 |
INTRODUCTION |
Macrophages express several scavenger receptors that bind modified
lipoproteins (1-5). The cloning and identification of individual
scavenger receptors have permitted a detailed assessment of their
functions. The type I and type II class A scavenger receptors (SR-A)1 were the first to be
cloned (6, 7). These receptors are trimeric integral membrane
glycoproteins generated by alternative splicing of a single gene
product (1, 6, 7). They both bind a wide variety of molecules,
including certain chemically modified lipoproteins such as oxidized low
density lipoproteins (LDL) and acetylated LDL (AcLDL), certain
polysaccharides such as fucoidin and dextran sulfate, and
polyribonucleotides such as poly I and poly G (1). In addition, the
SR-A binds a limited number of native proteins. We recently showed that
the
-secretase cleavage products of the three main isoforms of the
amyloid precursor protein (APP695, APP751, and APP770) are ligands for
the SR-A (8). The SR-A also binds the amyloid-
(A
) peptide (9), another enzymatic cleavage product of APP and a major component of
senile plaques in Alzheimer's disease (AD).
Macrophage scavenger receptors have been postulated to contribute to
the internalization of modified lipoproteins, intracellular cholesterol
ester accumulation, foam cell formation, and atherogenesis (10).
Numerous studies have suggested that the SR-A is critically involved in
the deposition of cholesterol in arterial wall macrophages during the
development of atherosclerotic lesions. Elimination of SR-A expression
in mice significantly reduces the uptake of modified LDL by peritoneal
macrophages from these animals and inhibits atherogenesis (11-13).
Although reduced atherosclerosis in
SR-A
/
mice is widely
assumed to result from reduced uptake of modified lipoproteins and
reduced accumulation of cholesterol esters in macrophages of the
arterial wall, this mechanism has not been proved experimentally. SR-A
may, in fact, contribute to atherogenesis in ways distinct from the
function of SR-A in the uptake of modified lipoproteins (14). The SR-A
also may play an important role in immune response and in cell adhesion
(11, 15-20). In fact, it has been suggested that the SR-A may interact
with components of the subendothelial space, thereby contributing to
the adhesion and retention of macrophages in the artery wall (11,
15-19).
To examine this possibility further, we tested the hypothesis that the
SR-A contributes to the adhesion of cells to the extracellular matrix.
We found that biglycan and decorin, components of the extracellular
matrix, as well as aggrecan, a proteoglycan that is similar to versican
(a prominent matrix component) are ligands of the SR-A and that the
SR-A contributes significantly to the divalent
cation-dependent and -independent adhesion of macrophages to extracellular matrix derived from both smooth muscle cells and
endothelial cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Bovine aggrecan, bovine biglycan, bovine decorin,
hyaluronan, and fucoidin were obtained from Sigma. Collagen IV
was obtained from ICN Biomedicals (Costa Mesa, CA). PD-10 columns and
Na125I were purchased from Amersham Biosciences.
Dulbecco's modified Eagle's medium, F12 medium, RPMI 1640 medium,
trypsin-EDTA solution, and penicillin-streptomycin solution were
purchased from Invitrogen. Fetal bovine serum was obtained from Hyclone
(Logan, UT). Tissue culture dishes and other plastic ware were obtained
from Falcon (Franklin Lakes, NJ), Corning (Acton, MA), and Nalge Nunc
International (Rochester, NY).
Lipoproteins and Proteoglycans--
Human LDL (d = 1.02-1.05 g/ml) were isolated from the plasma of normal fasted
donors by sequential ultracentrifugation at 4 °C (21). The LDL were
radiolabeled by the iodine monochloride method (22) to a specific
activity of 150-350 cpm/ng of protein. AcLDL was prepared by treating
LDL with acetic anhydride (23). Lipoproteins were dialyzed against 0.15 M NaCl and 0.01% EDTA, pH 7.2, before use. Aggrecan,
biglycan, and decorin were labeled with 125I using
iodobeads (Pierce) as recommended by the manufacturer. The specific
activities were 200-400 cpm/ng.
Cell Release Assay--
Control Chinese hamster ovary (CHO)
cells were grown in 95% air/5% CO2 at 37 °C in
Dulbecco's modified Eagle's medium/F12 medium containing 10% fetal
bovine serum. CHO cells stably transfected to express the type I SR-A
were grown in the same medium containing 400 µg/ml of G418. The
preparation and properties of CHO cells stably expressing the SR-A have
been described previously (24). The cells were grown to confluence
either in untreated 35- or 60-mm tissue culture plates or in plates
coated with the extracellular matrix from human smooth muscle cells
(HSMCs). The extracellular matrix was prepared as described below. The
plates were washed three times with phosphate buffered saline (PBS) and
incubated with 1 ml of trypsin-EDTA solution at room temperature on an
orbital platform. Trypsin activity was stopped at the times indicated with 1 ml of serum-containing medium. An aliquot of the medium was
taken, and the number of cells released was determined with a cell
counter (Coulter).
Cell Adhesion Assays--
HSMCs and bovine aortic endothelial
cells were grown to confluence in 96-well plates, and the extracellular
matrix was prepared as described (25). Briefly, the cells were washed
three times with PBS and incubated with 0.5% Triton X-100 in
H2O for 10 min at room temperature. The wells were then
washed three times with PBS and dried. Thioglycollate-elicited
peritoneal macrophages from SR-A+/+ or
SR-A
/
mice (11) were
obtained by peritoneal lavage, concentrated by centrifugation,
resuspended in RPMI 1640 medium, and counted with a cell counter
(Coulter). Macrophages (3 × 104) were seeded in wells
coated with extracellular matrix, incubated for 1.5 h at 37 °C
under the conditions indicated, and washed three times with PBS.
Adherent cells were fixed with 3% paraformaldehyde in PBS and their
nuclei stained with Sitox green (Molecular Probes, Eugene, OR) in PBS.
Fluorescence images were captured with a Nikon Eclipse TE 300 inverted
microscope equipped with a digital camera (Diagnostic Instruments SPOT
RT, Sterling Heights, MI). Image processing was used to isolate the
nuclei from background fluorescence produced by the extracellular
matrix, and then the nuclei in each image (a standard field captured
using a ×10 objective) were counted. Five or six images were captured
for each condition from triplicate wells of a 96-well plate. The images
were processed and quantitated with FoveaPro Version 1.0 (Reindeer
Graphics, Asheville, NC), running with PhotoShop 6.0 (Adobe, San Jose,
CA) on a Macintosh G4 computer.
Cell Association and Degradation of Proteoglycans and
Lipoproteins--
Control CHO cells and CHO cells expressing the SR-A
were grown in 12-well plates as indicated above, washed three times
with serum-free medium, and incubated for 5 h at 37 °C in
serum-free medium containing 125I-aggrecan, -biglycan, or
-decorin (1 µg/ml) alone or with the indicated competitors.
Experiments were also performed with 125I-AcLDL (2 µg/ml)
alone or with the indicated concentrations of competitors.
Cell-associated ligands (i.e. bound and internalized protein) and degraded ligands (trichloroacetic acid-soluble protein degradation products in the medium) were quantitated as described (26,
27).
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RESULTS |
SR-A Expression Contributes to Trypsin Resistance--
We observed
that SR-A-expressing cells were more resistant to trypsin release than
control cells. To quantitate this effect, cells were grown to
confluence in 35- or 60-mm plates, washed, treated with trypsin/EDTA
solution for different periods of time, and counted. CHO cells
expressing the SR-A were more resistant to trypsin release than control
CHO cells, which do not express the SR-A. Starting at 5 min of
incubation with trypsin, a striking difference in trypsin release of
the SR-A-expressing and nonexpressing cells was evident (Fig.
1). All control cells were released at 6 min of incubation with trypsin/EDTA, whereas it took 11-12 min for the
release of all of the SR-A expressing cells. This difference was even
greater when the experiment was performed with cells grown on plates
coated with extracellular matrix from HSMCs (Fig. 1B).
Expression of the SR-A therefore contributes to adhesion of the cells
either to the cell culture dishes or to the matrix elaborated by the
cells.

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Fig. 1.
Adherent CHO cells expressing SR-A are more
resistant to release by trypsin than control CHO cells. Control
CHO cells and CHO cells expressing the SR-A were grown to confluence on
tissue culture plates (A) or on plates coated with
extracellular matrix elaborated by HSMC (B). The cells were
washed with PBS and treated with trypsin-EDTA for the indicated times
at room temperature. Trypsin activity was stopped by the addition of
serum-containing medium, and the cells that were released into the
medium were quantitated. The data are the average of two independent
experiments in a series of four with similar results.
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|
SR-A Mediates Macrophage Adhesion to the Extracellular
Matrix--
We examined thioglycollate-elicited macrophages from
SR-A+/+ and
SR-A
/
mice for their
ability to adhere to the extracellular matrix produced by HSMCs. Under
all conditions tested, macrophages from SR-A
/
mice were less
effective in adhering to the coated wells than macrophages from
SR-A+/+ mice (Fig.
2). In the absence of serum, the adhesion
of SR-A
/
macrophages
to the HSMC extracellular matrix was 10% of the adhesion of
SR-A+/+ macrophages in the presence of EDTA (to
chelate divalent cations) and 16% in the absence of EDTA. In the
presence of serum, the adhesion of
SR-A
/
macrophages to
the HSMC extracellular matrix was ~35% of the adhesion of
SR-A+/+ macrophages in the absence of EDTA and
~5% in the presence of EDTA (Fig. 2). Similar results were obtained
when experiments were performed with an extracellular matrix from
bovine aortic endothelial cells (data not shown). The SR-A antagonist
fucoidin inhibited 70-90% of the adhesion of the
SR-A+/+ macrophages (Fig. 2). Taken together
these results demonstrate that the SR-A contributes significantly to
the divalent cation-dependent and -independent adhesion of
thioglycollate-elicited macrophages to extracellular matrix, both in
the presence and absence of serum.

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Fig. 2.
The SR-A mediates macrophage adhesion to the
extracellular matrix. Macrophages from
SR-A / (white
bars) and SR-A+/+ (black bars)
mice were seeded in 96-well plates coated with extracellular matrix
from HSMC, incubated for 1.5 h at 37 °C with or without serum
and EDTA as indicated, washed with PBS, and fixed with 3%
paraformaldehyde. Nuclei of adherent cells were counted after Sitox
green staining. Adhesion of SR-A+/+ macrophages
was also quantitated in the presence of 100 µg/ml fucoidin
(hatched bars). Results are the mean ± S.D. of five
independent fields from triplicate wells of a representative
experiment, in a series of three with similar results.
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|
Aggrecan, Biglycan, and Decorin Are Ligands for the SR-A--
We
next identified two components of extracellular matrix that are SR-A
ligands. Soluble biglycan and decorin were radioactively labeled and
tested for their ability to bind to the SR-A. Compared with control
cells, CHO cells stably expressing the SR-A showed a 5- to 6-fold
greater cell association (which represents bound and internalized
ligand) of 125I-labeled decorin and biglycan (Fig.
3). We also found that aggrecan, a
proteoglycan found in cartilage that is similar to the extracellular matrix component versican, is a ligand for the SR-A. SR-A-expressing cells showed an ~8-fold greater cell association of
125I-labeled aggrecan than control cells (Fig. 3).

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Fig. 3.
Aggrecan, biglycan, and decorin bind to the
SR-A. Control CHO cells (white bars) and CHO cells
expressing the SR-A (black bars) were incubated for 5 h
at 37 °C with 125I-aggrecan, 125I-biglycan,
or 125I-decorin (1 µg/ml). The cells were washed and the
cell association of labeled proteoglycans was determined. The results
are expressed as the -fold increase in cell association after
subtraction of the nonspecific binding obtained in the presence of 10 µg/ml fucoidin. Results are the mean ± S.D. of three
independent experiments performed in duplicate.
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|
To examine further the specificity of interaction of the proteoglycans
with the SR-A, the cell association and degradation of aggrecan,
biglycan, and decorin were tested in competition experiments with
unlabeled proteoglycan and fucoidin (Fig.
4). A 10-fold molar excess of aggrecan or
fucoidin competed for 80-90% of the cell association and 80-85% of
the degradation of 125I-aggrecan. Biglycan or fucoidin
(10-fold molar excess) also competed for 80-90% of the cell
association and ~80% of the degradation of
125I-biglycan, whereas a 10-fold excess of decorin or
fucoidin competed for only 50-60% of the cell association and ~50%
of the degradation of 125I-decorin. Higher concentrations
(as high as a 100-fold excess) of competitor did not result in a
substantially higher competition.

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Fig. 4.
Fucoidin and nonlabeled proteoglycans compete
for the cell association and degradation of
125I-aggrecan,
125I-biglycan, or
125I-decorin by CHO cells expressing the
SR-A. CHO cells expressing the SR-A were incubated for 5 h at
37 °C with 125I-aggrecan (A, B),
125I-biglycan (C, D), or
125I-decorin (E, F) (1 µg/ml) alone
(white bars) or in the presence of unlabeled competitor (10 µg/ml) (black bars). The cells were washed and the cell
association or degradation of labeled proteoglycans was determined.
Results are the mean ± S.D. of three independent experiments
performed in duplicate.
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Aggrecan, biglycan, and decorin also competed for the cell association
and degradation of 125I-AcLDL by CHO cells expressing the
SR-A (Fig. 5). Aggrecan, a high molecular
weight proteoglycan, was the most effective competitor for the
SR-A-mediated cell association and degradation of AcLDL, whereas
biglycan was as effective as AcLDL and fucoidin (Fig. 5). Decorin was a
less potent competitor.

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Fig. 5.
Aggrecan, biglycan, and decorin compete for
the cell association and degradation of
125I-AcLDL by CHO cells expressing the
SR-A. The cells were incubated for 5 h at 37 °C with
125I-AcLDL (2 µg/ml) alone or with increasing
concentrations of competitors. The cell association (left
panel) and degradation (right panel) of
125I-AcLDL were determined. In the absence of competitors,
the cell association and degradation of 125I-AcLDL were
192 ± 9 and 216 ± 13 ng/mg cell protein, respectively. Each
data point represents the mean ± S.D. of three independent
experiments performed in duplicate. The experimental error
bars are within the data points.
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We also examined the ability of hyaluronan and collagen IV, other
components of extracellular matrix, to compete for the SR-A-mediated cell association and degradation of AcLDL. We found that hyaluronan and
collagen IV did not compete with AcLDL even at concentrations of 100 µg/ml (data not shown).
 |
DISCUSSION |
This study shows that the SR-A contributes to the divalent
cation-dependent and -independent adhesion of macrophages
to extracellular matrix from HSMC and endothelial cells in the presence
and absence of serum. This adhesion was inhibited by the SR-A
antagonist fucoidin. Expression of SR-A by CHO cells delayed the
release of cells from tissue culture dishes and from HSMC extracellular
matrix by trypsin. Furthermore, we identified soluble biglycan and
decorin, proteoglycans of the extracellular matrix, as ligands for the
SR-A. In competition experiments, biglycan competed as effectively as
AcLDL and fucoidin for the SR-A-mediated cell association and
degradation of 125I-AcLDL. Decorin was less effective,
suggesting that it has a lower affinity for the SR-A than biglycan or
AcLDL. Unlabeled decorin or fucoidin competed only partially for
binding of 125I-decorin to CHO cells expressing the SR-A.
The binding of versican, another prominent component of the
extracellular matrix, was not examined directly. However, we determined
that aggrecan, a predominant proteoglycan in cartilage that is
analogous to versican (28, 29), is a ligand for the SR-A and a better
competitor for the cell association and degradation of AcLDL than
either biglycan or decorin. In contrast to biglycan and decorin, the
core proteins of both aggrecan and versican are extensively substituted
with chondroitin sulfate (28).
Macrophages exhibit both Ca2+-dependent and
-independent adhesion. Unlike other cell types, macrophages attach to
tissue culture plastic in the absence of divalent cations. In the
presence of EDTA, which chelates Ca2+ and Mg2+,
macrophages lose their spread morphology but remain adherent. The first
indication that the SR-A contributes to adhesion was the observation
that a monoclonal antibody to the SR-A (2F8) totally inhibits the
divalent cation-independent adhesion of murine macrophages to tissue
culture plastic in the presence but not in the absence of serum,
suggesting that a component of serum is necessary for this adhesion
(15, 16). Studies using
SR-A
/
and
SR-A+/+ macrophages showed that the SR-A
mediates 50-60% of the adhesion of macrophages to tissue culture
plastic in the presence of serum (11, 19). In the presence of EDTA to
chelate divalent cations, more than 85% of the adhesion is SR-A
dependent (11, 19).
The contribution of the SR-A to cell adhesion is dependent on the cell
type and the cellular activation state. In resident peritoneal
macrophages (not elicited) or resting Kupffer cells, cell adhesion does
not differ in SR-A+/+ and
SR-A
/
cells, whereas
in thioglycollate-elicited macrophages or phorbol ester-activated
Kupffer cells, the SR-A is responsible for about 85 and 35%,
respectively, of cell adhesion in the presence of serum (19). The SR-A
also mediates the adhesion of macrophages and SR-A-transfected
cells to glycated collagen type IV in the absence of serum and divalent
cations (17), to activated
lymphocytes (20), and to sections of
several mouse tissues (16, 18). The tissue components with which the
SR-A interacts were not identified.
Our data show that the SR-A contributes to both the
cation-dependent and -independent adhesion of
thioglycollate-elicited peritoneal macrophages to the extracellular
matrix from HSMCs, in either the presence or absence of serum. In the
presence of serum,
SR-A
/
macrophages
showed 65 and 95% reduced adhesion (in the presence and absence of
divalent cations, respectively) to extracellular matrix, compared with
the adhesion of SR-A+/+ macrophages. This
difference in adhesion is similar to data reported previously using
tissue culture plastic (11, 19). However, in the absence of serum the
difference in adhesion to the extracellular matrix between
SR-A
/
and
SR-A+/+ macrophages was much greater than the
effect when adhesion to plastic was studied.
SR-A
/
macrophages
showed 84 and 90% decreased adhesion to the HSMC extracellular matrix
(in the presence and absence of divalent cations, respectively)
compared with the decreases in adhesion to tissue culture plastic of 28 and 29% (19). These data demonstrate that the interaction of the SR-A
with components in the extracellular matrix contributes to the adhesion
of thioglycollate-elicited macrophages and that this interaction is
independent of serum and divalent cations.
We found that expression of the SR-A doubled the amount of time
necessary to release cells with trypsin from tissue culture plates or
tissue culture plates coated with extracellular matrix from HSMC. Our
studies complement and extend previous studies using a different
paradigm which suggested that the SR-A is partially responsible for the
trypsin-resistant adhesion of macrophages to tissue culture plastic
(16). In those studies, RAW 264 macrophages were gently trypsinized and
washed in serum-containing medium before adhesion assays. Trypsin
release after adhesion was not studied. Under those conditions, the
SR-A was determined to account for 15-20% of the trypsin-resistant
cell adhesion (16).
The contribution of the SR-A to the adhesion of macrophages in
vivo has not been demonstrated. However, our data and those reported previously suggest a role for the SR-A in adhesion under both
normal and pathological conditions in both the vasculature and the
central nervous system. The SR-A is highly expressed on activated
microglia in the vicinity of A
-containing senile plaques in brains
of patients with AD (30). The SR-A binds and internalizes microaggregates of the 42-amino acid form of A
in vitro
(9). In addition, we recently reported that the SR-A binds secreted forms of APP (8). The A
peptide and secreted APP are major constituents of senile plaques and cerebrovascular deposits in patients
with AD and Down's syndrome (31-33). The SR-A may contribute to the
clearance of both A
and secreted APP, which are produced continuously in normal and AD brains (32-35). However, the SR-A also
mediates the adhesion of microglia and human monocytes to
-amyloid
fibril-coated surfaces, leading to secretion of reactive oxygen species
and cell immobilization (36). The SR-A may, therefore, contribute to
the adhesion of cells to the senile plaques and/or to cells expressing
APP on their plasma membrane. In addition to A
and sAPP, AD plaques
contain a wide variety of molecules, including extracellular matrix
proteoglycans. It would be interesting to determine whether these
molecules have a role in the interaction with microglia and in the
progression of the disease.
The SR-A may also contribute to the adhesion of macrophages in
atherosclerotic lesions. Expression of the SR-A contributes to the
development of atherosclerosis.
SR-A
/
mice had
smaller atherosclerotic lesions than control mice (11-13). Because
SR-A
/
macrophages
exhibit a reduced uptake and degradation of modified LDL compared with
wild-type macrophages (11, 37), it is widely assumed that a decrease in
lipid accumulation in macrophages in the arterial wall is responsible
for the reduced atherosclerosis. However, this has not been proved.
Other aspects of SR-A biology have been suggested to contribute to the
reduced atherosclerosis (11, 15-19). Our results support a potential
alternative mechanism for the decrease in atherosclerosis in
SR-A
/
mice. These
data suggest that the adhesion and retention of
SR-A
/
macrophages in
the extracellular matrix may be lower than the retention of
SR-A+/+ macrophages, thereby resulting in
reduced development of atherosclerosis. Biglycan, versican, and
decorin, normally present in arteries, are substantially elevated in
atherosclerotic lesions (38-40). In atherosclerotic plaques, decorin
colocalizes with the macrophage-rich core (41, 42), whereas biglycan
and versican are prominent in the smooth muscle cell matrix adjacent to
macrophages (41, 43). Our data suggest that the interaction of the SR-A
with these proteoglycans or other components of the extracellular
matrix may contribute to the adhesion and retention of macrophages in atherosclerotic lesions, thereby enhancing lipid accumulation and the
progression of the disease.