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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:601-611

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:601-611.)
© 1995 American Heart Association, Inc.

Articles

Repression of the Macrophage Scavenger Receptor in Macrophage–Smooth Muscle Cell Heterokaryons

Matthias Rommeswinkel; Nicholas J. Severs; Mathias Köster; Horst Robenek

From the Institute for Arteriosclerosis Research, University of Münster, Münster, Germany, and the National Heart and Lung Institute (N.J.S.), London, England.

Correspondence to Professor Dr H. Robenek, Institute for Arteriosclerosis Research, Domagkstr 3, University of Münster, Münster, Germany.


*    Abstract
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 *Abstract
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Abstract Macrophage scavenger receptors mediate the uptake of chemically modified LDL in an unregulated manner, leading to massive intracellular accumulation of lipid and thus a foamy cellular morphology. In atherosclerotic lesions, foam cells originate not only from macrophages but also from smooth muscle cells, yet smooth muscle cells do not normally express scavenger receptors, and when exposed to chemically modified LDL in vitro, lipid accumulation does not occur. The mechanism of conversion of smooth muscle cells into foam cells in the arterial wall is thus still under discussion. To investigate whether direct interaction between macrophages and smooth muscle cells may be involved and to explore the effects of components of the two cell types on the expression of scavenger receptors, we report here experiments using somatic cell hybrids formed by fusion of the two cell types. Immunofluorescent labeling and confocal microscopic techniques were applied to investigate and measure (1) lipid accumulation (using Nile Red staining), (2) the binding and uptake of acetylated LDL (using 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–labeled acetylated LDL), and (3) receptor expression (assessed using a specific anti-receptor antibody) in smooth muscle cell–macrophage heterokaryons, macrophage-macrophage homokaryons, smooth muscle cell–smooth muscle cell homokaryons, and unfused macrophages and smooth muscle cells. The results demonstrate that scavenger receptor expression becomes repressed in macrophage–smooth muscle cell heterokaryons but not in macrophage-macrophage homokaryons. One possible explanation for the observed repression would be the existence of a negative regulatory cytoplasmic factor produced by smooth muscle cells.


Key Words: scavenger receptor • macrophages • smooth muscle cell • cell fusion • lipid accumulation • atherosclerosis


*    Introduction
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 *Introduction
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 down arrowDiscussion
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Foam cells have long been recognized as a characteristic feature of the atherosclerotic lesion, and these cells are known to originate both from macrophages and smooth muscle cells (SMCs).1 2 The genesis of macrophage foam cells is well understood: macrophages express scavenger receptors, of which two types have been cloned (termed type I and type II),3 and these receptors mediate the uptake of modified LDL in an unregulated manner, leading to massive intracellular accumulation of lipid.4 However, exposure of SMCs to chemically modified LDL in vitro does not normally lead to lipid accumulation.5 Although some recent reports indicate that scavenger receptors are expressed following exposure of SMCs to phorbol esters6 7 or growth factors,8 9 studies on the atherosclerotic plaque in situ indicate that SMCs do not appear to express scavenger receptors at any stage of atherosclerosis.10 11 12 Another line of work has shown that SMCs adopt a foam cell–like morphology in vitro when brought into intimate contact with macrophage-derived lipid droplets.13 The possibility exists, therefore, that direct interactions between macrophages and SMCs in vivo might provide an alternative possible mechanism by which the properties of the SMC are altered, such that they become susceptible to foam cell transformation. In theory, such interactions could take a variety of forms, but studies aimed at investigating this possibility further are lacking, so their nature has remained speculative.14

The use of hybrid cells, formed by fusion of two different cell types,15 16 potentially offers a new approach to these questions. After fusion, the influence of cytoplasmic and nuclear components of different cellular origin on gene expression can be explored since differentiated functions may cease17 18 19 20 or appear.21 22 23 24 Here we report experiments using somatic cell hybrids, formed by fusion of macrophages and SMCs, designed to establish the effects of direct interaction between components of the two cell types on scavenger receptor expression. We produced somatic cell hybrids by fusing porcine SMCs and mouse peritoneal macrophages using polyethylene glycol (PEG) treatment.25 Species-specific differences permitted, with the aid of a nuclear stain, characterization of the numbers and types of cell that constituted the hybrids, and also made possible the investigation of the expression of a further mouse macrophage–derived protein in addition to the scavenger receptor. The culture conditions chosen ensured that all cytoplasmic and nuclear components remained intact within the fused cells, excluding the possibility that any changes in gene expression could result from the loss of genetic information through cell division.16

Using confocal/immunofluorescence microscopic techniques to follow lipid accumulation, ligand binding and uptake, and receptor expression in cells exposed to chemically modified LDL, we show that the direct interaction between macrophages and SMCs resulting from fusion between these cell types in fact leads to repression of scavenger receptors rather than their expression. The findings raise the possibility that SMCs produce a cytoplasmic factor (or factors) that inhibit(s) expression of the scavenger receptor.


*    Methods
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 up arrowIntroduction
 *Methods
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Chemicals
Dulbecco's minimal essential medium (DMEM), fetal calf serum (FCS), penicillin, streptomycin, L-glutamine, PEG 1500, and trypsin-EDTA were obtained from Boehringer. Sodium pyruvate and nonessential amino acids were from GIBCO. Collagenase CLS (204 U/mg; Worthington) was purchased from Biochrom KG. Elastase type III (62 U/mg) was obtained from Sigma. 1,1'-Dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Molecular Probes. All other chemicals were from Sigma or Merck.

Cell Culture
SMCs were isolated from the thoracic aortas of healthy 6- to 8-month-old female pigs.26 27 Cells were released from the tunica media by collagenase digestion. Briefly, immediately after exsanguination, the thoracic aorta was excised and the tunica adventitia removed. The aorta was opened longitudinally and the tunica intima scraped off mechanically. The remaining tunica media was cut into small pieces. Samples of 0.3 g wet wt were first incubated with collagenase (3 mg/mL) followed by elastase (0.5 mg/mL) in serum-free medium at 37°C for 1 hour each. Isolated SMCs were obtained by a second collagenase treatment (5 mg/mL serum-free medium) in a moist atmosphere of 5% CO2 and 95% air at 37°C until the tissue was completely dispersed. The final digest was passed through a filter (mesh size 40x40 µm), and the freed cells were recovered by centrifugation at 200g for 10 minutes. Cells were cultivated (passages 2 through 5) in DMEM containing 10% FCS, L-glutamine (4 mmol/L), penicillin (100 IU/mL), streptomycin (100 µg/mL), sodium pyruvate (1 mmol/L), and nonessential amino acids. The growth medium was changed every second day, and cells were subcultured by trypsinization when they became confluent. Two days before cell fusion experiments, 10-5 mol/L cytosine arabinoside28 (Ara-C; an inhibitor of DNA synthesis) was added to the medium to block proliferation.

To determine and monitor the optimal conditions for blocking proliferation without cytotoxic damage, proliferation assays were first undertaken. Ara-C was added to the cell cultures 1 day after seeding. SMCs were detached by trypsinization, J774 macrophages were gently released by using a cell scraper, and the cells were counted daily using an electronic Coulter counter (Schaerfe System).

For the fusion and incubation experiments (see below) we employed murine macrophages freshly isolated from unstimulated mice by peritoneal lavage29 with 0.15 mol/L NaCl containing 0.5 U heparin/mL. The cells were washed with FCS-free medium and maintained on round coverslips (diameter, 13 mm) for subsequent incubation experiments.

Cell Fusion
The following conditions were determined for successfully achieving fusion between macrophages and SMCs; these were used routinely throughout the subsequent experimental work. Macrophages were allowed to adhere to bacteriological dishes for 1 hour. The dishes were washed 10 times to remove nonadherent cells. The remaining adherent macrophages were then carefully removed using a cell scraper. SMCs were prepared by trypsinization. Macrophages ({approx}107 cells) and SMCs ({approx}3x106 cells) were washed separately. The two cell types were then mixed and centrifuged, and the supernatant was discarded. Cell fusion was induced by adding 1 mL of PEG 1500 and, subsequently, FCS-free culture medium to the cell mixture.25 The fused cell preparation was centrifuged, resuspended in medium containing 10% FCS and 10-5 mol/L Ara-C, and placed in six-well plates with three coverslips per well at a density of 2x105 cells/well. The culture medium was changed the following day and then every second day.

Expression of an Additional Macrophage Protein, the Na-K-ATPase
Experiments on the Na-K-ATPase were conducted to determine whether a macrophage plasma membrane protein other than the scavenger receptor remains expressed after fusion. These experiments exploited the differential sensitivity of the enzyme in murine and porcine cells to ouabain, an inhibitor of the enzyme.

A proliferation assay was first carried out to investigate the function of the porcine and murine ATPase in the presence of 10-5 mol/L ouabain, following the procedure described for analyzing proliferation in cells exposed to Ara-C. The mouse macrophage J774 cell line was used for this assay.

After cell fusion as described in the preceding section, cells were cultured in the presence of 10-5 mol/L ouabain, and the survival of porcine SMCs, murine macrophages, and hybrids was assessed microscopically.

Preparation of Lipoproteins
LDL (d=1.019 to 1.063 g/mL) was isolated from human plasma of individual normolipidemic volunteers by sequential ultracentrifugation in a Beckman L7-65 ultracentrifuge using a 70 TI rotor operated at 59 000 rpm at 4°C for 24 hours.30 The d=1.063 g/mL top fraction was dialyzed at 4°C for at least 48 to 72 hours in 0.15 mol/L NaCl containing 0.3 mmol/L EDTA (pH 7.4).

To label the LDL with the fluorescent probe DiI,31 32 2 mL of lipoprotein-deficient serum and 50 µL of DiI in dimethyl sulfoxide (3 mg/mL) were added per milligram of LDL, with gentle agitation. The resulting mixture was incubated for 15 hours at 37°C. The density of the solution was raised to 1.063 g/mL by adding KBr, and the DiI-labeled LDL was separated from the free fluorochrome by centrifugation at 63 000 rpm for 24 hours at 4°C in a TLA 100.3 rotor using a Beckman TL-100 ultracentrifuge. The DiI-LDL was isolated by tube slicing and dialyzed against saline-EDTA.

LDL and DiI-LDL were acetylated following established procedures.32 33 Briefly, an equal volume of saturated sodium acetate was added to the continuously stirred lipoprotein in saline-EDTA while being kept chilled using an ice-water bath. Acetic anhydride in 2- or 5-µL amounts was added over a period of 1 hour until the total mass of anhydride equaled 1.5 times that of the lipoprotein protein. For small amounts of lipoprotein, four 1.5-µL aliquots of anhydride were used. After continuous stirring for another half hour, the acetylated lipoprotein (acLDL or DiI-acLDL) was dialyzed extensively against saline-EDTA. Acetylation of the lipoproteins was confirmed by agarose gel electrophoresis using a Corning apparatus and Universal Gel/8 according to the manufacturer's instructions. Protein content of lipoprotein solutions was determined according to Lowry et al34 as modified by Peterson.35

Incubation of Unfused and Fused Cells With acLDL
Experimental observations on lipid accumulation, ligand binding and uptake, and scavenger receptor expression were conducted in fused cells from days 2 through 9 after the fusion treatment, with appropriate comparisons with each individual cell type.

Lipid accumulation was examined by incubating each cell type, and fused cells, with acLDL at 37°C for 24 hours, fixing the samples with 4% paraformaldehyde (wt/vol) in phosphate-buffered saline (PBS), staining with 0.12 µg/mL Hoechst 3325836 overnight (to discriminate SMC and macrophage nuclei; see "Results"), followed by Nile Red37 for 10 minutes to detect intracellular lipid.

To investigate the binding and uptake of DiI-acLDL,31 32 each cell type, and fused cells, were incubated with the fluorescently labeled ligand at 4°C to establish binding characteristics, or at 37°C for 0.5 hour at various concentrations (for details, see Figs 4Down and 5Down) to follow uptake by receptor-mediated endocytosis. Control experiments were done in the presence of a 10-fold excess (by weight) of fucoidan,32 a competitive inhibitor of acLDL binding. After DiI-acLDL incubation, the cells were washed, fixed, and stained with Hoechst 33258 overnight for identification of nuclei.



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  		Figure 4. Comparison of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acetylated LDL (DiI-acLDL) binding (a and b) and uptake (c and d) in unfused macrophages (M{Phi}). Cells were incubated with 50 µg/mL DiI-acLDL for 4 hours at 4°C (a and b) or 30 minutes at 37°C (c and d). All analyzed cells were able to bind and internalize the lipoprotein ligand. Bars=25 µm.



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  		Figure 5. Line graphs showing quantitative confocal analysis of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acetylated LDL (DiI-acLDL) binding and uptake in unfused macrophages. The binding curve shows two plateaus consistent with the presence of two binding sites (presumed to correspond to the two forms of scavenger receptor43 ). Marked uptake of the ligand is apparent at concentrations as low as 2 µg/mL DiI-acLDL. Binding and uptake are completely abolished in controls simultaneously treated with fucoidan (a competitive ligand for the acLDL receptor32 ). The discontinuous line in the lower graph represents an extrapolation from the determined values. Data are mean±SEM.

For the direct labeling of scavenger receptors, a specific monoclonal antibody (mAb), 2F8,38 was used. This antibody detects both type I and type II scavenger receptors. Fused and unfused cells were fixed and stained with the Hoechst dye, washed and incubated with mAb 2F8 (20 µg/mL) followed by goat anti-rat fluorescein isothiocyanate (1:50 in PBS; Sigma). One series of preparations was permeabilized with 0.05% Nonidet P-40 in PBS before the antibody treatment; another series was antibody treated without prior permeabilization. Controls, in which the primary antibody was omitted, were in each case run in parallel. All preparations were mounted cell-side-up under coverslips suspended by spacers to avoid cell compression.39

Fluorescence Microscopy
The results of the above experiments were obtained by confocal and conventional fluorescence microscopy using Zeiss and Leitz objectives (numerical aperture, 0.9 and 1.3, respectively) in combination with a Leitz Orthoplan microscope. Quantitative measurements of the amount of fluorescence due to DiI-acLDL or mAb 2F8 binding/uptake were obtained for each set of experiments using a BioRad MRC-500 confocal microscope. The samples were scanned at zoom factor 1.5, and during recording, the confocal aperture was set at a constant position of 54% of its full adjustable range. The mean sum of pixels per cell or per square micrometer of cell area was determined from sets of images using the standard BioRad software (COMOS). The number of cells/image or the size of the cells investigated was determined from corresponding phase-contrast images.

Since the argon laser of the confocal microscope does not excite the Hoechst dye, nuclear composition of the labeled fused cells was analyzed by parallel conventional fluorescence microscopy of the same fields. The identity of the nuclei was recorded on the confocal images using outlined arrows to denote the position of SMC nuclei, and filled arrows to denote macrophage nuclei. For this, the confocal aperture was opened to 77% of its maximum range to increase the depth of the analyzed field.40 41

Owing to methodological limitations inherent in these analytical techniques, the approach should be considered semiquantitative, permitting comparative estimates of the extent of scavenger receptor expression rather than absolute values.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
The use of SMCs and macrophages from two different species (pig and mouse, respectively) for the fusion experiments was a specific feature of the experimental design to enable subsequent characterization of the hybrid cells. Characterization was accomplished by differential staining of nuclei with Hoechst 33258, a DNA stain that fluoresces with greatest intensity in adenine- and thymine-rich regions.36 Satellite DNA in the centromeric regions of mouse chromosomes is rich in poly(dAT); thus, mouse interphase nuclei revealed intense punctate staining with the fluorochrome (Fig 1aDown). Porcine nuclei, whose DNA does not contain adenine- and thymine-rich regions, showed uniform, less intense fluorescence and were markedly larger in size (Fig 1aDown). The number and type of parental nuclei in any given hybrid could thus be readily identified (Fig 1bDown and 1cDown), and this procedure could therefore be applied in all subsequent experiments on scavenger receptor expression in hybrid cells.



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  		Figure 1. Characterization of hybrid cells using Hoechst 33258 dye to identify murine macrophage and porcine smooth muscle nuclei. Smooth muscle cell nuclei (s) are large and show uniform low-intensity fluorescence, whereas the macrophage nuclei (m) are small and contain bright punctate fluorescence (a). These features make it possible to identify the parental nuclei of fused cells. b and c show corresponding views of the same cell, at the same magnification, after Hoechst staining and phase-contrast microscopy, respectively. Bars=25 µm.

The PEG treatment resulted in a mixed population of hybrid cells (homokaryons and heterokaryons) as well as cells that remained unfused. Under the conditions described, the proportion of fused cell products to unfused cells was in the order of 2:3. In a typical experiment, 19% of the fused cell population comprised homokaryons consisting of fused SMCs, 13% comprised homokaryons consisting of two or more fused macrophages, 10% comprised heterokaryons consisting of one macrophage plus one SMC, and 4% comprised heterokaryons consisting of two SMCs with one macrophage. Approximately 50% of fused cells could not be characterized with certainty, and these were not included in the experimental observations.

To maintain stability of the fused cells for the duration of the experiments on scavenger receptor expression, proliferation was blocked using Ara-C.28 This excludes the possibility that any changes in gene expression could result from the loss of cytoplasmic or nuclear components through cell division. It was determined that a concentration of 10-5 mol/L Ara-C was sufficient to block division in SMC cultures (Fig 2Down) and the mouse macrophage cell line J774 (data not shown). Cell cycle analysis of SMCs treated with Ara-C demonstrated that more than 70% of the cells were trapped in the G1 phase of the cell cycle (data not shown). Mouse peritoneal macrophages usually do not proliferate, but can be induced to do so after incubation with chemically modified LDL42 ; thus, it was important to establish conditions that eliminated the possibility of proliferation under the conditions of the experiments planned.



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  		Figure 2. Line graph shows inhibition of proliferation of smooth muscle cells by cytosine arabinoside (Ara-C). The cells were maintained in 10-5 mol/L Ara-C added 1 day after seeding. At this concentration, proliferation is blocked, both in smooth muscle cells and J774 macrophages (not shown). From these data, the same concentration of Ara-C was used to prevent division of fused cells. Data represent mean±SEM of three experiments each counted in triplicate. Ara.-C. indicates Ara-C.

The first step in applying the experimentally fused cells to investigate foam cell genesis was to examine the capacity of hybrids to accumulate lipid upon exposure to acLDL. As illustrated in Fig 3aDown through 3c, Nile Red staining disclosed that macrophage homokaryons avidly accumulated lipid droplets upon exposure to acLDL, but heterokaryons failed to do so and did not adopt a foam cell–like morphology (Fig 3dDown through 3f).



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  		Figure 3. Macrophage homokaryons (a through c), but not macrophage–smooth muscle cell heterokaryons (d through f), are converted to foam cells by incubation with acetylated LDL at 37°C for 24 hours, as revealed by Nile Red staining for lipid (c and f). The examples illustrated come from samples 6 days after fusion. Bars=25 µm.

The experiments then proceeded to examine the binding and uptake of acLDL tagged with the fluorescent probe DiI. Unfused SMCs did not bind or take up DiI-acLDL under any of the experimental conditions investigated (not shown). At 4°C, unfused macrophages all bound the ligand (Fig 4aUp and 4bUp); the binding curve, determined by quantitative analysis of confocal digital images, showed two plateaus (Fig 5Up), consistent with the presence of two binding sites (presumed to correspond to the two forms of scavenger receptor produced by alternative splicing43 ). Incubation of the macrophages at 37°C for 30 minutes led to marked uptake of the ligand (Fig 4cUp and 4dUp) that was apparent at concentrations as low as 2 µg/mL DiI-acLDL (Fig 5Up).

To establish the binding and uptake properties of DiI-acLDL in hybrid cells, a concentration of 50 µg/mL DiI-acLDL (see Fig 5Up) was used to ensure that the sensitivity for the detection of receptors was optimal, even if their expressions were low.

Macrophage-macrophage homokaryons, and single macrophages that remained unfused after the PEG treatment, revealed intense labeling upon incubation in DiI-acLDL. By contrast, however, macrophage-SMC heterokaryons did not bind or take up the ligand. This difference is strikingly demonstrated in Fig 6aDown through 6c, which illustrates an SMC-macrophage heterokaryon and a macrophage-macrophage homokaryon after incubation in DiI-acLDL, side by side in the same culture dish.



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  		Figure 6. Analysis of 1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acetylated LDL (DiI-acLDL) uptake in a fused cell population. Hoechst staining (a) and the corresponding phase-contrast image (b) reveal two hybrid cells. One (upper right field) is identified as a macrophage homokaryon, and the second (mid-to-lower part of the field) is a macrophage–smooth muscle cell heterokaryon. Inspection of image (c) demonstrates that marked uptake of DiI-acLDL occurs in the macrophage homokaryon, whereas there is no uptake of the ligand in the heterokaryon. The example illustrated comes from a sample examined 2 days after fusion. Bar=25 µm.

Fig 7Down presents the results from a series of experiments using mAb 2F8 (an anti–scavenger receptor type I and II antibody) on unfused macrophages and SMCs. Macrophages treated with this antibody followed by fluorescein-labeled secondary antibody revealed intense signal at the cell surface (Fig 8Down), and when the cells were permeabilized before primary antibody exposure, substantial signal was also apparent in intracellular compartments (eg, endosomes) (Figs 7aDown, 7bDown, and 8Down). The monoclonal antibody did not label the surfaces of SMCs (Fig 8Down), and no significant labeling occurred when these cells were prepermeabilized (Figs 7cDown, 7dDown, and 8Down).



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  		Figure 7. Investigation of scavenger receptor expression in unfused, permeabilized single cells as detected by confocal immunofluorescence microscopy with monoclonal antibody (mAb) 2F8. Macrophages (M{Phi}) show strong positive signal (b), whereas smooth muscle cells (SMC) are unlabeled (d). Detail of the whole cells is shown by the corresponding phase-contrast images (a and c). Bars=25 µm.



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  		Figure 8. Bar graphs showing semiquantitative confocal measurements of the immunofluorescently monoclonal antibody (mAb) 2F8–labeled samples shown in Fig 7Up for both nonpermeabilized and permeabilized macrophages and smooth muscle cells. Negligible labeling is seen in controls (secondary antibody only). Smooth muscle cells treated with mAb 2F8 show low levels of fluorescence comparable to controls. The positive signal detected in macrophages is markedly higher in permeabilized cells than in nonpermeabilized cells. Data are mean±SEM.

Fig 9Down shows the results obtained when fused cells (fixed and permeabilized) were exposed to mAb 2F8. Macrophage-macrophage homokaryons and unfused macrophages examined from 2 days postfusion onward revealed high levels of scavenger receptors detectable with mAb 2F8 (Fig 9aDown and 9bDown). In macrophage-SMC heterokaryons, however, no scavenger receptors were detectable, either at the cell surface or intracellularly (Fig 9aDown and 9bDown), the fluorescence intensity being comparable to that of unfused SMCs and SMC-SMC homokaryons (Fig 10Down). Similar results were obtained throughout the postfusion observation period (Fig 9cDown and 9dDown), though a slight decline in the amount of labeling on the macrophages was observed at 6 days postfusion compared with 2 days postfusion (Fig 10Down).



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  		Figure 9. Analysis of scavenger receptor expression as detected by monoclonal antibody (mAb) 2F8 labeling of permeabilized cells at 2 (a and b) and 6 (c and d) days after fusion. Phase-contrast images (a and c) show macrophages and heterokaryons containing nuclei of macrophage (filled arrows) and smooth muscle cell (outlined arrows) origin. In the corresponding fluorescence images, macrophages are strongly immunopositive to mAb 2F8 while heterokaryons are immunonegative. Note that in the phase-contrast image in (c), two smooth muscle cell nuclei and one macrophage nucleus can be seen in the heterokaryon. Bars=25 µm.



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  		Figure 10. Bar graphs showing semiquantitative confocal analysis of monoclonal antibody (mAb) 2F8 immunofluorescently labeled samples from the same experiment as the image shown in Fig 9Up. The results reveal that fluorescence of heterokaryons (SMC-M{Phi}) is comparable to that of unfused smooth muscle cells (SMC) and significantly lower than that of macrophages (M{Phi}). Data are mean±SEM.

An additional experiment was designed to examine the expression of a further mouse-derived protein in SMC-macrophage hybrids. Microscopic observations and cell counting revealed that a 10-5 mol/L ouabain concentration in the culture medium killed all porcine SMCs as a result of the inhibitory effect of this toxin on the Na-K-ATPase of the plasma membrane44 (Fig 11aDown and 11bDown). In contrast, mouse peritoneal macrophages survived at the same ouabain concentration, and mouse macrophages of the J774 cell line did not show any difference in their proliferation activity in comparison with untreated control cells (Fig 11cDown). Thus, the mouse macrophage enzyme remains functional under conditions that are toxic to its counterpart in porcine SMCs.



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  		Figure 11. Graphs showing species specific differences in response to ouabain treatment. a, Cell count of porcine smooth muscle cells exposed to 10-5 mol/L ouabain for 24 hours; b, corresponding untreated control. The cells do not survive the ouabain treatment, a finding confirmed by microscopic observation. Signal representing a cell diameter smaller than 11 µm is due to cell debris. In contrast, murine cells do survive treatment with ouabain at the same concentration as demonstrated by the unaffected proliferation of the mouse macrophage cell line J774 (c).

In a fused cell preparation treated with 10-5 mol/L ouabain 5 days after fusion, unfused peritoneal macrophages and macrophage homokaryons survived, but unfused SMCs and SMC homokaryons did not. Heterokaryons, in which SMC nuclei were visible, were able to survive the ouabain treatment (Fig 12aDown). As before, scavenger receptors were not detectable with mAb 2F8 in these hybrid cells (Fig 12bDown), and quantitative fluorescence analysis revealed no significant difference between heterokaryons and unfused macrophages in comparison with their counterparts in a cell population not treated with the agent (Fig 13Down; compare with Fig 10Up). These results demonstrate that repression of murine macrophage scavenger receptors occurred alongside continued expression of the murine macrophage Na-K-ATPase since the turnover rates of this protein and its mRNA are reported to be in the range of 10 to 18 hours45 46 and 3 to 12 hours,47 48 respectively.



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  		Figure 12. Survival of a hybrid cell (outlined arrow) in the presence of ouabain (a). Arrow indicates smooth muscle cell nucleus. The ouabain treatment was given 5 days after fusion for 24 hours. Analysis of cells fixed, permeabilized, and labeled 6 days after fusion with monoclonal antibody 2F8 demonstrated that no scavenger receptor expression occurred in such smooth muscle cell nucleus–containing hybrid cells, while unfused macrophages (cell to upper right) remained immunopositive (b). Bar=25 µm.



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  		Figure 13. Bar graph showing semiquantitative analysis of ouabain-treated and monoclonal antibody 2F8–labeled, permeabilized cells from the same sample as that shown in Fig 12Up. The analysis reveals a similar level of fluorescence to that of corresponding cells that were not treated with ouabain (compare with Fig 10Up). Data represent mean±SEM.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The use of interspecies heterokaryons, pioneered by Harris15 and further developed to a well-defined system by Blau and associates,16 23 24 provides an elegant approach by which two differentiated cell types can be combined so that the influence of one on the function of the other can be studied. In the present investigation, we have developed and applied this approach to examine the in vitro expression of scavenger receptors in SMCs, macrophages, and hybrids made from the two cell types, with the aim of furthering understanding of the possible mechanisms underlying genesis of foam cells in the atherosclerotic plaque.

Although foam cells derived from SMCs have been identified in the atherosclerotic plaque,1 2 the difficulty in inducing a comparable foamy appearance in SMCs exposed to ligands for the scavenger receptor in vitro5 has meant that the underlying mechanism for SMC/foam cell genesis has remained under discussion. From the information available to date, the possibility exists that the coexistence of macrophages and SMCs in the atherosclerotic plaque might lead to smooth muscle foam cells by mechanisms that involve direct or indirect interaction between the two cell types or components of them.13 Recent reports that a protein homologous to connexin43, the principal gap-junctional protein of SMCs,49 50 is expressed in the macrophage,51 and that macrophage/foam cells in the atherosclerotic plaque in situ express connexin43 messenger RNA,52 have opened the unexpected and intriguing possibility that direct interaction between the two cell types might occur via gap junctions, the organelles that mediate direct communication between the cytoplasmic compartments of neighboring cells.

One hypothesis that would explain genesis of smooth muscle–derived foam cells is that scavenger receptor expression is induced in SMCs as a result of specific conditions prevailing in the plaque. Expression of scavenger receptors in SMCs is reported to be induced in vitro after incubation with phorbol esters6 7 or growth factors such as platelet-derived growth factor8 or monocyte-colony stimulating factor.9 However, labeling of atherosclerotic lesions with monoclonal antibodies directed against scavenger receptors has failed to demonstrate the expression of these receptors on SMCs.10 11 Furthermore, another recent report suggests that scavenger receptor mRNA is not expressed in SMCs of atherosclerotic lesions.12

Several lines of work indicate that expression of proteins that are not normally expressed can be induced after in vitro formation of cell hybrids.16 21 22 23 24 However, our demonstration that heterokaryons failed to accumulate lipid upon exposure to acLDL, while macrophage homokaryons did so avidly, suggested that, rather than inducing expression of scavenger receptors, direct interaction of the components of SMCs and macrophages achieved by fusion of the two cell types might lead to receptor repression. We therefore proceeded to establish whether this was indeed the case. Accordingly, scavenger receptor activity was investigated more directly by examining the binding and uptake of one of its principal ligands, acLDL, which, for this purpose, was tagged with the fluorescent probe DiI. These experiments confirmed the absence of ligand binding and uptake in the heterokaryons, and demonstrated that these activities remained functional in macrophage-macrophage homokaryons. In contrast to the scavenger receptor, we found, in a separate series of experiments using DiI-labeled LDL as a probe, that the heterokaryons continued to express the classic LDL receptor (M.R. and H.R., unpublished data, 1994).

Our results thus showed that in heterokaryons either the expression of scavenger receptor protein is repressed after fusion, or the scavenger receptors remain present but are in some way inactivated or nonfunctional. To distinguish between these possibilities we used mAb 2F8, an antibody that recognizes both forms of the murine scavenger receptor,38 and that would therefore be expected to bind to it if present. By using this antibody on permeabilized cells, we were able to demonstrate that, while macrophage-macrophage homokaryons continued to express scavenger receptors, there was a complete absence of the receptors in heterokaryons, both at the surface and within the cytoplasm. From the latter it was clear that the absence of scavenger receptors at the cell surface was not due to failure of translocation of the receptor from a cytoplasmic compartment.

The receptor repression observed does not appear to be due entirely to a general repressive effect on all macrophage proteins. Rather, it shows a degree of specificity for the scavenger receptor. This was shown by the experiments in which fused and unfused cells were maintained in the presence of ouabain. The inhibitory action of ouabain on the Na-K-ATPase of porcine and human cells is markedly more potent than that on rodent cells,44 and at a concentration of 10-5 mol/L ouabain, we found that murine macrophages survived, whereas porcine SMCs, and homokaryons formed from SMCs, did not. When a porcine SMC was fused with a mouse macrophage, we found that the resultant hybrid could survive in the presence of 10-5 mol/L ouabain added 5 days after fusion. As the turnover of Na-K-ATPase protein is reported to be in the order of 10 to 18 hours,45 46 and of its mRNA in the range of 3 to 12 hours,47 48 our observation indicates that the macrophage Na-K-ATPase continues to be expressed in the hybrids.

Taken together, then, these results demonstrate that scavenger receptor expression is repressed in macrophages when SMCs are fused with them. This result does not appear to be caused by some incidental aspect of the experimental conditions used for cell culture and fusion. Although Ara-C has been reported to induce increased synthesis of proteins in some cell types,53 54 it had no effect on scavenger receptor expression under the conditions of our experiments, as shown by the continuing expression of the receptors in the macrophage homokaryons. Moreover, the ability of all macrophages before, and of those unfused single macrophages that remain after the fusion step, to express the receptors excludes the further possibility that the heterokaryons examined had been derived from non–receptor-active macrophages. Since loss of genetic information due to cell division was avoided in the fused cells by inhibition of DNA synthesis and proliferation using Ara-C, the evidence suggests that SMCs may contain a factor or factors that suppress expression of the macrophage scavenger receptor.

In other cell fusion systems, it has been shown that tissue-specific genes typical of muscle are readily induced in nonmuscle cell types that normally never express these genes, following fusion of such cells with muscle cells. Conversely, genes typical of the nonmuscle cell phenotype are often repressed.16 Our results conform to this pattern, and are consistent with the assumption that expression of scavenger receptors is not representative of the muscle phenotype. Formation of smooth muscle foam cells in the atherosclerotic plaque would, therefore, seem unlikely to be explicable by induction of scavenger receptor expression in the SMC via direct macrophage interaction.

Our observations open new insights into the specific physiological regulation of the scavenger receptor, which is known, in contrast to the classic LDL receptor, not to be downregulated after exposure to its ligand.4 Some extracellular factors, like monocyte-colony stimulating factor,55 markedly modify the amount of receptors expressed, but data on intracellular regulating factors, especially those that act on the molecular level, are sparse.56 Recent analyses of promoter sequences of the scavenger receptor gene failed to demonstrate binding sites for SP1, AP1, or other common transcription factors, indicating that novel factors are required for its expression.57

Though it is not yet determined whether the inhibitory effect observed is exerted at the level of transcription or translation, the putative inhibitor of scavenger receptor expression would appear to be a diffusible, cytoplasmic molecule, probably showing evolutionary conservation, since the heterokaryons were derived from two different species and maintained separate macrophage and SMC nuclei over the time course of the experiments. Identification of the molecule(s) involved in the observed repression of scavenger receptors in the heterokaryons would enable clarification of the molecular mechanisms governing scavenger receptor expression, and might ultimately open opportunities for inhibiting lipid accumulation in the atherosclerotic plaque.


*    Acknowledgments
 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 310 and SFB 223) and Nato Collaborative Research Grant No. CRG 910122. We thank Dr I. Fraser for the gift of mAb 2F8, Dr D. Troyer for outstanding help with computing, Dr K. Geering for helpful information on the Na-K-ATPase, and R. Fischer, L. Greune, M. Opalka, K. Poorthuis, and K. Schlattmann for their technical expertise. This work is part of a doctorate thesis by M. Rommeswinkel.

Received November 14, 1994; accepted February 10, 1995.


*    References
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