Download Immunohistochemical Detection of Macrophages and T

745
Immunohistochemical Detection of
Macrophages and T Lymphocytes in
Atherosclerotic Lesions of
Cholesterol-Fed Rabbits
Goran K. Hansson, Paul S. Seifert, Gun Olsson, and Goran Bondjers
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Human atherosclerotic plaques contain significant numbers of T lymphocytes and monocytederived macrophages. Cytokines released from activated T lymphocytes induce aberrant
expression of major histocompatibility complex class II (la) antigens by vascular smooth
muscle cells and may also regulate cell proliferation and metabolism in the vessel wall. We have
analyzed the arteries of cholesterol-fed rabbits to study the sequence of lymphocyte and
monocyte entry into the forming atherosclerotic lesion. Rabbits were fed 0.3% cholesterol for
1-10 weeks, and monoclonal antibodies to rabbit leukocyte differentiation antigens and la
antigen were applied to sections of the aorta. Monocytes were already observed 1 week after
initiation of cholesterol feeding, and they accumulated in the intima, where they formed the
bulk of the foam cell-rich lesion. T lymphocytes also adhered to the aortic surface from 1 week
onward, and also accumulated in the lesion, although in lower proportions than did monocytes.
In 10-week lesions, approximately 6% of cells expressed the T-lymphocyte marker LI 1/135. la
antigen expression was frequent throughout the lesion in all phases of its development, and
most of the la-expressing cells could be identified as monocyte-derived macrophages. These
data indicate that the cholesterol-fed rabbit is a useful model for studying the role of monocytes
and T lymphocytes in atherosclerosis. (Arteriosclerosis and Thrombosis 1991;ll:745-750)
T
he atherosclerotic plaque bears many similarities to chronic inflammatory lesions. One of
the hallmarks of inflammation is the activation of the immune system. This is reflected in the
infiltration of lymphocytes and monocytes and the
expression of class II major histocompatibility complex (MHC) antigens in the inflammatory lesion.1-2
We3 and others4-5 have recently found that these
phenomena also take place in atherosclerosis. T
lymphocytes as well as monocytes are present in the
human atherosclerotic plaque, and class II MHC
From the Department of Clinical Chemistry (G.K.H., P.S.S.)
and the Wallenberg Laboratory for Cardiovascular Research
(G.O., G.B.), Gothenburg University, Gothenburg, Sweden. P.S.S.
is currently at the Institute of Microbiology, University of Mainz,
Mainz, F.R.G.
Supported by the Swedish Medical Research Council (project
Nos. 6816 and 4531), the Swedish National Association Against
Heart and Chest Diseases, the Magnus Bergvall Foundation, the
Salus Foundation, and AB Hassle. P.S.S. was the recipient of a
Fogarty International Research Fellowship from the US National
Institutes of Health.
Guilio Gabbiani kindly acted as Guest Editor for this article.
Address for correspondence: Dr. Goran K. Hansson, Department of Clinical Chemistry, Gothenburg University, Sahlgren's
Hospital, S-413 45 Gothenburg, Sweden.
Received April 11, 1990; revision accepted November 28, 1990.
antigens are expressed by many cells of the lesion,
both macrophages and smooth muscle cells.67 However, it has not been possible to deduce the time
sequence of these inflammatory phenomena from
analyses of human surgical or autopsy material. We
have therefore turned to an animal experimental
model to investigate the temporal relation between
class II MHC expression, lymphocyte and monocyte
infiltration, and development of the atherosclerotic
lesion.
In rabbits, addition of moderate amounts of cholesterol to the diet results in rapid development of
arterial lesions.8-9 In the early stages of lesion formation, occasional foam cells are present in the subendothelial intimal space. The lesions progress into
fatty streaks and may with time even develop a
fibrous component of smooth muscle cells and extracellular matrix components, thus resembling the human atherosclerotic plaque.1011
We have studied the inflammatory and immunologic components of atherosclerosis in the diet-induced rabbit model. We present evidence for adhesion of monocytes and T lymphocytes to the arterial
intima at an early stage of hypercholesterolemia.
Expression of la antigen is high from the start of
746
Arteriosclerosis and Thrombosis
Vol 11, No 3 May/June 1991
TABLE 1. Antibodies Used in This Study
Antibody
RAM11
LI 1/135
12.C7
1.24
HHF35
2C4
Specificity
Type
Reference
Macrophages
Pan-T lymphocytes
Cytotoxic/suppressor
T lymphocytes
Leukocyte common
antigen
Smooth
muscle-specific
actin
MHC class Il/Ia
MAb Asc
MAblgG
MAb Asc
12
MAb Sup
14
MAb Asc
15
MAb Sup
16
13
14
MHC, major histocompatibility complex; MAb, monoclonal
antibody; Asc, ascites fluid; IgG, immunoglobulin G; Sup, supernatant from hybridoma cultures.
lesion formation. T lymphocytes are present, and
monocytes/macrophages dominate the fatty streaktype lesions in these rabbits.
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Methods
Animals
Four-month-old rabbits of the New Zealand White
strain were obtained from Lidkopings kaninfarm,
Lidkoping, Sweden. They were fed standard rabbit
chow supplemented with 0.3% cholesterol, and serum cholesterol was analyzed monthly by a standard
enzymatic procedure (Monotest Cholesterol, Boehringer Ingelheim, Ingelheim, F.R.G.). Groups of four
rabbits were killed after 1, 3, 6, and 10 weeks on the
diet. They were sedated with diazepam, anesthetized
with halothane, and fixed by perfusion with 1%
paraformaldehyde in 0.1 M phosphate buffer (pH
7.2) at a pressure of 100 cm H2O. Samples of the
descending part of the thoracic aorta were snap
frozen in liquid N2 as described.3-9
Antibodies
Monoclonal antibodies against cell type-specific
rabbit antigens were used for immunohistochemical
analysis. They are listed in Table 1. The Lll/135
hybridoma was obtained from the American Type
Culture Collection (Rockville, Md.) and grown in
Dulbecco's modified Eagle's medium (GEBCO, Paisley, Scotland) supplemented with 10% fetal bovine
serum, 100 units/ml penicillin G, 100 /ig/ml streptomycin, nonessential amino acids, glutamine, oxaloacetic acid, and insulin as described.13 Hybridoma cells
were grown in the mass culture system described by
Sjogren-Jansson and Jeansson,17 and immunoglobulins were purified from the supernatant by protein
A-Sepharose chromatography (Bio-Rad MAPS n,
Richmond, Calif.). All other antibodies were obtained
from the producer either as supernatants or as purified immunoglobulin (see Table 1). All antibodies
were used at optimal dilutions determined by checkerboard titrations on sections of rabbit spleen and
aorta. Staining of such sections was also performed to
verify the cellular specificity of each antibody.
Immunohistochemistry
Staining was performed essentially as described,37
except that alkaline phosphatase instead of peroxidase was used for immunoenzyme localization. In
brief, 10-p,m cryostat sections were fixed in ethanol,
incubated with monoclonal antibodies followed by
species-specific biotinylated F(ab')2 fragments of anti-mouse immunoglobulin G (IgG) (Amersham, Amersham, U.K.) and alkaline phosphatase-conjugated
avidin (Dakopatts, Copenhagen, Denmark). Antibody binding was visualized with an alkaline phosphatase substrate kit (AP substrate kit I, Vector,
Burlingame, Calif.). Controls included antibodies to
irrelevant antigens (species-specific anti-human leukocyte differentiation antigens) as well as omission of
primary antibody. The percentage of cells positive
with a certain antibody was determined by counting
100 cells in a segment from the surface to the internal
elastic lamina at the site of maximal lesion thickness.
Corresponding fields were chosen in serial sections.
Results
Rabbits were fed a diet supplemented with 0.3%
cholesterol for varying time periods ranging from 1 to
10 weeks. The changes in serum cholesterol levels
with time on diet are shown in Figure 1. After 10
weeks on the diet, the average serum cholesterol
concentration was 17.69±6.61 mmol/1 (mean±SD),
corresponding to 682±255 mg/dl. Small intimal foam
cell accumulations could be observed after 2-3
weeks on the cholesterol-rich diet, and macroscopically detectable lesions were found in rabbits treated
with cholesterol for 6 and 10 weeks.
We used a battery of cell type-specific monoclonal
antibodies to detect different hematopoietic cells in
these lesions (Table 1). RAM11 binds to a protein
that is expressed on monocyte-derived macrophages
in rabbits.12 It has recently been used to successfully
identify such cells in atherosclerotic lesions.12 Antibody 1.24 recognizes the majority of rabbit leukocytes
and appears to be analogous to the leukocyte common antigen in humans.14 Lll/135 is a pan-T lymphocyte marker, which recognizes T lymphocytes in
blood and tissues but does not cross-react with other
leukocytes or any other cell type.13 The cytotoxic/
suppressor subset of rabbit T lymphocytes is detected
by the CD8 equivalent, 12.C7.14 Finally, the 2C4
antibody binds to the rabbit homologue of the class II
MHC antigen.16
Occasional RAM11+ monocytes and Lll/135 + T
lymphocytes were seen at the endothelial surface
after 1-3 weeks of cholesterol feeding (Figure 2).
After 6 weeks on the diet, small clusters of cells were
observed in the intima (Figure 3). The majority of
them were always RAM11+ monocyte-derived macrophages (Figure 3, upper panel, Table 2). Smaller
amounts of LI 1/135 + T lymphocytes were also invariably present in these lesions (Figure 3, lower panel,
Table 2). Approximately two thirds of the macrophages expressed la, as judged from serial sections
Hansson et al
Inflammatory Cells in Rabbit Plaques
747
S-Cholesterol,
mmol/1
20
FIGURE 1. Serum (S-) cholesterol levels (mmol/l) in cholesterol-fed rabbits (mean±SD) as a function of days on
diet.
10
20
30
40
50
60
70
days on diet
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stained with RAM11 and 2C4 (Figure 3, middle
panel, Table 2). Occasional spindle-shaped cells that
were presumably intimal smooth muscle cells could
also be detected in these early lesions.
The lesions in rabbits fed cholesterol for 10 weeks
were of the fatty streak type. They were dominated
by lipid-laden foam cells, most of which expressed la,
and the macrophage marker RAM 11 (Figure 4,
Table 2). Expression of RAM11 was less intense in
the lower part of the lesion than in the subendothelium, and the 1.24 epitope was only expressed by
subendothelial macrophages, suggesting that there
may be phenotypic differences between macrophages
in different parts of the lesion.
Comparison of serial sections implied that most
of the Ia+ cells were RAM11+ macrophages. In
addition, there was a population of Ia + spindleshaped cells that were probably Ia+ smooth muscle
cells.
Lll/135 + T lymphocytes were also present in
these lesions but to a lower extent (Figure 4, Table
2). The frequency of T cells varied between regions
and was highest subendothelially and in the shoul-
FiGURE 2. T lymphocyte (arrow) adhering to endothelial
surface from a rabbit fed cholesterol for 2 weeks. Lll/135,
avidin-biotin—alkalinephosphatase stain, no counterstain. x88.
der regions. Only a small proportion of the cells
reacted with the 12.C7 monoclonal antibody, suggesting that very few CD8-type T lymphocytes were
present in the lesions.
Discussion
Rabbits respond to cholesterol feeding by developing arterial intimal lesions that resemble human fatty
streaks, that is, they largely consist of lipid-laden
foam cells.910 With time, these lesions may acquire a
fibrotic component consisting of smooth muscle cells
and extracellular matrix components and transform
into fibrous plaque-like lesions.11 Our present data
confirm the previous observation that the foam cells
that build up the early lesions are largely derived
from blood monocytes.8-912
The lesions of cholesterol-fed rabbits resemble
human atherosclerotic lesions in that they both contain large amounts of monocyte-derived macrophages. In contrast, the lesions that form after mechanical arterial injury in rats are dominated by
proliferating smooth muscle cells and contain very
few hematopoietic cells.1819 Human atherosclerotic
lesions exhibit a significant inflammatory component,
with large amounts of T lymphocytes as well as
macrophages3-5 and with a high frequency of la
antigen expression.6-7 The latter phenomenon suggests that an immune response may be taking place,
since la antigens (in humans, HLA-DR, -DQ, and
-DP) are pivotal for the recognition of foreign antigens by T lymphocytes.20 Our recent observation21
that T lymphocytes in human plaques express activation markers such as the interleukin-2 receptor further supports the idea that an immune response may
be taking place.
The similarities in macrophage accumulation between the human disease and the cholesterol-fed rabbit
model suggested to us that the latter could be useful
also for studies of inflammatory and immune aspects of
atherosclerosis. We have therefore analyzed monocytes/macrophages, T lymphocytes, and la expression in
748
Arteriosclerosis and Thrombosis
Vol 11, No 3 May/June 1991
FIGURE 3.
Monocyteslmacrophages and T lympho-
cytes (arrows) in la-expressing aortic intima of a
rabbit fed cholesterol for 6 weeks. Upper panel:
RAM11* monocyteslmacrophages; middle panel:
2C4+ la-expressing cells; lower panel: Lll/135+ T
lymphocytes. Hematoxylin counterstain. X160.
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TABLE 2. Frequency of Different Cell Types Defined by Monoclonal Antibodies in Intima and Lesions at Various Stages
Time on cholesterol diet
(weeks)
Cell type
T lymphocytes
Ia+ cells
Macrophages
MAb
LI 1/135
2C4
RAM11
10
7.4±5.6
41±14
73±13
5.7±4.7
62±6.8
63±4.8
Data represent percent antibody-positive cells in lesions of
different age (mean±SD, n=4 per group).
MAb, monoclonal antibody.
the early phases of atherogenesis in cholesterol-fed
rabbits. Immunohistochemical staining of serial cryostat sections was used to detect binding of cell typespecific monoclonal antibodies. The use of frozen
rather than paraffin-embedded sections was necessary
to avoid destruction of antigenic epitopes but inevitably
resulted in thicker sections and suboptimal morphology. This, in turn, made it difficult to compare reactivity
with different antibodies on the single-cell level. Instead, conclusions concerning macrophage la expression, for example, had to be based on comparison of
similar fields and on frequencies of positive cells.
*.
FIGURE 4. T lymphocytes (arrows) in fatty streaklike lesion of a rabbit fed cholesterol for 10 weeks.
LI 1/135 monoclonal antibody, hematoxylin counterstain. x80.
Hansson et al Inflammatory Cells in Rabbit Plaques
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Adhesion of monocytes and T lymphocytes to the
aortic surface was detectable already after 1 week of
cholesterol feeding, and intimal lipid-laden monocyte-derived macrophages were detected at 3 weeks.
Small lesions could be observed at 6 weeks, and by 10
weeks, large fatty streaks were present throughout
the aorta.
la antigen, the major rabbit class II MHC antigen,
was present on the majority of cells in the lesions at
all time points studied. la staining was strong and
uniform and could be detected intracellularly as well
as on cell surfaces, suggesting a high synthesis of la
protein. These observations indicate that la is expressed in experimental atherosclerosis from the
earliest detectable stage and onwards.
The majority of la-expressing cells were macrophages that expressed the RAM11 antigen and, at a
lower frequency, the 1.24 antigens. la antigens are
expressed constitutively in this type and are upregulated by y-interferon.1'22 This lymphokine can also
induce de novo expression of class II MHC molecules
in smooth muscle cells.6'19'23-24 Occasional la-expressing smooth muscle cells appeared to be present, but
the frequency of these cells could not be determined
since the smooth muscle contribution to the experimental lesions under study was very low.
The function of la molecules is to participate in
antigen recognition. T lymphocytes are activated by a
complex of foreign antigen and la molecule on the
surface of the antigen-presenting cell.20 The high
frequencies of la-expressing cells and T lymphocytes
indicate that the potential for antigen presentation,
and thus, for an immune response, is present in
dietary-induced experimental atherosclerosis.
The use of the rabbit T cell-specific monoclonal
antibody Lll/135 13 permitted us to detect T lymphocytes in rabbit vascular tissue by immunohistochemistry. At early stages of hypercholesterolemia, T cells
could be seen adhering to the endothelial surface
before any fatty streaks had developed. In fully
developed lesions, T cells constituted 5.7% of the cell
population. Although this frequency is slightly lower
than that of T cells in complicated human atherosclerotic plaques,3 it is possible that these cells may play
a biologically significant role in the development of
the rabbit lesions. Any further comparisons between
the human plaques previously studied by us and
others and the present rabbit model are obviously
limited by the differences in age of lesions and
species.
The findings of both T lymphocytes and la-expressing cells that may have the capacity to present
antigens to cells raise the possibility that a specific
immune response is taking place in cholesterolinduced atherosclerosis. It has been shown that modified lipoproteins such as glycosylated low density
lipoprotein can induce immune responses,25 but
there is no reason to assume that this particular
response was taking place in the present model.
In addition to their role in antigen recognition, T
lymphocytes are important producers of paracrine
749
factors that can regulate the functions of surrounding
cells.26 The activated T lymphocyte secretes -y-interferon, which can inhibit cell proliferation in vascular
smooth muscle cells27 and endothelial cells28 and also
activate macrophages.26 It also produces lymphotoxin, an analogue of tumor necrosis factor,29 and
we24 recently found that this protein modulates -y-interferon-induced gene expression in smooth muscle
cells.
Finally, it has recently been shown that type I
interferon, which is produced by virally infected cells,
can suppress lesion formation in cholesterol-fed rabbits.30 Although the secretory patterns and spectra of
effects differ in many respects between the two types
of interferons, they share some effects. It is therefore
possible that the effect of parenterally administered
type I interferon could, to some extent, mimic the
effect of endogenous type II (y-)interferon.
In conclusion, the present study shows that laexpressing cells, largely of monocytic origin, and T
lymphocytes are abundant in the aortic lesions of
cholesterol-fed rabbits. Both cell types were present
at all stages, initially adhering to the surface at
prelesion stages and later on as major components
of the fatty streak-like lesions. These observations
support our hypothesis that cells of the immune
system participate in the formation of atherosclerotic lesions. They open up the possibility to use the
cholesterol-fed rabbit for studies of immunologic
aspects of atherosclerosis.
Acknowledgments
We thank Marianne Haden and Monika Hellstrand for excellent technical assistance and Allen
Gown, Tom Kindt, Julie Knight, and Walter DeSmet
for kindly providing antibodies.
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KEY WORDS • hypercholesterolemia • histocompatibility
antigens • monoclonal antibodies • T lymphocytes •
experimental atherosclerosis • macrophages • rabbits
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Immunohistochemical detection of macrophages and T lymphocytes in atherosclerotic
lesions of cholesterol-fed rabbits.
G K Hansson, P S Seifert, G Olsson and G Bondjers
Arterioscler Thromb Vasc Biol. 1991;11:745-750
doi: 10.1161/01.ATV.11.3.745
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