© 1995 American Heart Association, Inc.
Articles |
Expression of the Macrophage Scavenger Receptor in Atheroma
Relationship to Immune Activation and the T-Cell Cytokine Interferon-
From the Departments of Laboratory Medicine (Y-j.G., S.N., M.B.) and Surgery (J.H.), Gothenburg University, Gothenburg, Sweden, and King Gustaf V Research Institute (M.B., S.S., G.K.H.), Karolinska Institute, Stockholm, Sweden.
Correspondence to Prof Göran K. Hansson, King Gustaf V Research Institute, Karolinska Hospital, S-17176 Stockholm, Sweden.
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Abstract |
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Abstract Scavenger receptors mediate internalization of modified lipoproteins and foam cell transformation of monocyte-derived macrophages. Their expression is independent of the intracellular cholesterol content but is modulated by immune-derived cytokines. We investigated macrophage scavenger receptor (MSR) expression in monocyte-macrophages from human peripheral blood and in atherosclerotic lesions and analyzed its relationship to T lymphocytes and immunoregulatory cytokines by immunohistochemistry and polymerase chain reaction (PCR). Antibodies specific for the two MSR isoforms were generated by immunizing rabbits with isoform-specific synthetic peptides conjugated to keyhole limpet hemocyanin. In human atherosclerotic plaques, these antibodies stained macrophages and foam cells in a pattern that corresponded to the distribution of the macrophage marker CD68. CD3-positive T cells and
-actin–positive smooth muscle
cells exhibited no reactivity to the anti-MSR antibodies. The frequency
of cells stained with antibodies to MSR type I was equal to that
of cells stained for type II, suggesting that most macrophages
coexpress both isoforms. Reverse transcription (RT)–PCR
analysis confirmed that both MSR isoforms were expressed in all
plaques examined. There was, however, a tendency toward a lower
immunohistochemical staining intensity for MSR type I and a decreased
number of lipid-rich foam cells in T cell–rich areas. The
mRNAs for interleukin-2 and interferon-
, two major products of
activated T cells, were detected by RT-PCR in all plaques
tested. This indicates that activation of T lymphocytes occurs in
atherosclerotic plaques. Since interferon- downregulates MSR
expression, these observations suggest a potential mechanism for local
regulation of MSR expression in the atherosclerotic plaque.
Key Words: atherosclerosis • scavenger receptor • LDL • LDL oxidation • macrophages • T lymphocytes • immunohistochemistry • PCR
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Introduction |
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Intracellular cholesterol accumulation in foam cells is one of the hallmarks of the atherosclerotic lesion. Most foam cells express phenotypic markers of macrophages and are probably derived from circulating monocytes that enter the developing atherosclerotic lesion.1 Cells of the monocyte-macrophage lineage express a special type of lipoprotein receptor, the MSR, which mediates uptake of chemically modified LDLs, such as acLDL and oxLDL, and certain other proteins with increased negative surface charges.2 3 In contrast to the receptor for native LDL, MSR is not downregulated by increases in the intracellular cholesterol content.2 Macrophages will therefore continue to take up and process modified LDL via MSR as long as they are present in the extracellular milieu. This results in an intracellular cholesterol accumulation and the transformation of the macrophage into a foam cell.
MSR cDNAs have been cloned from bovine,4 5 human,6 rabbit,7 and murine sources.8 Two MSR isoforms were identified, both of which are integral membrane glycoproteins that bind their ligands via a collagen-like domain.3 4 Both isoforms are derived from a single MSR gene and generated by alternative splicing.9 The only difference between them is found in their extracellular carboxy-terminal parts. MSR-I contains a highly conserved scavenger receptor cysteine-rich domain in its C-terminus, but in the type II isoform, this domain is replaced by a short peptide of 17 amino acids without cysteines. However, both MSR isoforms bind and internalize modified LDL with similar affinities.4 5 8 10
MSR expression is associated with the differentiation stage of
the monocyte-macrophage and is modulated by
cytokines. Circulating monocytes express low or undetectable
levels of both isoforms, and differentiation into macrophages
is associated with upregulation of MSR, in particular the type I
isoform.11 This isoform, however, is downregulated by the
macrophage-activating, T lymphocyte–derived
cytokine IFN-.12 Tumor necrosis factor and
endotoxin, which also activate macrophages, also
downregulate MSR expression.13 Therefore, there appears to
be a direct relationship between macrophage differentiation and
MSR expression but an inverse relationship between macrophage
activation and MSR expression.
It has been demonstrated that MSR mRNA and proteins are expressed in
atherosclerotic plaques,14 15 but their regional
distribution and relationship to immunocompetent cells are unclear. We
therefore mapped MSR isoform expression in different plaque regions and
compared it with the distribution of monocyte-macrophages,
T lymphocytes, and HLA-DR. MSR isoforms were detected by
immunohistochemistry using a new set of peptide antibodies and by
RT-PCR analysis of MSR mRNA. Our data confirm that both MSR
isoforms are expressed by macrophages in atherosclerotic
plaques. We further observed that MSR-I expression is low and foam cell
formation less pronounced in T cell–rich regions. Both IFN- and
another important Th1-type T cell cytokine, IL-2, were detected
in all plaques, implying that T-cell activation is invariably taking
place in atherosclerosis. Together, our results suggest
that T cells may modulate MSR expression and foam cell development in
atherosclerotic lesions by local cytokine secretion.
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Methods |
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Atherosclerotic Plaques
Plaques were obtained from 13 patients, 9 men and 4 women, between 51 and 72 years of age, who underwent carotid endarterectomy for transitory ischemic attacks. None of the patients suffered from any known chronic disease other than atherosclerosis. The specimens represented advanced lesions, which have been described in detail in previous reports.16 They were immersed in sterile saline immediately after removal and rapidly transported to the laboratory. After thorough rinsing, the tissue was divided into two pieces, one for immunohistochemistry and one for RNA isolation.
Monocyte-Macrophages
Mononuclear cells were isolated by Ficoll-paque
centrifugation of peripheral blood from
healthy blood donors and from patients with
atherosclerosis. Monocytes were further isolated by
adherence on culture dishes, and nonadherent lymphocytes were removed
by PBS washing.11 12 The purity of the monocyte
preparation was >90%, with a viability of >95%, as determined by
anti-CD14 staining and trypan blue exclusion, respectively. Monocytes
were induced to mature into macrophages by incubation in
RPMI-1640 cell culture medium (Gibco) containing 10% fetal bovine
serum (Gibco) and 10% pooled normal human serum.
Antibodies
MSR isoform–specific antibodies were prepared by
immunization of rabbits with synthetic peptides.17
Peptides were synthesized by use of FMOC-protected amino acid esters
and a Milligen 9050 peptide synthesizer and purified by
high-performance liquid chromatography. The
peptide CSHSEDAGVTCTL is located in the C-terminal part of the
extracellular scavenger receptor cysteine-rich domain of MSR-I, and
the peptide CRPVQLTDHIRAGPS represents the C-terminal of the
MSR-II isoform, with the exception of the cysteine residue that was
added as a linker. The peptides were sequenced for confirmation and
conjugated with keyhole limpet hemocyanin by an
m-maleimidobenzoyl-N-hydroxysuccinimide
ester–based conjugation kit (Imject Maleimide Activated
Immunogen Conjugation Kit, Pierce). New Zealand White rabbits were
immunized subcutaneously with 200 µg of peptide–keyhole limpet
hemocyanin conjugate mixed with Freund's complete adjuvant (Pierce)
and again after 2 weeks with the same amount of immunogen in Freund's
incomplete adjuvant.17 They were boosted with 20 µg of
immunogen mixed with aluminum hydroxide. Sera were collected 1 week
after booster injections and assayed for anti-MSR antibodies by ELISA.
IgG was isolated from high-titer antisera by affinity
chromatography on protein G–Sepharose (HiTrap Protein
G, Pharmacia) and used for experiments. In addition, the following
monoclonal antibodies were used: Leu-4 (anti-CD3), Leu-M3 (anti-CD14),
and anti–HLA-DR from Becton Dickinson; EBM11 (anti-CD68) from Dako;
and anti–-smooth muscle actin, which was a gift from Dr G.
Gabbiani, Geneva, Switzerland.
ELISA of MSR Antibodies
Polyvinyl chloride–based microtiter plates (Nunc)
were coated with synthetic peptides at 10 µg/mL in a 0.1 mol/L
carbonate buffer, pH 9.6, for 18 hours at +4°C. After being rinsed
with 0.05% Tween-20 in PBS, the wells were incubated with antisera at
various dilutions. Bound antibody was detected with alkaline
phosphatase–conjugated goat anti-rabbit IgG (Dako) followed by
an alkaline phosphatase substrate solution (Dako). Plates were
analyzed by spectrophotometry at 405 nm.
Lipoprotein Uptake and Lipid Staining
MSR ligand uptake by monocyte-macrophages was
determined by incubation of cultured monocytes with acLDL conjugated
with a fluorescent dye, DiI (Biogenesis), as previously
described.11 12 Briefly, 5 µg/mL of DiI-acLDL was added
to monocyte cultures and incubated for 3 hours at 37°C. After
incubation, cells were washed with PBS, fixed, and subjected to
fluorescence analysis on a FACScan (Becton Dickinson)
flow cytometer. For analysis of cellular lipid content, cells
and tissue sections were stained with 3% oil red O in 60% isopropanol
for 1 hour, rinsed in PBS, and mounted for light microscopic
examination.
Flow Cytometry (FACS)
Anti-MSR peptide antibodies at concentrations between 1 and 50
µg/mL were incubated with peripheral blood mononuclear
cells and with monocyte-derived macrophages, both prepared
as described previously.12 After being rinsed, the cells
were stained with FITC-labeled anti-rabbit IgG (Dako) and
analyzed in a Becton Dickinson FACScan flow cytometer using the
LYSYS II software package. Double staining with anti-MSR
antibodies and DiI-acLDL was performed as described.11
Immunohistochemistry
Cryostat sections were prepared as described,16
fixed in 4% formaldehyde in phosphate buffer for 15 minutes and 95%
ethanol for 5 minutes, rinsed, and preincubated with normal goat serum
(1:50 in PBS with 2% fat-free dry milk). Sections were then
incubated with peptide antibodies and monoclonal antibodies at optimal
dilutions determined by checkerboard titration. After PBS rinses, they
were incubated with biotinylated anti-rabbit IgG (Jackson Lab) or
biotinylated anti-mouse IgG (BioGenex) followed by alkaline
phosphatase–labeled avidin and a fast red alkaline phosphatase
substrate solution (Link-Label system, BioGenex). Incubations with a
nonspecific monoclonal antibody (MOPC21 hybridoma IgG) and with
preincubation solution alone were used to control mouse monoclonal
antibody staining. As controls for the peptide antibodies, we used
protein G–Sepharose–purified IgG fractions of preimmune sera that
did not contain any anti-MSR antibody activity. For double staining,
sections were first incubated with monoclonal antibodies and stained
with biotinylated anti-mouse IgG followed by an
avidin-peroxidase conjugate (Vectastain ABC kit, Vector
Laboratories) and diaminobenzidine. They were then incubated with
peptide antibodies followed by biotinylated anti-rabbit IgG and the
alkaline phosphatase detection system described above.
RNA Isolation
Plaque tissue was homogenized in 4 mol/L guanidinium
thiocyanate, 25 mmol/L sodium citrate pH 7.0, 0.5%
N-laurylsarcosine, and 0.1 mol/L 2-mercaptoethanol with an
UltraTurrax homogenizer, and total RNA was isolated by
the method of Chomczynski and Sacchi.18 The RNA
concentration was determined by spectrophotometry at 260 nm, and its
integrity was assessed by agarose gel electrophoresis.
Reverse Transcription–Polymerase Chain Reaction
A total of 250 ng RNA was reverse transcribed by Moloney's
murine leukemia virus reverse transcriptase (Boehringer
Mannheim) by use of random hexamer priming as described.11
PCR amplification was carried out by 30 to 35 cycles of denaturation,
annealing, and elongation in a PCR buffer11 containing
Taq DNA polymerase. Products were analyzed by
agarose gel electrophoresis in the presence of ethidium bromide. The
primers used for MSR amplification have been described.11
To determine differential expression of the two MSR transcripts, a
common 5' primer was used together with two 3' primers that were
specific for the two isoforms.
The primers used for analysis of IL-2 were
5'-GAATGGAATTAATAATTACAAGAATCCC-3' (sense) and
5'-TGTTTCAGATCCCTTTAGTTCCAG-3'
(antisense); for IFN-, 5'-ATGAAATATACAAGTTATATCTTGGCTTT-3' (sense)
and 5'-GATGCTCTTCGACCTCGAAACAGCAT-3' (antisense); and for GAPDH,
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (sense) and
5'-CATGTGGGCCATGAGGTCCACCAC-3' (antisense).
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Results |
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Generation and Characterization of Anti-MSR Peptide Antibodies
A new set of MSR antibodies was generated by immunization with synthetic peptides corresponding to the C-terminals of the two MSR isoforms. Their specificities and titers were determined by analysis of reactivities to the purified MSR peptides used for immunization and to cultured monocyte-derived macrophages known to express MSR. ELISA analysis showed high-titer immunoreactivity of the hyperimmune sera to the peptides used for immunization, and there was no cross-reactivity between isoforms (data not shown). The reactivity of the peptide antibodies to MSR proteins was verified by FACS analysis of monocyte-derived macrophages. As shown in Fig 1
, both MSR-I and MSR-II antibodies stained cultured
monocytes that internalized the MSR ligand DiI-acLDL. Lymphocytes, in
contrast, neither internalized DiI-acLDL nor were stained by the
antibodies. Together, the ELISA and FACS analyses indicate that
the peptide antibodies were specific for the MSR peptide sequences and
reacted with cells exhibiting MSR activity.
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Immunohistochemical Analysis of Plaques
Immunohistochemical staining of carotid
endarterectomy specimens demonstrated the presence
of both MSR-I and MSR-II in a large proportion of the cells in the
plaque (Fig 2 and Table 1). A comparison
of the staining patterns for MSR isoforms and cell type–specific
markers confirmed that MSRs were expressed by CD68-positive
macrophages (Fig 2, Table 1). MSR-I and -II were localized in
the same areas (Fig 2b and 2c), suggesting that both isoforms were
expressed by the same cells. No significant difference could be
observed in the number of cells expressing MSR-I and MSR-II (Tables 1 and 2), but the intensity of staining was usually
higher for MSR-II (Fig 2b and 2c). This suggests that both isoforms are
expressed by macrophages of the plaque but at individually
variable levels.
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MSR distribution in different regions of the plaque closely followed
the distribution of CD68, with the highest frequency of positive cells
in the lipid core and the lowest in the shoulder region (Table 2
).
However, not all CD68-positive macrophages expressed MSR, since
the number of CD68-positive cells was larger than that of
MSR-I–positive or MSR-II–positive cells (Table 2). Furthermore, the
frequency of HLA-DR–expressing cells was higher than the frequency of
CD68-positive, MSR-I–positive, or MSR-II–positive cells (Fig 2,
Tables 1 and 2). This is in line with our previous observation that in
addition to macrophages, certain T lymphocytes and smooth
muscle cells also express HLA-DR.19 The intensity and
frequency of MSR-I staining were lower in areas with a high level of
HLA-DR expression (Fig 2b and 2d). In contrast, MSR-II staining was
relatively prominent in these areas (Fig 2c and 2d).
"Aberrant" HLA-DR expression, ie, expression by nonimmune
cells, is induced by IFN-, a product of activated T
cells,19 which also downregulates MSR-I
expression.12 Immunohistochemical double staining was
therefore performed to evaluate the relationship between T cells and
macrophages expressing MSR-I. As shown in Fig 3,
a number of T cells were found in the fibrous cap and shoulder regions,
where MSR-I staining was low and few MSR-I-positive foam cells were
found. T cells were often localized to the
subendothelium, whereas CD68-positive,
MSR-I–positive macrophages were more abundant in deeper layers
of the fibrous cap and within the lipid core (Figs 2b, 2e, 2f, and 3;
Table 2). Conversely, –smooth muscle actin–positive smooth
muscle cells dominated the fibrous cap but were sparse in the lipid
core region (Fig 2b, Table 2). Thus, the localization of
MSR-I–positive lipid-laden macrophages differed from that
of T cells and smooth muscle cells. This was further confirmed by
double immunostaining with a combination of anti-CD3
and anti-CD68, which showed that the distribution of CD3-positive T
cells was not identical to that of CD68-positive macrophages
(Fig 3c).
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To determine whether MSR expression is associated with foam cell
formation in the plaque, we combined lipid histochemistry with MSR
immunohistochemistry. Fig 4 shows an area at the border
between the fibrous cap and lipid core of a plaque. MSR-I (Fig 4a) and
CD68 (Fig 4c) were found in all oil red O–stained foam cells (Fig 4d).
In contrast, only a few foam cells stained with MSR-II (Fig 4b).
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Expression of MSR mRNA in the Plaque
To confirm that MSR is expressed in atherosclerotic plaques, RNA
from endarterectomy samples was analyzed
for MSR mRNA by RT-PCR. After reverse transcription of RNA, the
resulting MSR cDNA was amplified with a 5' primer corresponding to a
sequence shared between MSR-I and -II and two 3' primers, one
complementary to MSR-I and the other to MSR-II. Hence, both MSR isoform
transcripts could be analyzed simultaneously in a
single tube. As illustrated in Fig 5, both MSR mRNA
isoforms were detected by this method. The MSR-I bands were more
intense than those representing MSR-II. This could imply
that MSR-I was more abundantly expressed; however, the PCR conditions
did not permit quantitative conclusions regarding mRNA concentrations.
We found MSR mRNA transcripts in all 10 plaques analyzed (Fig 5). Very little MSR mRNA was observed in monocytes freshly isolated
from the peripheral blood of the same patients, suggesting
that MSR-I was upregulated after the monocytes had migrated into the
arterial wall (data not shown).
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mRNA Expression of the T-Cell Cytokines IL-2 and
IFN-
MSR expression in cultured cells is regulated by the T-cell
cytokine IFN-,12 and there is an inverse
relationship between T-cell activation and MSR-I expression of
macrophages (see above and References 20 and 2120 21 ). We therefore
speculated that T cells activated in the plaque could secrete
IFN-, which would be expected to modulate MSR expression in vivo as
well. As shown in Fig 6, IL-2 mRNA was detected by
RT-PCR in all plaque samples analyzed, indicating that
activated T cells are invariably present in
atherosclerosis. IFN- was also detected in all these
plaques, although to a variable extent (Fig 6). This suggests that
infiltrating T cells are activated and secrete MSR-regulating
cytokines in the plaque.
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Discussion |
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MSR plays a key role in the lipid deposition and foam cell transformation of macrophages. The data of the present study confirm and extend previous reports6 14 15 that MSR mRNA and proteins are expressed in atherosclerotic plaques. MSR proteins colocalized with the macrophage marker CD68 but not with the smooth muscle cell marker
–smooth muscle actin or the
T-lymphocyte antigen CD3. Furthermore, MSR was present in most of
the lipid-laden foam cells, strongly suggesting that MSR-expressing
macrophages form the bulk of the foam cell population. There
was no significant difference in the number of cells stained with MSR-I
and MSR-II antibodies, which confirms previous observations that both
MSR isoforms are expressed by the same cell.14 15 MSR expression could, however, be regulated at the cellular level. Thus, we recently observed that the two MSR isoforms are differentially regulated during monocyte-macrophage differentiation.11 MSR type I mRNA and proteins were found to be upregulated during differentiation, whereas MSR-II remained constitutively expressed at the same level.11 In the present study, foam cells were strongly stained with anti–MSR-I antibodies, suggesting that MSR-I is expressed at a high level in these cells. Together, these observations are compatible with the notion that MSR-I expression is associated with macrophage differentiation and foam cell formation.
MSR-I–expressing foam cells were preferentially localized to areas
devoid of T cells, suggesting that the latter may exert a regulatory
influence on MSR expression and foam cell formation. This is in line
with previous in vitro observations that T cell–derived
cytokines reduce cholesterol
accumulation.20 21 It is likely to be accomplished by the
release of IFN-, which downregulates MSR-I in cultured human
macrophages.12
Previous immunohistochemical studies have shown that T cells are
present in plaques16 and that some of them produce
IFN- and the IL-2 receptor.22 The hypothesis that
paracrine regulation of gene expression in the plaque is accomplished
by cytokine release was supported by the observation that
plaque smooth muscle cells express the IFN-–inducible gene
HLA-DR.19 We now show, by analyzing mRNA for IL-2 and
IFN- by RT-PCR, that all plaques examined contained these
cytokines. This provides direct evidence for the presence of
activated, cytokine-producing T cells in the
plaque.
IFN-, which is a potent activator of
macrophages, has been reported to inhibit lipid accumulation in
vitro in murine23 and human
macrophages.12 In vivo, administration of IFN was
reported to suppress aortic atherosclerosis in
rabbits.24 Although this has not been confirmed in vivo in
humans, it is tempting to speculate that the local synthesis of IFN-
and other cytokines released during immunological activation
might affect the uptake of modified lipoproteins and foam cell
transformation of macrophages in the plaque.
The mechanisms by which T cells are activated and induced to
secrete cytokines in the atherosclerotic plaque have not been
completely clarified. Most plaque T cells are phenotypically memory
cells derived from the blood.25 By cell cloning and T-cell
antigen receptor gene analysis26 and also by
direct PCR analysis of T-cell antigen receptor genes in
plaques,27 it has been demonstrated that plaque T cells
are heterogeneous; they may consequently recognize a
variety of antigenic epitopes. OxLDL28 and heat-shock
proteins29 may be two of the antigens that
activate plaque T cells, since both are generated in the plaque
and elicit the production of autoantibodies, which can also be
found in the plaque. Recently, Stemme et al30 discovered
that CD4-positive plaque T cells immunospecifically recognize oxLDL.
This strongly suggests that oxLDL is an important local antigen that
elicits cellular immune responses in the plaque. As shown in
this article, the ensuing IFN- secretion is likely to downregulate
MSR expression, thereby reducing oxLDL uptake by the
antigen-presenting macrophage. MSR has been shown to
target ligands for antigen presentation,31 and
the cellular immune response to oxLDL may therefore elicit a negative
feedback control on antigen presentation to T cells.
The presence of T lymphocytes in atherosclerotic lesions has been well
documented by this and other laboratories.16 19 32 33
However, the pathophysiological role of T
lymphocytes in atherosclerosis has not been completely
clarified. IFN- has been found to inhibit arterial
smooth muscle cell proliferation after endothelial
injury34 and to induce the production of nitric
oxide,35 36 a potent vasodilator and antagonizer of
platelet adhesion and aggregation. Therefore,
activated T cells might play a protective role in
atherogenesis. This notion is supported by the recent observation that
CD8-deficient mice develop larger fatty-streak lesions than
immunocompetent controls.37 However, some of the
consequences of T-cell activation may be atherogenic rather than
protective. For instance, the secretion of
macrophage-activating factors and the inhibition of
collagen38 and -actin formation39 may
reduce the mechanical strength of the arterial wall, thus
increasing the risk for rupture of the advanced plaque. To clarify the
role of immunocompetent cells in atherosclerosis will
be important for understanding the pathogenetic process and for
designing new therapeutic strategies for this disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Swedish Medical Research Council (project 6816) and the Swedish Heart-Lung Foundation. We thank Qi Wu for excellent technical assistance. Y-j.G. is currently in the Department of Medicine, Brigham and Women's Hospital, Boston, Mass.
Received June 30, 1994; accepted July 28, 1995.
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