Expression of Class A Scavenger Receptor Inhibits
Apoptosis of Macrophages Triggered by Oxidized Low
Density Lipoprotein and Oxysterol
Hai-Sun Liao, Tatsuhiko Kodama, Yong-Jian Geng
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Abstract—The class A macrophage scavenger receptor (MSR-A) is a multifunctional trimeric glycoprotein involved in
innate immune response as well as the development of lipid-laden foam cells during atherosclerosis. The MSR ligand,
oxidized low density lipoprotein (oxLDL), is known to be cytotoxic to macrophages and other cell types. This study
examined whether MSR mediates or modulates oxLDL-induced apoptosis. Treatment with oxLDL and its cytotoxic
oxysterol, 7-ketocholesterol (7-KC), reduced viability and increased DNA fragmentation in human THP-1 cells, Chinese
hamster ovary cells, and mouse peritoneal macrophages. However, cell death and DNA fragmentation were markedly
diminished in the phorbol ester– differentiated MSR-expressing THP-1 cells and Chinese hamster ovary cells, with
stable expression of MSR-AI after cDNA transfection when exposed to the same concentrations of oxLDL and 7-KC.
Moreover, treatment with oxLDL and 7-KC induced much greater death and DNA fragmentation in MSR-A– deficient
peritoneal macrophages compared with wild-type macrophages. Thus, MSR-A does not act as a receptor responsible for
the apoptotic effect of oxLDL, and instead, expression of this receptor confers resistance of macrophages to the
apoptotic stimulation by oxLDL and its cytotoxic lipid component. These results suggest that by preventing apoptosis,
MSR-A may contribute to the long-term survival of macrophages and macrophage-derived lipid-laden foam cells in
atherosclerotic lesions. (Arterioscler Thromb Vasc Biol. 2000;20:1968-1975.)
Key Words: macrophages 䡲 scavenger receptors 䡲 atherosclerosis 䡲 lipoproteins 䡲 cell death
A
therosclerosis is a chronic arterial disease with 2 lifethreatening complications, myocardial and cerebral infarcts. The long-lasting process of atherogenesis involves
dramatic alterations in cellularity, extracellular matrix, and
lipid components of the arterial wall, resulting in intimal
thickening, vessel lumen narrowing, and increased susceptibility to thrombosis. Cell proliferation has been traditionally
regarded as a key factor that increases the number of cells in
the arterial intima. However, analysis of proliferative markers
has revealed a low degree of cell replication.1 On the other
hand, recent studies have documented that deregulation of
apoptosis, a form of genetically programmed cell death,
occurs in atherosclerotic lesions.2,3 Imbalance between cell
survival and death may contribute to dramatic alterations in
cellularity of the arterial wall with atherosclerosis.
Formation of lipid-laden foam cells from macrophages
and, to a less extent, from smooth muscle cells represents a
landmark for atherosclerosis. The development of foam cells
is mainly due to overloading of lipids, particularly cholesterol
and cholesterol ester, into the cells through a scavenger
receptor–mediated process. It is believed that the lipids within
the foam cells largely come from the lipid-rich proteins in
blood, particularly LDLs, which enter the cells after chemical
modification, such as oxidation and converting to the ligand
for macrophage scavenger receptors (MSRs). Three different
classes of MSR have been identified.4 Among them, the class
A MSR (MSR-A) is the first one to be cloned and characterized for its ligand binding specificity and capacity.5 Its
expression is limited to differentiated or mature monomyeloid cells and is thus characterized for the monocyte-to-macrophage differentiation. Our previous studies have shown that
circulating monocytes elaborate MSR-A at undetectable levels but that when the cells differentiate into tissue macrophages, they express high levels of MSR-A.6,7
The MSR ligand, oxidized LDL (oxLDL), has been identified as an apoptosis-promoting agent to a variety of cell
types, including vascular smooth muscle cells,8,9 endothelial
cells,10 –12 and macrophages.13,14 Other modified LDLs, such
as enzymatically degraded LDLs (E-LDL), can selectively
induce the expression of monocyte chemotactic protein-1 and
exert cytotoxic effects on human macrophages.15,16 In vitro
treatment with oxLDL may activate the cysteinyl protease,
caspase-3,9 which acts as an effector in the downstream
caspase death cascade. However, despite the well-defined
Received December 31, 1999; revision accepted January 17, 2000.
From the Cardiovascular and Pulmonary Research Institute (H.-S.L., Y.-J.G.), Allegheny General Hospital, Pittsburgh, Pa; Molecular Biology and
Medicine (T.K.), University of Tokyo, Tokyo, Japan; and the Cardiology Division (Y.-J.G.), Department of Internal Medicine, University of Texas School
of Medicine at Houston.
Correspondence to Yong-Jian Geng, MD, PhD, Cardiology Division, Department of Internal Medicine, University of Texas School of Medicine at
Houston, 6431 Fannin St, MSB 1.240, Houston, TX 77030. E-mail [email protected]
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1968
Liao et al
Scavenger Receptor Inhibits Macrophage Apoptosis
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ligand-receptor relationship between oxLDL and MSR, it
remains largely unclear as to whether MSR-A mediates or
modulates apoptotic effects of oxLDL and associated lipids.
Our recent work17 has shown that differentiated MSR-A–
expressing human THP-1 macrophages become resistant to
apoptosis triggered by fluoride anions, a global activator of G
protein. Chinese hamster ovary (CHO) cells with stable
expression of MSR-A also exhibit increased resistance to the
fluoride apoptotic stimulation. Moreover, recent studies have
shown that expression of a scavenger receptor–like gene, the
cellular stress response gene, prevents cellular damage by UV
irradiation and oxidative stress18 and that thymocytes lacking
the apoptosis inhibitor of macrophages, a member of the
scavenger receptor cysteine-rich domain family, are more
susceptible to apoptosis.19 Thus, these data seem argue
against the notion that oxLDL triggers apoptosis in an
MSR-A– dependent manner.
The present study was aimed to determine (1) whether the
expression of MSR-A prevents oxLDL-induced apoptosis in
differentiated human THP-1 monocytic macrophages, (2)
whether overexpression of MSR-A in CHO cells reduces
apoptosis triggered by oxLDL and oxysterol, and (3) whether
resident peritoneal macrophages from the MSR-A–null
(MSR⫺/⫺) mice are more sensitive to oxLDL and oxysterol.
Methods
Reagents
Free cholesterol and 7-ketocholesterol (7-KC) were purchased from
Sigma Chemical Co and dissolved in dimethyl sulfoxide at a stock
solution of 10 mg/mL. Native LDL and oxLDL were obtained from
Perlmmune Inc. Acridine orange and ethidium bromide (nucleic
acid– binding dyes) were obtained from Sigma. RPMI-1640 and F-12
medium were purchased from GIBCO. Phorbol 12-myristate 13acetate (PMA) was obtained from Sigma and dissolved in dimethyl
sulfoxide at a stock concentration of 10 mmol/L.
Cell Culture and Treatment
THP-1 and CHO cells were obtained from American Type Culture
Collection. THP-1 cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS, 100-U/mL penicillin, and
100 ␮g/mL streptomycin. CHO cells were maintained in F-12
medium supplemented with 10% heat-inactivated FBS, 100 U/mL
penicillin, and 100 ␮g/mL streptomycin. THP-1 cells were pretreated
with or without 100 nmol/L of PMA, washed with PBS, and then
treated with various concentrations of oxLDL and 7-KC, respectively. Control experiments were performed by using the same
concentration of the vehicle, dimethyl sulfoxide.
Resident Peritoneal Macrophage Isolation
and Treatment
Resident peritoneal macrophages were isolated from wild-type
(MSR⫹/⫹) and homozygous MSR-deficient (MSR⫺/⫺) mice by peritoneal lavage as described previously.20 Briefly, mice injected with
0.2 mL of ketamine (100 ␮g/mL) underwent lavage with 8 mL of
serum-free RPMI-1640 media into the peritoneal cavity. Peritoneal
macrophages were obtained by centrifugation of the lavage fluid
collected from the peritoneal cavity. The collected macrophages
were resuspended in RPMI-1640 with 10% heat-inactivated FBS and
incubated at 1⫻105 cells per milliliter in 8-well chamber slides for 48
hours. The cells were then stimulated with oxLDL or 7-KC. After
stimulation, the cells were fixed for terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining as shown below.
1969
Cell Viability Assay
Cell viability was determined by staining with a combination of the
nucleic acid– binding dyes acridine orange (10 ␮g/mL) and ethidium
bromide (10 ␮g/mL) for 5 minutes on ice. After staining, viable and
nonviable cells were counted on the basis of color and appearance
under a fluorescence microscope. Acridine orange and ethidium
bromide dyes can intercalate into DNA. Viable cells are stained with
green nuclei by acridine orange, whereas nonviable cells are stained
with ethidium bromide, producing a red or orange color in nuclei. At
least 200 cells were counted. The percentage of dead cells was
calculated by dividing the number of dead cells by total cell number.
Analysis of DNA Fragmentation
DNA was extracted from the cells stimulated by oxLDL and 7-KC in
various concentrations for 24 hours in 6-well plates. After treatment,
5⫻106 cells were lysed in 1 mL of DNA extract solution containing
100 mmol/L NaCl, 20 mmol/L Tris-HCl (pH. 8.0), and 10 mmol/L
EDTA at room temperature for 15 minutes. The cell lysates were
incubated overnight with proteinase K (200 ␮g/mL) at 37°C,
extracted for DNA with phenol/chloroform isoamyl alcohol, and
precipitated with the same volume of isopropanol. Remaining RNA
was removed by digestion with RNase A (25 ␮g/mL) at 37°C for 30
minutes. After reextraction and precipitation, DNA concentration
was determined by spectrophotometer at a wavelength of 260
nmol/L. DNA at 20 ␮g per lane was loaded into 2% agarose gels
with 0.5 ␮g/mL ethidium bromide and separated by electrophoresis
at a constant voltage of 100 V for 1 to 2 hours at room temperature.
The DNA ladder was visualized under UV light.
Total RNA Isolation and RT-PCR
Total RNA was extracted from THP-1 cells treated with or without
PMA (100 nmol/L) for 48 hours by using the phenol/guanidinium
thiocyanate method.21 cDNA was generated by reverse transcription
(RT) from 1 ␮g of total RNA and a 30-cycle polymerase chain
reaction (PCR) with specific primers for MSR-A types I and II
(MSR-AI and MSR-AII, respectively), as described previously.6
RT-PCR for ␤-actin mRNA was used as an internal control. The
PCR products were analyzed by electrophoresis on 2% agarose gels
containing 0.5 ␮g/mL ethidium bromide.
In Situ 3ⴕ End Labeling of DNA
Fragments (TUNEL)
Mouse peritoneal macrophages treated with and without oxLDL or
7-KC in 8-well chamber slides for 24 hours were fixed with 3%
paraformaldehyde in PBS. After they were washed in PBS, the cells
were incubated with 2% H2O2 in methanol to inactivate endogenous
peroxidase. Digoxigenin-conjugated dUTP was incorporated into 3⬘
ends of DNA with TdT by using an ApoTag in situ apoptosis
detection kit (Oncor, Inc). The presence of digoxigenin-labeled DNA
fragments was determined by using a peroxidase-conjugated antibody against digoxigenin. The chromogenic substance diaminobenzidine was used as a substrate for visualization of the immunostaining. The percentage of TUNEL-positive cells was calculated by
dividing the number of positive cells by the total cell number.
CHO Cell Line With Stable Overexpression
of MSR-A
CHO cells were transfected with MSR-A type I cDNA by a standard
calcium phosphate precipitate method.17 After transfection, CHO
cells were selected with G-418 medium for 4 weeks. The establishment of the CHO cell line with stable expression of MSR-A was
confirmed by Northern and Western blotting.
Flow Cytometry
THP-1 cells were analyzed by flow cytometry for the expression of
different isoforms of MSR, such as MSR-A, CD36, and CD68. The
cells were pretreated with PMA and then exposed to 25 ␮g/ml
oxLDL at 37°C for 24 hours. After they were washed in PBS, the
cells were stained with monoclonal antibodies against MSR-A,
CD36, and CD68, followed by incubating with FITC-conjugated
anti-mouse IgG. The immunostains were detected by using a
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Arterioscler Thromb Vasc Biol.
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Difference in Viability of MSRⴙ and MSRⴚ THP-1 Cells Treated
With and Without OxLDL and 7-KC
% Cell Death
Treatment
MSR⫺
MSR⫹
P
OxLDL, ␮g/mL
0
3.6⫾1.2
5.8⫾1.4
ND
50
18.3⫾5.1
11.9⫾1.2
⬍0.05
100
29.3⫾5.6
16.3⫾2.5
⬍0.05
200
36.3⫾4.5
23⫾4
⬍0.05
0
2.6⫾0.6
5.6⫾0.4
ND
5
23⫾2
12.5⫾1.3
⬍0.05
10
28⫾4
18⫾1
⬍0.05
20
48⫾4
29⫾6
⬍0.05
7-KC, ␮g/mL
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Values are mean⫾SD (n⫽3). ND indicates not determined. THP-1 cells
pretreated with or without PMA (100 nmol/L) were exposed to oxLDL or 7-KC
in various concentrations for 24 hours. Cell viability was determined by staining
with acridine orange and ethidium bromide.
Figure 1. RT-PCR for MSR mRNA and agarose gel electrophoresis of DNA in human THP-1 cells. Panel A, RT-PCR analysis of MSR-AI and MSR-AII mRNA in PMA-treated and
untreated THP-1 cells. Total RNA was isolated from THP-1 cells
treated with or without 100 nmol/L PMA. cDNA reversetranscribed from RNA was amplified by 30-cycle PCR with a set
of primers that detect MSR-AI and MSR-AII. ␤-Actin primers
were used as an internal control (panel a, bottom). M indicates
DNA size markers; B, no template (blank). Panel B, Agarose gel
electrophoresis of DNA isolated from PMA-pretreated THP-1
cells after exposure to oxLDL (200 ␮g/mL) for 24 hours. Lanes
are as follows: 1, DNA from control cells treated with oxLDL; 2,
DNA from PMA-differentiated cells treated with oxLDL; and 3,
DNA from cells treated without oxLDL.
FACScalibur flow cytometer (Becton Dickinson) with Cytoquest
software.
Statistic Analysis
The experimental data were analyzed by using Excell software
(Microsoft), and ANOVA was used for statistical analysis of
multiple groups. Significant difference was established at a value
of P⬍0.05.
Results
ⴙ
MSR THP-1 Cells Resist Apoptosis Triggered
by OxLDL
Human THP-1 monocytic cells expressed high levels of
mRNA for MSR-AI and MSR-AII after stimulation with
PMA (Figure 1). To determine the extent of apoptosis in the
MSR⫹ THP-1 cells, the cells were pretreated with PMA and
then treated with various concentrations of oxLDL for 24
hours. We observed that treatment with oxLDL significantly
reduced the viability of MSR⫺ and MSR⫹ THP-1 cells in a
concentration-dependent manner, as determined by staining
with acridine orange and ethidium bromide (Table). In
contrast, under the same concentration, native LDL did not
alter cell viability. Interestingly, the number of nonviable
MSR⫹ THP-1 cells appeared significantly lower than the
number of nonviable MSR⫺ THP-1 cells (Table). Because
internucleosomal DNA fragmentation biochemically characterizes apoptosis, we analyzed the sizes of DNA isolated from
oxLDL-treated and untreated THP-1 cells by agarose gel
electrophoresis. As shown in Figure 1b, PMA-treated THP-1
cells showed decreased fragmentation of DNA at the internucleosomal sizes compared with the untreated MSR⫺ THP-1
cells. Morphological changes, including cell shrinkage, blebbing, chromatin condensation, and nucleus fragmentation,
also occurred, suggesting that the cells underwent apoptosis.
MSRⴙ THP-1 Cells Resist Apoptosis Triggered
by 7-KC
OxLDL contains many different lipid components, eg, lipid
peroxides, hydroxides, and oxidized cholesterol derivatives
(eg, oxysterol), many of which are potentially cytotoxic. We
examined whether 7-KC, a major oxysterol component found
in oxLDL,22,23 promotes apoptosis of THP-1 cells. We
observed that similar to oxLDL, 7-KC also reduced the
viability of THP-1 cells in a concentration-dependent fashion
(Table). Increasing concentrations of 7-KC from 5 ␮g/mL to
20 ␮g/mL led to 50% cell death (Table). Thus, 7-KC
appeared to exert a cytotoxic effect on THP-1 cells. The
7-KC–induced cell death occurred via an apoptotic mechanism that was evidenced by internucleosomal DNA fragmentation, as shown by the appearance of DNA ladder in agarose
gel electrophoresis in the 7-KC–treated cells (Figure 2). In
control experiments, we treated the cells with the same
concentrations of free cholesterol. We observed no effect of
free cholesterol on the cell viability, because it neither
increased the percent cell death (4.3⫾0.4% in the free
cholesterol–treated cells versus 3.9⫾0.5% in the control cells,
n⫽3) nor caused DNA fragmentation (data not shown). To
determine whether MSR-expressing cells resist apoptosis
induced by the oxysterol, we analyzed the differences in
viability and DNA fragmentation between MSR⫺ and MSR⫹
THP-1 cells. We found that the percentage of cell death and
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Scavenger Receptor Inhibits Macrophage Apoptosis
1971
Figure 2. Agarose gel electrophoresis of DNA from PMAdifferentiated THP-1 cells after exposure to 7-KC. DNA was
extracted from THP-1 cells pretreated with (lanes 4 to 6) and
without (lanes 1 to 3) 100 nmol/L PMA for 48 hours and then
exposed to 7-KC for 24 hours. DNA (20 ␮g per lane) was
loaded and fractionated on 2% agarose gels containing 0.5
␮g/mL of ethidium bromide. Lanes are as follows: 1 and 4, control; 2 and 5, 10 ␮g/mL 7-KC; and 3 and 6, 20 ␮g/mL 7-KC.
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the internucleosomal DNA fragmentation were diminished in
the MSR⫹ cells compared with the MSR⫺ THP-1 cells (Table
and Figure 2). These results indicate that PMA-pretreated
MSR-A– expressing THP-1 macrophages were resistant to
apoptosis induced by 7-KC.
To determine whether other isoforms of MSR participate in
the regulation of apoptosis induced by oxLDL and oxysterols,
we analyzed the expression of CD36 and CD68, 2 isoforms of
MSR, in the PMA-differentiated THP-1 cells by flow cytometry after stimulation with oxLDL. We observed that compared with untreated control cells, there were no significant
increases in the numbers of CD36-positive cells (11% versus
12% in control cells) and CD68-positive cells (20% versus
19% in control cells) in the PMA-pretreated cultures 24 hours
after stimulation with oxLDL (50 ␮g/mL). Similarly, we
found that the 7-KC–treated cells exhibited no changes in the
expression of the 2 proteins. This suggests that MSR-A other
than CD36 and CD68 contributed to the resistance of the cells
to apoptosis induced by oxLDL and 7-KC.
Overexpression of MSR-A by cDNA Transfection
Inhibits Apoptosis Induced by OxLDL in
CHO Cells
To further confirm that expression of MSR itself prevents
apoptosis induced by oxLDL and 7-KC, we recently established a CHO cell line with stable overexpressed MSR-AI by
transfection with the MSR-AI cDNA. Under baseline conditions, CHO cells expressed little MSR activities, but transfection with a cDNA coding for this receptor dramatically
increased MSR expression in the cells, as shown previously.5,24 Figure 3a demonstrates that the CHO cells with stable
expression of MSR showed significantly higher viability than
untransfected wild-type CHO cells when treated with the
same concentrations of oxLDL. Agarose gel electrophoresis
of DNA revealed a decrease in internucleosomal fragmentation of DNA in the MSR-A– expressing CHO cells compared
with wild-type control cells (Figure 3b). These results suggest
that overexpression of MSR-AI might increase the resistance
of CHO cells to apoptosis induced by oxLDL.
Expression of MSR by cDNA Transfection Inhibits
Apoptosis Induced by 7-KC in CHO Cells
To determine whether MSR expression confers resistance
of CHO cells to apoptosis induced by the oxysterol, 7-KC,
Figure 3. CHO cell death and internucleosomal DNA fragmentation after treatment with oxLDL. Wild-type and MSR-A cDNAtransfected CHO cells were stimulated with oxLDL in various
concentrations for 24 hours. a, Cell viability. After they were
stained with nucleic acid– binding dyes acridine orange (10
␮g/mL) and ethidium bromide (10 ␮g/mL) for 5 minutes on ice,
cells were observed under a fluorescence microscope. Cell viability was determined by dividing the number of dead cells by
the total number of cells. Data indicate mean⫾SD of at least 3
independent experiments. *P⬍0.05 (ANOVA). b, Internucleosomal DNA fragmentation. DNA extracted from CHO cell transfected with or without MSR-A cDNA after exposure to oxLDL
was loaded on 2% agarose gel containing ethidium bromide for
electrophoresis. Lanes are as follows: 1 and 4, control; 2 and 5,
100 ␮g/mL oxLDL; and 3 and 6, 200 ␮g/mL oxLDL.
we examined the viability of wild-type and MSR-AI–
transfected CHO cells after treatment with 7-KC. We
observed a concentration-dependent increase in the percentage of cell death in untransfected wild-type cells
treated with 7-KC for 24 hours (Figure 4a). However, the
percentage of cell death was much lower in the transfected
cells than in the wild-type control cells. Agarose gel
electrophoresis also showed a reduction in DNA cleavage
in the CHO cells with overexpressed MSR (Figure 4b).
Thus, the MSR-overexpressing CHO cells were also resistant to apoptotic stimulation by 7-KC.
MSR-A–Null Macrophages Exhibit Increased
Sensitivity to Apoptosis Induced by OxLDL
and 7-KC
To further verify the antiapoptotic effect of MSR-A on
macrophages, we treated resident peritoneal macrophages
isolated from wild-type (MSR⫹/⫹) and MSR-knockout
(MSR⫺/⫺) mice with oxLDL and 7-KC. We observed that
treatment with oxLDL led to increased numbers of cell death
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Arterioscler Thromb Vasc Biol.
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Figure 5. Death of oxLDL-treated peritoneal macrophages from
MSR⫺/⫺ and MSR⫹/⫹ mice. Peritoneal macrophages from
MSR⫺/⫺ and MSR⫹/⫹ mice were treated with and without oxLDL.
The numbers of nonviable cells were determined by staining
with acridine orange and ethidium bromide. The percentage of
dead cells was calculated by dividing the number of dead cells
by the total cell number. Data represent mean⫾SD (n⫽3).
*P⬍0.05 (ANOVA).
Figure 4. CHO cell death and internucleosomal DNA fragmentation after treatment with 7-KC. a, Cell viability. CHO cells transfected with or without MSR-A cDNA were treated with 7-KC for
24 hours and stained with acridine orange and ethidium bromide. After the staining procedure, the cells were observed
under a fluorescence microscope. The percentage of cell death
was determined by dividing the number of dead cells by the
total number of cells. Data represent mean⫾SD of at least 3
independent experiments. *P⬍0.05 (ANOVA). b, Internucleosomal DNA fragmentation. DNA extracted from 7-KC–treated and
untreated CHO cells with (lanes 4 to 6) or without (lanes 1 to 3)
overexpressed MSR-AI was loaded onto 2% agarose gel containing 0.5 ␮g/mL ethidium bromide. After electrophoresis, lanes
were as follows: 1 and 4, untreated control; 2 and 5, 10 ␮g/mL
7-KC; and 3 and 6, 20 ␮g/mL 7-KC.
in MSR⫺/⫺ macrophages compared with wild-type cells in a
concentration-dependent manner (Figure 5), as determined by
staining with acridine orange and ethidium bromide.25,26 In
situ 3⬘ end labeling of DNA fragments by use of the TUNEL
technique demonstrated an increase in nuclear DNA fragmentation in the treated cells (Figure 6). There were significantly
higher numbers of TUNEL-positive nuclei present in the
MSR⫺/⫺ cells compared with wild-type macrophages treated
with the same concentration of oxLDL (Figure 7a). The
MSR⫺/⫺ macrophages also exhibited higher TUNEL positivity than did the wild-type cells when exposed to 7-KC for 24
hours (Figures 6 and Figure 7b). Thus, the MSR-A deficiency
appeared to increase the susceptibility of the peritoneal
macrophages to apoptotic stimulation by oxLDL and the
oxysterol 7-KC.
Discussion
As part of the innate immune system, macrophages recognize
and remove denatured macromolecules, pathogenic microor-
ganisms, and damaged tissue or apoptotic cells through their
scavenger system. Despite the high frequency of exposure to
those environmental cytostatic or cytotoxic factors, the lifespan of tissue macrophages remains considerably longer. This
is particularly the case in the atherosclerotic lesions, in which
numerous macrophages survive an extremely harsh microenvironment that contains numerous toxic substances, such as
oxLDL and E-LDL.15,16,27 This mystery has led us to explore
the possibility that tissue macrophages may express a protein(s) with the potential of preventing apoptosis, a form of
genetically programmed cell death.
MSR, an abundant glycoprotein expressed on the surface
of macrophages, mediates the uptake of chemically modified
LDL by macrophages. This raises an interesting question as
to whether oxLDL exerts its cytotoxic effect through the
scavenger receptor pathway. In the present study, we used 3
different in vitro models to define the role for MSR-A in the
modulation of macrophage apoptosis triggered by oxLDL and
its cytotoxic lipid component, 7-KC. The first model uses
PMA-differentiated human THP-1 cells, which express
MSR-A at high levels. The second model uses a CHO cell
line with stably overexpressed MSR-A. The third or last
model focuses on peritoneal macrophages from the MSR-A
knockout mice. Our observations clearly indicate that MSR-A
does not act as a signaling receptor for oxLDL and 7-KC to
achieve their cytotoxic effects. Instead, the expression of
MSR-A reduces the extent of apoptosis in macrophages
exposed to oxLDL and 7-KC, whereas MSR-A deficiency
increases the sensitivity to apoptosis. Thus, MSR-A may
function as an inhibitor of apoptosis triggered by the oxidized
lipoprotein and its toxic lipid component rather than as a
signal transducer responsible for the oxLDL-induced cell
death. The cytotoxicity of oxLDL appears to be nonselective
or independent of MSR-A, inasmuch as it causes apoptosis in
a broad range of cell types, including MSR-A–negative and
–positive cells.
However, we cannot exclude other scavenger receptor
families, which may serve as death-signaling receptors for
oxLDL. In fact, MSR-A has a relatively lower affinity to
Liao et al
Scavenger Receptor Inhibits Macrophage Apoptosis
1973
Figure 6. In situ detection of DNA fragments by
TUNEL in MSR⫹/⫹ and MSR⫺/⫺ peritoneal macrophages treated with 7-KC. Wild-type (WT, a and b)
and MSR⫺/⫺ (c and d) peritoneal macrophages
treated with 7-KC in an 8-well chamber slide were
labeled with digoxigenin-conjugated dUTP and TdT
by use of an ApoTag in situ apoptosis detection kit
(Oncor, Inc). The presence of digoxigenin-labeled
DNA fragments was determined with a peroxidaseconjugated antibody against digoxigenin, with diaminobenzidine as a substrate. a and c, Phasecontrast microscopy. b and d, Light microscopy.
Note an increased number of MSR⫺/⫺ macrophages with shrunken and TUNEL-positive nuclei
stained brown.
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oxLDL compared with other forms of MSR, such as CD3628
and CD 68.29,30 The major ligand used for evaluating MSR
activity is acetylated LDL, an artificially modified lipoprotein
that may not exist in vivo. Hence, the physiological ligand for
Figure 7. Quantification of the TUNEL positivity in MSR⫹/⫹ and
MSR⫺/⫺ peritoneal macrophages treated with oxLDL and 7-KC.
Peritoneal macrophages treated with or without oxLDL and
7-KC, respectively, were fixed with 3% formaldehyde in PBS.
TUNEL staining was performed with an ApoTag in situ apoptosis detection kit (Oncor, Inc). The percentage of TUNEL-positive
cells was calculated by dividing the number of positive cells by
the total cell number. a, Macrophages treated with oxLDL. b,
Macrophages treated with 7-KC. Data represent mean⫾SD of at
least 3 independent experiments. *P⬍0.05 (ANOVA).
MSR-A has not been characterized at this time point. Nevertheless, the observation that oxLDL and 7-KC induce
apoptosis of THP-1 and CHO cells confirms and extends
previous findings8,9 by other groups that oxLDL treatment
can trigger vascular cell apoptosis. Because we observed no
major change in the expression of CD36 and CD68 in the
PMA-differentiated THP-1 cells after stimulation with oxLDL and because oxLDL can trigger apoptosis in MSRnegative cells, it is unlikely that CD36 and CD68 are the
principal contributors to the resistance of macrophages to
apoptosis induced by oxLDL and oxysterols.
The mechanism underlying the inhibitory effect of MSR on
oxLDL-mediated apoptosis remains to be determined.
OxLDL is composed of various cytotoxic components, including oxysterols. 7-KC is the second most abundant oxysterol found in human atherosclerotic lesions after 27hydroxycholesterol. Whether 7-KC is a major cytotoxic
component in oxLDL remains to be investigated. The intracellular domains of MSR do not contain the sequences of any
known signaling factor– binding sites. Although ligand transporting to lysosomes via the MSR pathway can help to
remove and degrade oxLDL, the MSR-mediated endocytosis
may not be the sole or major mechanism by which MSR
prevents apoptosis. This notion is supported by the fact that
there is a low affinity between oxLDL and MSR-A and no
7-KC binding to MSR-A. Our recent studies17 have provided
an additional interpretation for the protective effect of MSR,
showing that expression of MSR-A blocks apoptosis induced
by the activation of membrane GTP-binding proteins. In this
regard, MSR-A may participate in G-protein–mediated crossmembrane signal transduction in macrophages.
THP-1 cells, a human monocytic leukemia cell line, can
differentiate into macrophage-like cells, after exposure to the
transforming agent, phorbol esters. The differentiated THP-1
macrophages express high levels of MSR and behave in a
manner highly similar to that of native monocyte-derived
macrophages.31 PMA is a potent activator of protein kinase C.
Regulation of apoptotic death by the protein kinase C
pathway has been recently reported in the cells.32–35 However,
in the present study, we observed no difference in cell death
1974
Arterioscler Thromb Vasc Biol.
August 2000
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between PMA-treated and untreated THP-1 cells. Very recently, we have shown increased expression of apoptosispromoting genes, such as FasL, and CPP-32 (caspase-3) in
PMA-differentiated THP-1 cells, but there is no increase in
the number of cells bearing markers of apoptosis.17 It is
reasonable to expect that the expression of MSR-A plays a
role in the regulation of apoptosis of macrophages. Nishio
and Watanabe9 reported that oxysterols induced apoptosis in
cultured smooth muscle cells through CPP-32 activation and
bcl-2 protein downregulation. However, it is unclear whether
7-KC exerts a similar effect on macrophages and whether
MSR can block the regulatory effects of 7-KC on the cells.
Munn et al36 reported that macrophage colony-stimulating
factor (CSF)– differentiated macrophages are resistant to
apoptosis, whereas granulocyte-macrophage CSF– differentiated and interferon-␥– differentiated macrophages are sensitive to apoptosis. Interestingly, macrophage CSF increases
but interferon-␥ and granulocyte-macrophage CSF decrease
the expression of MSR.37–39
Recently, several proteins with the MSR cysteine-rich
domain (SRCR), such as cellular stress response elements18
and apoptosis inhibitors of macrophages,19 have been reported to exert protective effects on apoptosis of fibroblasts
and macrophages, respectively. The mechanism underlying
the protective effects of the SRCR-containing proteins is
unknown. The mRNA and protein levels of MSR-AI with the
SRCR domain are not appreciable in most peripheral blood
monocytes, but MSR-AI expression elevates dramatically
when monocytes differentiate into macrophages. The increased expression of MSR-AI concurs with the development
of resistance to apoptosis in the monocyte-derived macrophages. Further investigation is needed to delineate the
relationship of MSR-AI expression with the macrophage
resistance to apoptosis.
It has been shown that macrophages of atherosclerotic
lesions express high levels of MSR-A,6 suggesting that the
expression of MSR is associated with maturation of monocytes and resistance of certain macrophages to apoptosis in
atherosclerosis. Most of the plaque macrophages contain
substantial amounts of lipids, mainly cholesterol, that are
derived from modified lipoproteins internalized via the scavenger pathway. The increased MSR-A expression may not
only promote the uptake of those lipid components but also
contribute to the longevity of the lipid-rich foam cells by
increasing the resistance of the cells to cytotoxic effects of
oxLDL and oxysterols. Recently, Kockx and colleagues40,41
reported lower levels of apoptosis in certain atherosclerotic
lesions than previously thought, suggesting that nonspecific
staining for the cells undergoing apoptosis or attenuation of
apoptosis might occur in the lesions. Our present data appear
to support the notion that in atherosclerotic plaque, there is a
mechanism that is responsible for the inhibition of apoptosis
and that leads to long-term accumulation of lipid-laden
macrophage-derived foam cells during atherogenesis.
Acknowledgments
This study was supported by National Institutes of the Health grant
HL-59249 – 01 (Y.-J. Geng) and an American Heart Association,
Pennsylvania affiliate, Grant-in-Aid award (Y.-J. Geng). We thank
Dr Hiroshi Suzuki, Chugaii Pharmaceutical Co, Japan, for the
generation of MSR-A knockout mice. We also thank Tamara Pittman
and Qi Wu for their skilled technical assistance.
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Expression of Class A Scavenger Receptor Inhibits Apoptosis of Macrophages Triggered
by Oxidized Low Density Lipoprotein and Oxysterol
Hai-Sun Liao, Tatsuhiko Kodama and Yong-Jian Geng
Arterioscler Thromb Vasc Biol. 2000;20:1968-1975
doi: 10.1161/01.ATV.20.8.1968
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