© 1995 American Heart Association, Inc.
Articles |
Repression of the Macrophage Scavenger Receptor in Macrophage–Smooth Muscle Cell Heterokaryons
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.
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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
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Introduction |
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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.
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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 (
107 cells) and
SMCs (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 4
and 5) 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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). 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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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.
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Results |
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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 1a
). Porcine nuclei, whose DNA does not contain adenine-
and thymine-rich regions, showed uniform, less intense fluorescence and
were markedly larger in size (Fig 1a). The number and type of parental
nuclei in any given hybrid could thus be readily identified (Fig 1b and 1c), and this procedure could therefore be applied in all subsequent
experiments on scavenger receptor expression in hybrid cells.
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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 2) 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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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 3a 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 3d through 3f).
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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 4a
and 4b); the binding curve, determined by
quantitative analysis of confocal digital images, showed two
plateaus (Fig 5), 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 4c and 4d) that was apparent at concentrations as low as 2
µg/mL DiI-acLDL (Fig 5).
To establish the binding and uptake properties of DiI-acLDL in hybrid
cells, a concentration of 50 µg/mL DiI-acLDL (see Fig 5) 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 6a 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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Fig 7 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 8), and
when the cells were permeabilized before primary antibody exposure,
substantial signal was also apparent in intracellular compartments (eg,
endosomes) (Figs 7a, 7b, and 8). The monoclonal antibody did not label
the surfaces of SMCs (Fig 8), and no significant labeling occurred when
these cells were prepermeabilized (Figs 7c, 7d, and 8).
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Fig 9 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 9a and 9b). In macrophage-SMC heterokaryons, however, no
scavenger receptors were detectable, either at the cell surface or
intracellularly (Fig 9a and 9b), the fluorescence intensity being
comparable to that of unfused SMCs and SMC-SMC homokaryons (Fig 10). Similar results were obtained throughout the
postfusion observation period (Fig 9c and 9d), though a slight decline
in the amount of labeling on the macrophages was observed at 6 days
postfusion compared with 2 days postfusion (Fig 10).
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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 11a and 11b).
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 11c). Thus, the mouse macrophage enzyme
remains functional under conditions that are toxic to its counterpart
in porcine SMCs.
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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 12a). As before,
scavenger receptors were not detectable with mAb 2F8 in these hybrid
cells (Fig 12b), 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 13; compare with Fig 10). 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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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.
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Acknowledgments |
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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.
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