© 1999 American Heart Association, Inc.
Original Contributions |
The Role of Interleukin 12 in the Development of Atherosclerosis in ApoE-Deficient Mice
From the Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica (T-S.L., C-C.P., L-Y.C.), and Graduate Institute of Immunology, College of Medicine, National Taiwan University (H-C.Y.), Taipei, Taiwan, R.O.C.
Correspondence to Lee-Young Chau, Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, R.O.C. E-mail lyc{at}mail.ibms.sinica.edu.tw
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Abstract |
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Abstract—The cytokine profile of atherosclerotic aortas from apoE-deficient mice was assessed by reverse transcriptase-polymerase chain reaction. The results clearly showed that the expression of mRNA for IL-12p40 was evident in aortas from 3-month-old apoE-deficient mice. The mRNA for IL-10 was detected in aorta from these mice at the age of 6 months, indicating that expression of IL-12 is earlier than that of IL-10 in these animals. Concurrent with IL-12p40, the mRNA for the T-cell cytokine IFN-
, but not IL-4, was detected in aortas of mice at young and old
ages. Both in situ hybridization and immunostaining
further demonstrated the localization of IL-12 in macrophages
of atherosclerotic lesions. Immunohistochemistry also demonstrated the
expression of costimulatory molecules B7–1 and B7–2 in
macrophages, suggesting that activation of T lymphocytes by
macrophages may occur via surface antigens in lesions. When the
immunoglobulin isotype of the antioxidized LDL antibodies in sera of
apoE-deficient mice was determined, it revealed that both IgM and IgG
were present. Furthermore, IgG2a is predominant and comprises
50% of the antioxidized LDL IgG in sera from young mice (3 months),
but decreased to lower levels (35%) in older mice (6 months). Daily
administration of IL-12 led to an increase in serum levels of
antioxidized LDL antibodies and accelerated
atherosclerosis in young apoE-deficient mice compared
with control mice injected with PBS alone. Taken together, these data
suggest that IL-12 plays an active role in regulating the immune
response during the early phase of atherosclerosis in
apoE-deficient mice.
Key Words: interleukin 12 • atherosclerosis • oxidized LDL
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Introduction |
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Atherosclerosis is a chronic pathological process which eventually leads to the occlusion of large arteries.1 Although the pathogenesis of this disease is not yet fully understood, histological assessment has shown that monocytes/macrophages and T lymphocytes, are present in atherosclerotic lesions, implicating the involvement of the immune system in atherogenesis.2 3 4 5 6 This notion is further supported by a number of observations. For example, the deposits of complement factors and immunoglobulins are localized in atherosclerotic lesions.7 8 9 Autoantibodies specific for oxidized LDL are present in both sera and lesions of humans and experimental animals developing atherosclerosis,10 11 12 13 and both MHC class I and II molecules are expressed on endothelial cells and smooth muscle cells of lesions.3 14 15 Furthermore, immunosuppression of rabbits and mice,16 17 or MHC class I-deficient mice,18 developed more severe atherosclerosis after feeding with a high-cholesterol diet. Nevertheless, how the local immune reaction is initiated and propagated to affect the progression of atherosclerotic lesions remains unclear.
Recently, a study by Uyemura et al demonstrated that IL-12 and IL-10,
which are 2 principal cytokines influencing the differential
development of T lymphocytes to the Th1 and Th2
phenotypes,19 20 are expressed in
macrophages of human atherosclerotic lesions.21
Because the expression of IFN-, but not IL-4, was preferentially
detected in lesions, it was suggested that the Th1 cell is the
predominant T lymphocyte present in atherosclerotic plaques.
However, the interplays between different cytokines as well as
the interactions between macrophages and T cells in relation to
the development of atherosclerosis is not yet
well-characterized. In an attempt to understand the mechanisms by which
the immune response is triggered during the development of
atherosclerosis, we used apoE-deficient mice, which
spontaneously develop atherosclerosis with features
similar to those seen in humans,22 23 24 25 as the animal model
to investigate the potential function of IL-12 in atherogenesis. In
addition to its primary role in initiation of cell-mediated immunity,
IL-12 has been shown to influence the humoral immune response by
affecting the immunoglobulin isotype switch to IgG2a in
mice.26 27 To examine whether IL-12 exerts an effect on
the humoral immune response in apoE-deficient mice, we also assessed
the subclass distribution of the antioxidized LDL IgG in these mice,
which will provide important evidence to support the pathological
relevance of IL-12 in this disease.
Because 1 major function of the macrophage is to serve as an antigen-presenting cell (APC) to T cells, it is conceivable that macrophages can regulate T cell activation via interactions between cell surface molecules. It is known that one of the early events occurring in the subendothelial space prone to the formation of atherosclerotic lesions is the oxidation of LDL.28 The oxidized LDL is subsequently taken up by macrophages via the scavenger receptors to form the lipid-laden foam cells. A recent study by Stemme et al29 has shown that T-cell clones isolated from human atherosclerotic lesions are activated by oxidized LDL in the presence of autologous APCs. It is envisaged that the antigenic peptides derived from oxidized LDL processed in macrophages may be recognized by T cells in atherosclerotic lesions via TCR/MHC complex engagement. In addition to the signal generated through the binding of the MHC/antigen peptide complex to the T-cell receptor, the costimulatory signal delivered by the interaction of the CD28 receptor on T cells and its counter ligand B7 molecules on APC has been shown to be required for the optimal activation of T lymphocytes.30 31 In view of the importance of B7 molecules in the activation of T cells by APCs, the expression of the B7 molecules, B7–1 and B7–2, on macrophages of atherosclerotic lesions was also examined.
In the present report, we demonstrate that IL-12 is expressed in aortas of young mice as early as 3 months, suggesting that IL-12 may play an active role in the initial phase of atherosclerosis. The detection of the costimulatory molecules B7–1 and B7–2 on macrophages further supports the regulatory role of macrophages in activation of T lymphocytes in plaques. The observation that IgG2a is the predominant subclass of antioxidized LDL IgG in the sera of these mice suggests that IL-12 plays a role in the modulation of the humoral response. The pathophysiological link between IL-12 and the development of atherosclerosis was further supported by the observation that daily injections of IL-12 into mice for a month augmented the progression of atherosclerosis and the production of antioxidized LDL antibody in these mice.
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Materials and Methods |
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Animals
Homozygous apoE-deficient mice on C57BL/6J background (C57BL/6J-Apoetm1Unc) were purchased from the Jackson Laboratory (Bar Harbor, Me). The C57BL/6 mice were from the National Animal Center of Taiwan. The mice fed with normal chow diet were killed between 3 and 6 months of age. For the assessment of IL-12 treatment on lesion formation, male C57BL/6 or apoE-deficient mice, 4 months of age, were divided into 2 groups. One group received intraperitoneal injections of PBS containing 1% mouse serum daily for 30 days; the other group received 300 ng recombinant murine IL-12 (R & D Systems) in the same buffer daily for 30 days. The animals were killed and blood was collected from the vena cava. After perfusion of the aortic tree with ice-cold PBS, the heart, aorta, spleen, and liver were dissected, removed, quickly frozen in liquid nitrogen, and stored at -70°C for RNA extraction. To prepare tissue sections for in situ hybridization and immunocytochemistry, the entire aorta was removed intact and immersed in fresh 4% paraformaldehyde for 3 hours, embedded in paraffin, and cut in 5-µm sections. Before use, sections were deparaffinized in xylene and rehydrated in graded ethanols. To prepare cryosections for lipid staining, the paraformaldehyde-fixed aorta segments were incubated at 37°C in 5% gelatin for 2 hours, and10% gelatin for 2 hours, followed by incubation 25% gelatin overnight. The tissues were allowed to harden at 4°C then were stored in 10% paraformaldehyde at 4°C. Before sectioning of the tissue, excess gelatin was removed and the tissue was embedded in OCT compound (Tissue-Tek) and frozen in liquid nitrogen.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA from each tissue specimen was isolated according
to the method described by Chomczynski and Sacchi.32 The
quality and purity of the RNA was determined by absorbance at 260 nm
and the ratio of 260 nm to 280 nm. The integrity of the RNA was
monitored by ethidium bromide staining of the 28S and 18S ribosomal
RNAs analyzed by electrophoresis on 1% agarose gel. The cDNA
was synthesized from 2 µg of total RNA by reverse transcription using
0.2 µg random hexamers (Promega) and 200 U Moloney Murine Leukemia
Virus reverse transcriptase (Gibco Life Technologies) in the presence
of 0.4 mmol/L of each deoxynucleotide triphosphate,
10 mmol/L dithiothreitol, and 10 U RNasin (Promega) in a final
volume of 20 µL. After 1 hour incubation at 37°C, the reaction was
terminated by heating at 95°C for 5 minutes, immediate cooling on
ice, followed by dilution with DEPC-H2O to 100
µL. Aliquots of the synthesized cDNAs were then used for PCR. The
primers used for analysis of ß-actin were
5'-TGGAATCCTGTGGCATCCATG-3' (sense), and 5'-AACGCAGCTCAGTAACAGTCC-3'
(antisense); for IL-12p40, 5'-CAGAAGCTAACCATCTCCTGGTTTG-3' (sense), and
5'-TCCGGAGTAATTTGGTGCTTCACAC-3' (antisense); for IL-10,
5'-CCAGTTTTACCTGGTAGAAGTGATG-3' (sense), and
5'-TGTCTAGGTCCTGGAGTCCAGCAGAC-TCAA-3'
(antisense); for IFN-, 5'-AGCGGCTGACTGAACTCAGATTG-3' (sense),
and 5'-GTCACAGTTTTCAGCTGTATAGG-3' (antisense); and for IL-4,
5'-CGAAGAACACCACAGAGAGTGAG-3' (sense), and
5'-GACTCATTCATGGTGCAGCT- TATCG-3' (antisense). All PCR
amplifications were performed in a 25-µL reaction mixture containing
0.5 mmol/L each of sense and antisense primers, 0.2 mmol/L
deoxynucleotide triphosphates, and 1 U Thermus aquaticus
DNA polymerase (Promega). The reaction proceeded for 30 cycles in a
programmable DNA thermal cycler (Perkin-Elmer Cetus Thermocycler 480),
with denaturation at 94°C for 1 minute, annealing at 55°C for 1
minute, and extension at 72°C for 1 minute, followed by an extension
at 72°C for 7 minutes. The PCR products were visualized by
electrophoresis on 1.5% agarose gel containing ethidium bromide. To
confirm the PCR products of expected cytokines, amplified
cDNA was electrophoresed on a 1.5% agarose gel and excised from the
gel. The DNA was purified from the gel with a DNA purification kit
(Qiagen). DNA sequencing was performed on an ABI 373A automated DNA
sequencer. Sequence analysis was conducted using the GenBank
Database in NCBI (USA).
Histological Staining
Histology was examined on paraffin sections with hematoxylin and
eosin (H&E) staining. Sections were stained with Mayer's hematoxylin
solution (Sigma) for 3 minutes, washed, and stained with eosin Y
(Sigma) for 10 minutes. Slides were viewed by light microscopy. For
lipid staining, the cryosections were fixed with 50% alcohol for 10
minutes, then stained with 1% Sudan III (Sigma) for 1 hour at room
temperature. The sections were briefly washed with 50% alcohol and
counterstained with hematoxylin.
In Situ Hybridization
IL12p40 cDNA, which contains 394 bp, was subcloned into pCRII
vector (Invitrogen). The antisense and sense IL12p40 RNAs were then
synthesized by T7 RNA polymerase and SP6 RNA polymerase, respectively,
and labeled with digoxigenin (DIG)-UTP according to manufacturer's
instructions (Boehringer Mannheim Biochemica). Paraffin
sections were treated with 1 µg/mL proteinase K for 15 minutes at
37°C and acetylated with 0.25% acetic anhydride in 0.1 mol/L
triethanolamine and 0.9% NaCl for 10 minutes. Sections were then
washed with 2x SSC (1x SSC consists of 150 mmol/L NaCl, 15
mmol/L Na-citrate, pH 7.0), dehydrated with increasing concentrations
of ethanol, and air dried for 30 minutes. Before hybridization,
sections were prehybridized in a humid chamber with 100 µL
prehybridization solution containing 5x SSC, 5x Denhardt's solution,
50% deionized formamide, 250 µg/mL yeast tRNA, 250 µg denatured
salmon sperm DNA, and 4 mmol/L EDTA for 3 hours at 50°C.
Hybridization was performed at 42°C for 16 to 24 hours in a humid
chamber with 25 µL/section prehybridization solution containing 10
ng/µL RNA probe. After the hybridization, the sections were washed at
42°C twice in 2x SSC, once each in 0.2x SSC and 0.1x SSC, for 15
minutes per wash. Sections were then blocked for 30 minutes with PBS
containing BSA, incubated with alkaline phosphatase-conjugated anti-DIG
antibody for 30 minutes, and detected with color solution containing
nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl-phosphate for
90 minutes according to the manufacturer's instructions. After
completion of the color reaction, each section was counterstained with
carmine.
Immunocytochemistry
Immunostaining was carried out using the
following antibodies: rat anti-mouse Mac-1 monoclonal antibody
(Boehringer Mannheim Biochemica); rabbit anti-human CD3
polyclonal antibody (Dako); rat anti-mouse CD80 (B7–1) monoclonal
antibody (PharMingen); rat anti-mouse GL1(B7–2) monoclonal antibody
(PharMingen); goat anti-mouse IL-12 polyclonal antibody (R&D Systems).
The rabbit anti-human CD3 polyclonal antibody exhibits cross-reactivity
to mouse antigen.33 Tissue sections were pretreated with
3% H2O2 for 10 minutes at
room temperature to exhaust endogenous peroxidase
activities. Unless specified, sections were blocked in PBS containing
1% BSA and 1% goat serum at 37°C for 30 minutes, followed by
treatment with the primary antibody for another 30 minutes. Sections
were then incubated with horseradish peroxidase–conjugated secondary
antibody for 30 minutes or biotin-conjugated secondary antibody
followed by peroxidase-conjugated streptavidin for 30 minutes each at
37°C. After 3 washes in PBS, color was developed with 0.1%
3,3'-diaminobenzidine (DAB) and 0.01%
H2O2 in Tris-HCl, pH 7.0.
In some experiments, color was developed with 0.1% DAB in 0.1 mol/L
Na-acetate, pH 6.0, containing 0.2% ß-D-glucose, 130 U
of glucose oxidase, and 2.5% NiCl3 to enhance
the signals.34 A negative control was performed by
incubating the sections with secondary antibody only (omission of
primary antibody).
Measurement of Oxidized LDL Antibody
Human LDL was isolated as described previously.35
Oxidized LDL was prepared by incubating LDL (1 mg/mL) with 5
µmol/L CuCl2 in PBS overnight at 37°C. An
enzyme-linked immunosorbent assay technique was used to determine
antibody titers. Ninety-six–well polyvinylchloride microtitration
plates were coated with native LDL (5 µg/mL) or oxidized LDL (5
µg/mL) in PBS overnight at 4°C followed by blocking with 1% BSA
for 2 hours at room temperature. The plates were freshly prepared
before all binding assays. Serial dilutions of sera from apoE-deficient
mice were added into duplicate wells and incubated for 2 hours at room
temperature. The amount of immunoglobulin (Ig) bound was quantified by
incubating with a goat anti-mouse IgM conjugated with alkaline
phosphatase (Sigma) or a rabbit anti-mouse IgG conjugated with alkaline
phosphatase (Sigma) at room temperature for 2 hours. After 3 washes,
the alkaline phosphatase activity was determined using
p-nitrophenyl phosphate as substrate. Color development was
measured at 405 nm. Specific antibody to oxidized LDL was defined as
the difference between 405 nm readings obtained from the binding to
oxidized LDL and to native LDL. Serum IgG isotypes of oxidized LDL
antibodies were determined using a MonoAb-ID EIA KIT (Zymed
Laboratories). In brief, 50 µL of sera (1:40) from mice was added
into wells of the antigen-coated plate and incubated for 2 hours at
room temperature. After washing, subclass-specific rabbit anti-mouse
Igs were added and incubated for another 2 hours. After washing,
incubation continued with a goat anti-rabbit IgG conjugated with
alkaline phosphatase. Color was developed and data were calculated as
described above. The standard curves for Ig isotypes were determined
using plates coated with goat anti-mouse Igs (light chains) antibody
and known concentrations of mouse Ig isotypes which were then assayed
as described above for serum samples. The optical density readings of
mouse sera were converted to Ig concentrations by extrapolation from
the standard curves for each isotype. To analyze the total Ig
isotypes, plates coated with goat anti-mouse Igs were incubated with
serum samples (1:500 dilution), followed by incubation with
subclass-specific rabbit anti-mouse Igs antibodies as described
above.
Quantification of Aortic Atherosclerotic Lesions
For the quantitation of atherosclerotic lesions of
apoE-deficient mice, 45 serial sections from aortic sinus or arch of
each mouse were collected. Total 10 to 12 sections sampled from every 4
consecutive sections were H&E stained and the photomicrographs were
taken. The cross-sectional area of a given photomicrograph was
analyzed using a computer imaging graphic software (NIH Image
1.5). The lesion size of a particular location was then calculated from
the average of the area quantitated from the 10 to 12 sections.
Statistical analysis of the results was performed using
Student's t test for grouped data.
Lipoprotein Analysis
Plasma samples of 6 mice were pooled and subjected to density
gradient ultracentrifugation for the fractionation of
lipoproteins. SDS-PAGE analysis was performed on a 4% to 15%
gradient gel. Protein was visualized by Coomassie Blue R-250
staining.
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Results |
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Immunocompetent Cells in Atherosclerotic Lesions of ApoE-Deficient Mice
It has been shown that the apoE-deficient mice develop atherosclerotic lesions in aortic roots as early as 3 months.24 25 These lesions gradually extend throughout whole aortas and coronary arteries in older animals.24 25 With the use of antibodies specific against Mac-1 of macrophages and the CD3 antigen of T lymphocytes, immunostaining of the aorta sections of apoE-deficient mice revealed that macrophages are the predominant cell types present in early lesions as shown in Figure 1C
. The presence of extracellular lipid
and intracellular lipid accumulation in macrophages was also
demonstrated by Sudan III staining (Figure 1A and 1B). Likewise,
the infiltration of T lymphocytes in atherosclerotic lesions was also
evident (Figure 1E and 1F).
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Cytokine Expression in Different Tissues From
ApoE-Deficient Mice
The cytokine gene expression as analyzed by RT-PCR
in different tissues of apoE-deficient mice of young (3 months) and
older (6 months) ages was performed. As shown in Figure 2, mRNA encoding for IL-12p40, which is
one of the subunits of IL-12, and its expression have been shown to be
correlated with the secretion of the active
cytokine,19 was evident in aortas of apoE
-deficient mice at the age of 3 months. The expression of mRNA for
IFN-, but not IL-10 and IL-4, was also observed in aortas of these
mice. Because these cytokines were not detectable in aortas of
control C57BL/6 mice of the same age, they were presumably products
derived from the atherosclerotic lesions in aortas of apoE-deficient
mice. When the cytokine pattern was examined in older
apoE-deficient mice, it was very interesting to find that the
expression of IL-10 mRNA was evident in aortas. The IL-4 mRNA, however,
remained undetectable in aortas of the older mice. These results
clearly demonstrate the preferential expression of IL-12 and IFN- in
aortas of apoE-deficient mice in the earlier stage of the disease.
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Localization of IL-12 in Macrophages of
Atherosclerotic Lesions
To further confirm the cellular source of the IL-12p40 mRNA, in
situ hybridization was performed. As illustrated in Figure 3A and 3B, the IL-12p40 mRNA was detected
in macrophage-derived, foam-cell–like regions but not in
the spindle-shape cells of the thickened intima. Hybridization using
sense IL-12p40 RNA probe was virtually negative (Figure 3C). The
presence of IL-12 protein in lesions was further examined by
immunostaining of the serial sections with antibodies
to murine IL-12 and macrophage surface antigen. As demonstrated
in Figure 4, the immunoreactivity with
IL-12 antibody was localized to the macrophage-rich area.
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Expression of B7–1 and B7–2 Molecules on Macrophages of
Atherosclerotic Lesions
To assess whether macrophages in atherosclerotic lesions
may function as the antigen-presenting cells to activate
the adjacent infiltrated T cells after the interaction of surface MHC
molecule and TCR, the expression of B7 molecules, which are required
for generating costimulatory signal for T-cell activation on binding to
CD28 receptor of T cells, was examined. As shown in Figure 5, immunostaining of the
serial sections of an atherosclerotic lesion with specific antibodies
to Mac-1, B7–1, and B7–2 revealed that both B7–1 and B7–2
immunoreactivities were detectable in regions that were enriched in
macrophages. This observation suggests that macrophages
may regulate the T-cell activation and subsequent immune response via
surface antigens in atherosclerotic lesions.
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Immunoglobulin Isotype Distribution of Antioxidized LDL Antibodies
in Sera of ApoE-Deficient Mice
The presence of antioxidized LDL antibodies in sera and lesions
was previously demonstrated in humans with
atherosclerosis.10 11 A similar
observation was later reported in apoE-deficient mice.13
As shown in Figure 6A, both IgM and IgG
to oxidized LDL were detected in sera of these mice. Determination of
the subclasses of oxidized LDL IgG revealed the predominance of IgG2a,
which represented 50% of the total antioxidized LDL IgG
in sera of young mice. The subclasses IgG2b and IgG1 accounted for
another 30% and 15%, respectively, whereas IgG3 was only 5%.
However, when the sera of the older mice (6 months) were examined, the
amount of IgG2a had declined to 35%, which was accompanied by an
increase in IgG3 13%, with no significant changes in both IgG2b (33%)
and IgG1 (18%) (Figure 6B). Because the level of total IgG2a in
these mice was not significantly changed with age (Figure 6C),
the decrease in antioxidized LDL IgG2a in older mice was not due to an
alteration in the abundance of this subclass.
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Augmentation of Atherosclerosis in Mice
Administered Recombinant IL-12
To further elucidate the role of IL-12 in the progression of
atherosclerosis, young mice were injected with
recombinant murine IL-12 daily for 30 days, and the effects on the
severity of the atherosclerotic lesions and titers of antioxidized LDL
antibodies were examined. C57BL/6 mice treated with IL-12 did not
develop atherosclerotic lesions (data not shown), indicating that IL-12
alone is not sufficient to induce atherosclerosis in
normal animals under these experimental conditions. When apoE-deficient
mice were examined, the body weight of mice treated with IL-12 was not
significantly different from that of control littermates (26.8±1.8 g
versus 27.2±1.7 g, n=9). Likewise, the serum cholesterol
level of IL-12-treated animals was comparable with that of control mice
(614.2±86.6 mg/dL versus 684.0±125.1 mg/dL, n=9). SDS-PAGE
analysis of the lipoproteins isolated from plasma of these
animals did not reveal significant changes in levels of apoB, apoAIV,
and apoAI after IL-12 treatment (Figure 7). However, when the area of
atherosclerotic lesions was assessed, the IL-12-treated group had
lesion areas that were >100% larger that of the control group in the
aortic sinus and arch, respectively (P<0.005) (Figure 8). Furthermore, the lesions of the
IL-12-treated group of animals were more advanced and contained more
CD3-positive cells, as shown in Figure 9A. Examination of cytokine gene
expression by RT-PCR revealed that the expression of IFN- mRNA in
atherosclerotic aorta from IL-12-treated apoE-deficient mice was
substantially higher (Figure 9B), indicating that the
CD3-positive cells detected are primarily Th1 cells. The effect of
IL-12 on the humoral immune response was also assessed. As shown in
Figure 10, the antioxidized LDL IgM and
IgG2a, but not other isotypes, detected in sera of the IL-12-treated
mice, were also significantly higher than those of the control animals
(P<0.025). Because the level of antioxidized LDL antibodies
in C57BL/6 mice are significantly lower than that of the apoE-deficient
mice, the effect of IL-12 on antioxidized LDL antibodies in C57BL/6
mice was not considered significant.
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Similar to observations in humans and rabbits, immunocytochemistry revealed that macrophages and T lymphocytes were present within the atherosclerotic lesions of apoE-deficient mice. These lipid-laden foam cells originated from the Mac-1 positive macrophages and appear to be the predominant cell types in early lesions. Infiltrated lymphocytes, as detected by immunostaining with specific antibody for CD3 surface antigen, were also present in lesions from these mice, which was consistent with recent reports by others.36 37 The coexistence of macrophages and T lymphocytes in lesions of apoE-deficient mice makes these animals a superb murine model for studying the role of the immune system in the development of atherosclerosis. Because the cytokines derived from these cells are important for immune function, we examined the cytokine profile in aortas of apoE-deficient mice using a highly sensitive RT-PCR method. Our data showed that the mRNAs encoding IL-12p40 and IFN-
were readily detectable in aorta of 3-month-old
apoE-deficient mice. The mRNA for IL-10 was not evident in younger mice
but was detectable in older mice. Furthermore, the level of message
encoding IL-4 was undetectable in aorta from either young or old mice.
These results are consistent with a recent report by Uyemura et
al21 who demonstrated the expression of IL-12 and IFN-
in human atherosclerotic lesions. In their study, the expression of
IL-10 mRNA was also detected in some of the human plaques. Furthermore,
an in vitro cell culture study showed that IL-10 inhibits the
LDL-induced IL-12 synthesis in monocytes, suggesting that the balance
between IL-12 and IL-10 produced in lesions may influence the immune
response in atherosclerosis.21 Together
with previous observations that T lymphocytes in human atherosclerotic
lesions are activated and produce IFN- and
IL-23 38 (which are the characteristics of Th1 cells), the
coexpression of IL-12 and IFN- in atherosclerotic lesions implies
that the Th1-mediated immune response is involved in the pathogenesis
of this vascular disease. In addition to cytokines, activation of T lymphocytes by macrophages can be mediated through the interactions between surface molecules. It is theorized that the degradation of oxidized LDL by macrophages may result in the generation of some antigenic epitopes on small peptides carrying oxidation-modified amino acid residues, such as malondialdehyde-lysine,39 which then activate T lymphocytes via MHC-TCR engagement. Although direct evidence is lacking, the recent report that T-cell clones isolated from human atherosclerotic lesions are activated by oxidized LDL29 may be the first evidence to support this notion. Because full activation of T cells requires signals from the activation of both TCR and coreceptor CD28, we were interested in examining the expression of the ligand for CD28, B7 molecules, on macrophages of these lesions. The immunostaining results clearly demonstrated that both B7–1 and B7–2 molecules were present on macrophages, further supporting the potential regulatory role of macrophages on the immune function of T lymphocytes in atherosclerotic lesions. Recently, evidence has accumulated which demonstrates that IL-12 synergizing with CD28/B7 interaction is important for the activation of quiescent T cells and induction of Th1 cells.40 41 It is conceivable that in coordination with B7/CD28 interaction and the presence of IL-12, T cells present in lesions preferentially undergo differentiation toward Th1 type cells, which may then assist with the activation of B cells and the production of antioxidized LDL antibodies.
Experiments were further conducted to investigate the potential role of IL-12 in humoral immune response in atherosclerosis. When the distribution of the isotypes of antioxidized LDL IgG in the sera of apoE-deficient mice was examined, we found that all 4 types of IgG subclasses were present. Furthermore, a higher titer of IgG2a compared with other subclasses was observed in younger mice. This result was consistent with a recent study by Zhou et al42 who reported the preferential production of IgG2a isotype to MDA-LDL in the early phase of the disease in apoE-deficient mice fed with chow diet. These observations suggest that the humoral response was primarily regulated by the cytokines favoring Th1 cell development in the early phase of atherosclerosis. However, the predominance of IgG2a becomes less evident in older mice. Zhou et al42 were able to show that, in severe hypercholesterolemia induced by feeding a high-cholesterol diet to apoE-deficient mice, the expression of IL-4 as well as a switch to Th2-dependent IgG1 isotype was evident, indicating that the cytokine expression and immune response in these animals are subjected to modulation by hypercholesterolemia. This was also revealed in a study43 showing that immunodeficiency by Rag-1 gene knockout in apoE-deficient mice reduced the foam cell lesion formation in these animals when they were placed on a chow diet, supporting the theory that immune response plays a role at least in the early stage of lesion development. However, this effect was eliminated in mice that were fed a high-cholesterol diet to accelerate lesion development. Similar results were reported in immunodeficient Rag-2/apoE double-gene knockout mice who were placed on a cholesterol-enriched diet for 3 months; they developed fibrous plaques to the same extent as apoE-deficient mice.44 It will be of great importance to discover whether the switch to Th2 response after the high-cholesterol feeding in these mice was associated with the reduced impact of the immune system on lesion development. Because the level of serum cholesterol did not fluctuate significantly between 3- and 6-month-old apoE-deficient mice,24 25 the underlying mechanism responsible for the alteration in antioxidized LDL IgG isotypes with age is unclear. Whether IL-10 and/or other cytokines produced in older animals has an impact on the immunoglobin isotype switch in the later stage of the disease awaits further investigation. A recent study on the isotypes of oxidized LDL antibodies in humans demonstrated that both IgG1 and IgG3 predominate.45 Because the effect of Th1/Th2 cytokines on immunoglobulin isotypes observed in the murine system is not yet established in humans, the results from humans and mice are not comparable. Nevertheless, it is of great interest to note that the IgG1/IgG3 and IgG2a/IgG2b are the most potent immunoglobulin subclasses to activate the classical complement pathway in humans46 and mice,47 respectively.
When the young apoE-deficient mice were injected with recombinant
IL-12 daily for a month, the progression of
atherosclerosis was accelerated. The infiltration of
CD3-positive T cells and the expression of IFN- mRNA were markedly
increased in lesions of IL-12-treated animals. Apparently, this
observation is quite different from the report by Roselaar et
al,37 who demonstrated that T-cell density tends to
decrease with disease progression in apoE-deficient mice. Because the
mice in their study were placed on a high-cholesterol diet
which would promote the T-cell response toward the Th2
type,42 it is conceivable that the type of immune
activation as well as the effect on lesion progression in those animals
is different from that of the IL-12-treated animals. Whether the
density of T cells in atherosclerotic lesions is influenced by the
Th1/Th2 ratio remains to be clarified. In conjunction with the severity
of the disease, the relative abundance of the antioxidized LDL IgG2a
subclass in IL-12-treated mice was significantly higher than that in
control mice. These results clearly demonstrate that IL-12 augments the
immune response of the T cells and modulates the subsequent humoral
antibody production in atherosclerosis in
experimental animals. It is generally believed that this effect of
IL-12 is mediated by the production of IFN- by Th1 cells. In
addition to its role in regulating the humoral immune response, IFN-
has also been shown to up-regulate the expression of VCAM-1, MHC II,
and scavenger receptor in vascular cells,48 49 50 which
conceivably would exacerbate the lesion progression. This may explain
the observation that the T cell–independent IgM titer to oxidized LDL
was also significantly increased in IL-12-treated mice. Apparently, the
present observation was consistent with a recent study by
Gupta et al,51 who showed that apoE/IFN- receptor
double-knockout mice exhibited substantial reduction in lesion
formation. The detrimental role of IFN- in the pathogenesis of
arterial disease was also demonstrated in a study showing
that IFN- deficiency prevents coronary
arteriosclerosis in transplanted mouse
hearts.52 In apoE/IFN- receptor double-knockout mice,
the expression of atheroprotective apoA-IV was found to be
up-regulated, suggesting that IFN- may promote
atherosclerosis through effects on vascular walls and
plasma lipoproteins.51 However, in the present study
we did not find a significant alteration in the level of apoA-IV in
serum lipoproteins of apoE-deficient mice after IL-12 treatment. It is
possible that the amount of IFN- produced in control apoE-deficient
mice already caused maximal suppression of apoA-IV gene expression. In
contrast to its ability to protect against infectious diseases and
tumors, IL-12 apparently has a deleterious effect on vascular
atherosclerotic disease. This negative role of IL-12 has also
been reported recently in other murine disease models. Administration
of IL-12 accelerates autoimmune diabetes in NOD mice53 and
glomerulonephritis in MRL/lpr mice,54 and induces severe
arthritis in DBA/1 mice.55
In summary, this study clearly demonstrates that IL-12 is produced in
the early stage of atherosclerosis in apoE-deficient
mice. The observation of coexpression of IL-12 and B7 molecules in
macrophages suggests that macrophages can play an
active role in activating T lymphocytes to undergo preferential
differentiation to the Th1 phenotype in atherosclerotic
lesions. On the other hand, IL-12 together with IFN- produced by Th1
cells may also exert an effect on the isotype switch of antioxidized
LDL antibodies in these mice. Although atherosclerosis
is a disease with a complicated etiology, the immune system obviously
has an important impact on the development of the disease. We suggest
that the blockade of IL-12 function or/and alteration in the Th1/Th2
balance may serve as a potential therapeutic intervention for the
progression of atherosclerosis.
|
Acknowledgments |
|---|
Financial support from the Institute of Biomedical Sciences, Academia Sinica, and the National Science Council of Taiwan (NSC-87-2316-B-001-003-M26) are appreciated.
Received January 20, 1998; accepted October 12, 1998.
|
References |
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C. Buono, H. Pang, Y. Uchida, P. Libby, A. H. Sharpe, and A. H. Lichtman B7-1/B7-2 Costimulation Regulates Plaque Antigen-Specific T-Cell Responses and Atherogenesis in Low-Density Lipoprotein Receptor-Deficient Mice Circulation, April 27, 2004; 109(16): 2009 - 2015. [Abstract] [Full Text] [PDF]
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Z. Chen, M. Sakuma, A. C. Zago, X. Zhang, C. Shi, L. Leng, Y. Mizue, R. Bucala, and D. I. Simon Evidence for a Role of Macrophage Migration Inhibitory Factor in Vascular Disease Arterioscler Thromb Vasc Biol, April 1, 2004; 24(4): 709 - 714. [Abstract] [Full Text] [PDF]
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K. Y. Stokes, E. C. Clanton, J. L. Gehrig, and D. N. Granger Role of interleukin 12 in hypercholesterolemia-induced inflammation Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2623 - H2629. [Abstract] [Full Text] [PDF]
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O. J de Boer, A. E Becker, and A. C van der Wal T lymphocytes in atherogenesis--functional aspects and antigenic repertoire Cardiovasc Res, October 15, 2003; 60(1): 78 - 86. [Full Text] [PDF]
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B. OSTERUD and E. BJORKLID Role of Monocytes in Atherogenesis Physiol Rev, October 1, 2003; 83(4): 1069 - 1112. [Abstract] [Full Text] [PDF]
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 M. Cattaruzza, W. Slodowski, M. Stojakovic, R. Krzesz, and M. Hecker Interleukin-10 Induction of Nitric-oxide Synthase Expression Attenuates CD40-mediated Interleukin-12 Synthesis in Human Endothelial Cells J. Biol. Chem., September 26, 2003; 278(39): 37874 - 37880. [Abstract] [Full Text] [PDF]
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Z. Mallat, A. Gojova, V. Brun, B. Esposito, N. Fournier, F. Cottrez, A. Tedgui, and H. Groux Induction of a Regulatory T Cell Type 1 Response Reduces the Development of Atherosclerosis in Apolipoprotein E-Knockout Mice Circulation, September 9, 2003; 108(10): 1232 - 1237. [Abstract] [Full Text] [PDF]
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P. Davenport and P. G. Tipping The Role of Interleukin-4 and Interleukin-12 in the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice Am. J. Pathol., September 1, 2003; 163(3): 1117 - 1125. [Abstract] [Full Text] [PDF]
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Y. Matsui, S. R. Rittling, H. Okamoto, M. Inobe, N. Jia, T. Shimizu, M. Akino, T. Sugawara, J. Morimoto, C. Kimura, et al. Osteopontin Deficiency Attenuates Atherosclerosis in Female Apolipoprotein E-Deficient Mice Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 1029 - 1034. [Abstract] [Full Text] [PDF]
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 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
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A. Aicher, C. Heeschen, M. Mohaupt, J. P. Cooke, A. M. Zeiher, and S. Dimmeler Nicotine Strongly Activates Dendritic Cell-Mediated Adaptive Immunity: Potential Role for Progression of Atherosclerotic Lesions Circulation, February 4, 2003; 107(4): 604 - 611. [Abstract] [Full Text] [PDF]
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A. S. Major, S. Fazio, and M. F. Linton B-Lymphocyte Deficiency Increases Atherosclerosis in LDL Receptor-Null Mice Arterioscler Thromb Vasc Biol, November 1, 2002; 22(11): 1892 - 1898. [Abstract] [Full Text] [PDF]
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L. Zhao, C. A. Cuff, E. Moss, U. Wille, T. Cyrus, E. A. Klein, D. Pratico, D. J. Rader, C. A. Hunter, E. Pure, et al. Selective Interleukin-12 Synthesis Defect in 12/15-Lipoxygenase-deficient Macrophages Associated with Reduced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia J. Biol. Chem., September 13, 2002; 277(38): 35350 - 35356. [Abstract] [Full Text] [PDF]
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U. Schonbeck, G. K. Sukhova, N. Gerdes, and P. Libby TH2 Predominant Immune Responses Prevail in Human Abdominal Aortic Aneurysm Am. J. Pathol., August 1, 2002; 161(2): 499 - 506. [Abstract] [Full Text] [PDF]
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A. Daugherty and D. L. Rateri T Lymphocytes in Atherosclerosis: The Yin-Yang of Th1 and Th2 Influence on Lesion Formation Circ. Res., May 31, 2002; 90(10): 1039 - 1040. [Full Text] [PDF]
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L. J. Pinderski, M. P. Fischbein, G. Subbanagounder, M. C. Fishbein, N. Kubo, H. Cheroutre, L. K. Curtiss, J. A. Berliner, and W. A. Boisvert Overexpression of Interleukin-10 by Activated T Lymphocytes Inhibits Atherosclerosis in LDL Receptor-Deficient Mice by Altering Lymphocyte and Macrophage Phenotypes Circ. Res., May 31, 2002; 90(10): 1064 - 1071. [Abstract] [Full Text] [PDF]
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S. C. Whitman, P. Ravisankar, and A. Daugherty Interleukin-18 Enhances Atherosclerosis in Apolipoprotein E-/- Mice Through Release of Interferon-{gamma} Circ. Res., February 8, 2002; 90 (2): e34 - e38. [Abstract] [Full Text] [PDF]
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T. Luft, M. Jefford, P. Luetjens, H. Hochrein, K.-A. Masterman, C. Maliszewski, K. Shortman, J. Cebon, and E. Maraskovsky IL-1{beta} Enhances CD40 Ligand-Mediated Cytokine Secretion by Human Dendritic Cells (DC): A Mechanism for T Cell-Independent DC Activation J. Immunol., January 15, 2002; 168(2): 713 - 722. [Abstract] [Full Text] [PDF]
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Z. Mallat, A. Corbaz, A. Scoazec, S. Besnard, G. Leseche, Y. Chvatchko, and A. Tedgui Expression of Interleukin-18 in Human Atherosclerotic Plaques and Relation to Plaque Instability Circulation, October 2, 2001; 104(14): 1598 - 1603. [Abstract] [Full Text] [PDF]
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J. George, A. Afek, B. Gilburd, Y. Shoenfeld, and D. Harats Cellular and humoral immune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice J. Am. Coll. Cardiol., September 1, 2001; 38(3): 900 - 905. [Abstract] [Full Text] [PDF]
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E. Laurat, B. Poirier, E. Tupin, G. Caligiuri, G.K. Hansson, J. Bariety, and A. Nicoletti In Vivo Downregulation of T Helper Cell 1 Immune Responses Reduces Atherogenesis in Apolipoprotein E-Knockout Mice Circulation, July 10, 2001; 104(2): 197 - 202. [Abstract] [Full Text] [PDF]
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S. C. Whitman, P. Ravisankar, H. Elam, and A. Daugherty Exogenous Interferon-{{gamma}} Enhances Atherosclerosis in Apolipoprotein E-/- Mice Am. J. Pathol., December 1, 2000; 157(6): 1819 - 1824. [Abstract] [Full Text] [PDF]
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J. George, D. Harats, B. Gilburd, A. Afek, A. Shaish, J. Kopolovic, and Y. Shoenfeld Adoptive Transfer of {beta}2-Glycoprotein I-Reactive Lymphocytes Enhances Early Atherosclerosis in LDL Receptor-Deficient Mice Circulation, October 10, 2000; 102(15): 1822 - 1827. [Abstract] [Full Text] [PDF]
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R. A. Terkeltaub IL-10: An "Immunologic Scalpel" for Atherosclerosis? Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2823 - 2825. [Full Text] [PDF]
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L. J. Pinderski Oslund, C. C. Hedrick, T. Olvera, A. Hagenbaugh, M. Territo, J. A. Berliner, and A. I. Fyfe Interleukin-10 Blocks Atherosclerotic Events In Vitro and In Vivo Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2847 - 2853. [Abstract] [Full Text] [PDF]
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S. C. Whitman, P. Ravisankar, and A. Daugherty Interleukin-18 Enhances Atherosclerosis in Apolipoprotein E-/- Mice Through Release of Interferon-{gamma} Circ. Res., February 8, 2002; 90 (2): e34 - e38. [Abstract] [Full Text] [PDF]
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