organs, such as hepatic Kupffer cells and alveolar macrophages, have been shown to be derived from bone marrow monocytes 5,5' and to participate in disease processes. The lineage of a significant proportion of the cell population in lesions is therefore not a mere semantic problem having to do mainly with definitions of cell differentiation. The participation of circulating monocytes in lesion evolution is accepted as a possibility but has not been given adequate attention. Mononuclear phagocytes in lesions could release elastase and collagenase 52 and secrete factors affecting arterial cell growth and function,52-5 thereby exerting profound influence on lesion induction, remodeling, and regression. In addition, the marked lysosomal activity which some foam cells 10,11,18 appear to share with mononuclear phagocytes 52 may contribute to the catabolism of lipids and thereby to the lipid content and composition of lesions. Further characterization of the phagocytes described in this report and establishment of their derivation and regulation are likely to help illuminate further the mechanisms of atherogenesis and regression. References 1. Haust MD, More RH, Movat HZ: The mechanism of fibrosis in arteriosclerosis. Am J Pathol 1959, 35:265-273 2. Parker F: An electron microscopic study of experimental atherosclerosis. Am J

Pathol 1960, 36:19-53 3. Geer JC, McGill HC Jr, Strong JP: The fine structure of human atherosclerotic lesions. Am J Pathol 1961, 38:263-287

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4. French JE, Jennings MA, Poole JCF, Robinson DS, Florey H: Intimal changes in the arteries of aging swine. Proc Roy Soc Lond [Biol] 1963, 158:24-42 5. Wissler RW: The arterial medial cell, smooth muscle or multifunctional mesenchyme? J Atheros Res 1968, 8:201-213 6. Wissler RW, Vesselinovitch D: Experimental models of human atherosclerosis. Ann NY Acad Sci 1968, 149:907-922 7. Wissler RW, Vesselinovitch D, Getz GS: Abnormalities of the arterial wall and its metabolism in atherogenesis. Prog Cardiovasc Dis 1976, 18:341-369 8. Geer JC: Fine structure of human aortic intimal thickening and fatty streaks. Lab Invest 1965, 14:1764-83 9. Newman HAI, Murad TM, Geer JC: Foam cells of rabbit atheromatous lesion. Identification and cholesterol uptake in isolated cells. Lab Invest 1971, 25:586-595 10. Stary HC, Strong JP: The fine structure of nonatherosclerotic intimal thickening, of developing, and of regressing atherosclerotic lesions at the bifurcation of the left coronary artery. Adv Exp Med Biol 1975, 67:89-108 11. Haley NJ, Shio H, Fowler S: Characterization of lipid-laden aortic cells from cholesterol-fed rabbits: I. Resolution of aortic cell populations by metrizamide density gradient centrifugation. Lab Invest 1977, 37:287-296 12. Balint A, Veress B, Nagy Z, Jellinek H: Role of lipophages in the development of rat atheroma. Atherosclerosis 1972, 15:7-15 13. Adams CWM, Bayliss OB, Turner DR: Phagocytes, lipid-removal and regression of atheroma. J Pathol 1975, 116:225-238 14. Adams CWM, Bayliss OB: Detection of macrophages in atherosclerotic lesions with cytochrome oxidase. Br J Exp Pathol 1976, 57:30-36 15. Clowes AW, Breslow JL, Karnovsky MJ: Regression of myointimal thickening following carotid endothelial injury and development of aortic foam cell lesions in longterm hypercholesterolemic rats. Lab Invest 1977, 36:73-81 16. Schaffner T, Bekermeier M, Fischer-Dzoga K: Histochemical localization of acid lipase in developing primate atheroma. Circulation 1976, 54(Suppl 2):85 Gaton 17. E, Wolman M: The role of smooth muscle cells and hematogenous macrophages in atheroma. J Pathol 1977, 123:23-128 18. Schaffner T, Elner VM, Bauer M, Wissler RW: Acid lipase: A histochemical and biochemical study using triton X-100-naphtylpalmitate micells. J Histochem Cytochem 1978, 26:696-712 19. Bartucci EJ, Schaffner T, Fischer-Dzoga K, Wissler RW: Outgrowth of Fc-Receptor bearing cells from primary aortic explants of NZW rabbits and of M arctoides monkeys fed an atherogenic diet. Fed Proc 1978, 37:933 20. Schaffner T, Vesselinovitch DV, Wissler RW: Macrophages in experimental and human atheromatous lesions: Immunomorphologic identification. Fed Proc 1979, 38:1076 21. Taylor K, Schaffner T, Wissler RW, Glagov S: Immunomorphologic identification and characterization of cells derived from experimental atherosclerotic lesions. Scan Electron Microscopy 1979, 3:815-822 22. Fowler S, Haley NJ: Investigation of macrophage-like properties of rabbit aortic foam cells. Fed Proc 1979, 38:1076 23. Van Furth R: An approach to the characterization of mononuclear phagocytes involved in pathological processes. Agents Actions 1976, 6:91-98 24. Howard JG, Benacerraf B: Properties of macrophage receptors for cytophilic antibodies. Br J Exp Pathol 1966, 47:193-200 25. Lay WH, Nussenzweig V: Receptors for complement on leukocytes. J Exp Med 1968, 128:991-1009 26. Fischer-Dzoga K, Jones RM, Vesselinovitch D, Wissler RW: Ultrastructural and

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immunohistochemical studies of primary cultures of aortic medial cells. Exp Mol Pathol 1973, 18:162-176 Beeson JH, Wissler RW: The use of agarose-bound pepsin for the preparation of F(ab')2 fragments of IgG. Immunochemistry 1977, 14:305-311 Spicer SS, Frayser R, Virella G, Hall BJ: Immunocytochemical localization of lysozymes in respiratory and other tissues. Lab Invest 1977, 36:282-295 Jolles P: Lysozymes from rabbit spleen and dog spleen. Methods Enzymol 1962, 5:137-140 Taylor K, Glagov S, Lamberti J, Vesselinovitch D, Schaffner T: Surface configuration of early atheromatous lesions in controlled-pressure perfusion-fixed monkey aortas. Scan Electron Micros 1978, 2:449-457 Jones RM, Schaffner TJ, Chassagne G, Glagov S, Wissler RW: Comparison of coronary with aortic fatty streaks in rhesus monkeys. Scan Electron Micros 1979, 3:829834 Chamley-Campbell J, Campbell GR, Ross R: The smooth muscle cell in culture. Physiol Rev 1979, 59:1-61 Peterson M, Day AJ, Tume RK, Eisenberg E: Ultrastructure, fatty acid content and metabolic activity of foam cells and other fractions separated from rabbit atherosclerotic lesions. Exp Mol Pathol 1971, 15:157-169 Imai H, Lee KT, Pastori S, Panlilio E, Florentin R, Thomas WA: Atherosclerosis in rabbits: Architectural and subcellular alterations of smooth muscle cells of aortas in response to hyperlipemia. Exp Mol Pathol 1966, 5:273-310 Reaven EP, Axline SG: Subplasmalemmal microfilaments and microtubles in resting and phagocytizing cultivated macrophages. J Cell Biol 1973, 59:12-27 Hartwig JH, Stoessel TP: Isolation and properties of actin, myosin and a new actinbinding protein in rabbit alveolar macrophages. J Biol Chem 1975, 250:5696-5705 Kerbel RS, Davies AJS: The possible biological significance of Fc-receptors on mammalian lymphocytes and tumor cells. Cell 1974, 3:105-112 Shadforth MF, Cunningham PH, Andrews BS: The demonstration of complement Clq and Fc-IgG receptors on the surface of human endothelial cells. Fed Proc 1979, 38:1075 Fowler S, Shio H, Wolinsky H: Subcellular fractionation and morphology of calf aortic smooth muscle cells: Studies of whole aorta, aortic explants and subcultures grown under different conditions. J Cell Biol 1977, 75:166-184 Karnovsky ML: Metabolic basis of phagocytic activity. Physiol Rev 1962, 42:143168 Atkinson JP, Michael JM, Chaplin H Jr, Parker CW: Modulation of macrophage C3b receptor function by cytochalasin-sensitive structures. J Immunol 1977, 118:1292-1299 Stossel TP: Phagocytosis: recognition and ingestion. Sem Hematol 1975, 12:83-116 Streuli RA, Chung J, Scanu AM, Yachnin S: Inhibition of human lymphocyte Erosette formation by oxygenated sterols. J Immunol 1979, 123:2897-2902 Zucker-Franklin D, Grusky G, Marcus A: Transformation of monocytes into "fat" cells. Lab Invest 1978, 38:620-628 Myrvik QN, Leake ES, Fariss B: Lysozyme content of alveolar and peritoneal macrophages from the rabbit. J Immunol 1961, 86:133-136 Cohn ZA, Wiener E: The particulate hydrolases of macrophages: I. Comparative enzymology, isolation and properties. J Exp Med 1963, 118:991-1008 Spraragen SC, Giordano AR, Poon TP, Hamel H: Participation of circulating mononuclear cells in the genesis of atheromata. Circulation 1969, 40 (Suppl 3):24 Poole, JCF, Florey HW: Changes in the endothelium of the aorta and the behavior of macrophages in experimental atheroma of rabbits. J Pathol Bacteriol 1958, 75:245-251

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49. Gerrity RG, Naito HK, Richardson M, Schwartz CJ: Dietary induced atherogenesis in swine: Morphology of the intima in prelesion stages. Am J Pathol 1979, 95:775785 50. Thomas ED, Ramberg RE, Sale GE, Sparkes RS, Golde DW: Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science 1976, 192:10161018 51. Gale RP, Sparkes RS, Golde DW: Bone marrow origin of hepatic macrophages (Kupffer cells) in human. Science 1978, 201:937-938 52. Unanue ER: Secretory function of mononuclear phagocytes. Am J Pathol 1976, 83:396-418 53. Leibovich SJ, Ross R: A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro. Am J Pathol 1976, 84:501-514 54. Gospodarowicz D, Greenburg G, Bialecki H, Zetter BR: Factors involved in the modulation of cell proliferation in vivo and in vitro: The role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells. In Vitro 1978, 14:85-118

Acknowledgments We thank Ms. Mira Jerkovic and Ms. Carol Allen for excellent technical assistance and Ms. Willa Henderson and Ms. Dorothy Peoples for their assistance in the preparation of the manuscript.

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[Illustrations follow]

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Figure 1-Typical fatty streak lesions of rabbit thoracic aorta after 4 months on atherogenic diet. Large round foam cells occupy the intima, and many lipid vacuolated cells are present in the inner media. Bar equals 100 ,. Epon-embedded section, stained with methylene blue, basic fuchsin, and azure II. Figure 2-Intimal lesion from the carotid artery of a cynomolgus monkey fed an atherogenic diet for 6 months. Large foam cells are prominent in the upper (luminal) portion of the lesion (U). Smaller lipid vacuolated cells are present in the less cellular lower portion of the lesion (L). Bar equals 100 A. Epon-embedded section, stained with methylene blue, basic fuchsin, and azure II. Figure 3-Glass-adherent foam cells dislodged from rabbit aortic lesion and incubated with complement-coated erythrocytes. Erythrocytes are bound to the cells and appear as black dots. The immunoreactive cells are variable in size, shape, and degree of spreading. Some exhibit pseudopodia and appear to have been fixed while in motion (arrows). Scale line indicates 100 /. Hematoxylin and eosin stain.

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Figures 4-7-Properties of receptor-positive glass-adherent foam cells. Bars equal 10 ,. Figure 4-Rhesus monkey foam cell EAIgG rosette viewed by polarized light; lipid droplets are birefringent, and some show the Maltese cross configuration indicative of the liquid Figure 5-Transmission electron micrograph of an crystalline state of cholesteryl esters. Fc-receptor-positive rabbit aortic multinucleate foam cell. Cell is surrounded by bound erythrocytes and has numerous membrane ruffles; lipid inclusions are abundant, but no basal lamina or bundles of microfilaments are evident. Figure 6-Rabbit aortic foam cell EAIgG rosettes stained for acid lipase activity; staining is intense after 2 hours of incubation. Figure 7-Scanning electron micrograph of cynomolgus glass-adherent foam cell EAIgG rosette with attached erythrocytes; some of the erythrocytes are being phagocytized, and several are already engulfed (asterisks). Bar equals 10 ,u.

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Figure 8-Cells in the Figures 8-1l1-Properties of cells growing out of intimal lesion explants. immediate vicinity of an explant after 3 weeks in culture and after exposure to IgG-coated erythrocytes. Small, scattered round cells (arrows) have bound many IgG-coated erythrocytes, which appear as dark spots obscuring the cells. The spindle-shaped cells that have not bound erythrocytes are smooth muscle cells arranged in interlacing bundles that are focally several cell layers thick. Figure 9-Transmission electron miScale bar indicates 200 IL; hematoxylin and eosin stain. crograph of a round cell that was found in the outgrowth from a cynomolgus carotid artery lesion explant cultured for 52 days. The cell is completely surrounded by attached IgG-coated erythrocytes, and many have already been phagocytized; lipid vacuoles are greatly reduced in number afFigure 10-A different area from the same ter the extended period in culture. Bar equals 10 ,t. culture shown in Figure 9. A macrophagelike cell with bound and phagocytized erythrocytes (top) is without basal lamina or myofilaments. An adjacent smooth muscle cell below it has abundant peripheral microfilaments, dilated endoplasm reticulum, and a fuzzy surface covering that is probably Figure 11basal lamina; it has no attached or phagocytized erythrocytes. Bar equals 10 ,u. Flat polygonal cells at the margin of a cynomolgus artery lesion explant after 4 weeks in culture. The closely apposed cells, sharply outlined by silver staining, did not bind erythrocytes coated with IgG; these may be endothelial cells. Bar equals 100 It.

12

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Figure 12-Detection of lysozyme in the foam cells of a cynomolgus artery lesion. A-The primary antibody, goat antilysozyme, was diluted 1: 40; foam cells near the lumen stain intensely. B-The same antibody dilution but with added partially purified human lysozyme results in marked blocking of antilysozyme binding. Bars equal 200 ,u. Figure 13-Acid lipase activity and IgG receptors studied simultaneously in a 48-hour culture of an explant from a cynomolgus artery lesion. Large, lipid vacuolated, stellate and fusiform cells (arrows) fail to bind IgG-coated erythrocytes and are acid-lipase-negative. Smaller, rounded foam cells bind many erythrocytes and stain strongly for acid lipase activity. The large fusiform cells proved to be smooth muscle. The preparation was incubated for 6 hours and viewed by 25% phase contrast. Bar equals 200 ,u.

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Figures 14-16-Frozen sections of 3 lesions near those used for mechanical disruption or explant culture. Figures 14A, 15A, and 16A show the three samples after staining for acid lipase activity. The companion figures, 14B, 15B, and 16B show oil red 0 stains of immediately adjacent sections of the corresponding blocks. Bars equal 200 A-Acid lipase activity is limited to large foam ,u Figure 14 Rabbit aorta. B-The lipid-filled smooth muscle cells of the media do not stain cells in the intima. A-Foam cells near Figure 15-Cynomolgus carotid artery. for acid lipase. B-Smaller, lipid-filled cells are the lumen are intensely positive for acid lipase. Figure seen at the bottom of lesion (arrows). These did not stain for acid lipase. A-Cluster of foam cells stains for acid lipase activ16-Rhesus femoral artery. B-Lipid-filled smooth muscle in both intima and media adjacent to the foam ity. cell cluster did not stain for acid lipase.

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[End of Article]