© 1995 American Heart Association, Inc.
Articles |
Oxidized LDL Induces Enhanced Antibody Formation and MHC Class II–Dependent IFN-
Production in Lymphocytes From Healthy Individuals
From the Department of Medicine, Division of Rheumatology, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.
Correspondence to Johan Frostegård, Department of Medicine, Karolinska Hospital, Karolinska Institute, S-17176 Stockholm, Sweden.
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Abstract |
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Abstract The early stages of atherosclerosis are characterized by penetration into the arterial intima by both T lymphocytes and monocytes. Some of these T lymphocytes show signs of activation, though the mechanisms by which they become activated are not known. The monocytes develop into macrophages and subsequently into foam cells filled with oxidized LDL (oxLDL)-derived lipids. OxLDL has been found to exert several proinflammatory effects, including enhanced adhesiveness of endothelial cells and monocytes, chemotaxis of monocytes and T cells, and T-cell activation. The enzyme-linked immunospot (ELISPOT) assay has been shown to be a sensitive method for detection of single cells secreting antibodies or cytokines. Here we have used this method to characterize the T-cell cytokine secretion pattern after exposure to oxLDL in vitro. In peripheral blood mononuclear cells from healthy donors (n=27), a significantly enhanced number of INF-
–producing cells
was detected by ELISPOT (P<.001) after stimulation with 5
µg/mL oxLDL. In contrast, production of interleukin-4 was not
significantly enhanced after stimulation with oxLDL. OxLDL-induced
IFN- secretion and T-cell proliferation were completely inhibited by
major histocompatibility complex (MHC) class II antibodies.
Furthermore, oxLDL was found to enhance the antibody secretion,
indicating B-cell activation. Our results indicate that oxLDL
activates T cells by an MHC class II–dependent mechanism. In
healthy individuals, oxLDL induces IFN-, which is produced by T
helper type 1–like cells. These findings demonstrate that oxLDL
induces a cell-dependent immune reaction, which may play an
important role in the development of atherosclerosis.
Key Words: T lymphocytes • antibodies • oxidized LDL • atherosclerosis • IFN-
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Introduction |
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During recent years, it has become clear that atherosclerosis has many characteristics in common with chronic inflammatory diseases. At the earliest stages of the disease, monocytes and T lymphocytes adhere to the endothelium and penetrate into the arterial intima. The monocytes differentiate into macrophages and subsequently develop into foam cells, filled with oxLDL or other forms of modified LDL–derived lipids. Some T lymphocytes in the lesion also become activated, with an enhanced expression of interleukin-2 (IL-2) receptors and HLA-DR. Both CD4+ and CD8+ T cells have been described in the atherosclerotic lesion.1 2 3
OxLDL has been shown to exert several potentially proatherogenic effects on the cells participating in the development of the atherosclerotic lesion. Different forms of oxLDL induce enhanced adhesiveness both in endothelial cells4 5 and monocytes.6 OxLDL is chemotactic for monocytes7 and T lymphocytes,8 stimulates differentiation of monocytes,6 and induces activation of T lymphocytes.9 OxLDL also has cytotoxic properties, and an important role of the macrophage scavenger receptor may be to remove this cytotoxic substance.10
LDL may be oxidized by endothelial cells, macrophages, smooth muscle cells,11 and T lymphocytes.9 The oxidation is believed to take place mainly in the artery wall, but the mechanisms by which cells oxidize LDL are not clear.11 In experiments with animals, antioxidants such as butylated hydroxytoluene12 have been found to inhibit development of atherosclerosis. Furthermore, oxLDL has been demonstrated in atherosclerotic lesions.13 Taken together, these findings favor the notion that oxLDL may play an important role in the development of atherosclerosis.
T cells are generally believed to be primary participants in the
pathogenesis of several autoimmune diseases, including rheumatoid
arthritis14 and multiple sclerosis.15 A
preferential induction of Th1 response in rheumatoid
arthritis16 and multiple sclerosis17 has been
described, with expression of IFN- and IL-2, but no or little
expression of IL-4 or IL-10. T-cell cytokines and antibody
secretion may be determined by use of the ELISPOT technique, which has
been reported as more sensitive than ELISA.18 Still little
is known concerning the role of T cells in
atherosclerosis. In the present article we report
that exposure of PBMCs to low concentrations of oxLDL results in
enhanced antibody formation and production of IFN-, which is
MHC class II–dependent. The possible implications for oxLDL-mediated
immune reactions in atherogenesis are discussed.
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Methods |
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Cell Culture
PBMCs were isolated from human buffy coats obtained from healthy blood donors by sterile technique. The buffy coat was diluted in PBS at a ratio of one to four, layered onto Ficoll-Hypaque, and centrifuged for 20 minutes at 1500g. Interface cells were washed twice in PBS. They were then counted and resuspended in RPMI 1640 medium supplemented with HEPES buffer, penicillin, and streptomycin and 10% fetal calf serum (complete medium) at a cell concentration of 1x106 cells/mL. Cell viability was determined by trypan blue exclusion and exceeded 95% in all experiments.
Determination of DNA Synthesis
PBMCs were prepared as described above, suspended in complete
medium, and diluted to a cell concentration of 2x105
cells/mL. After the addition of native or oxLDL to the relevant
cultures, 200 µL cell suspension was added to each well in
round-bottomed 96-well cell culture plates (Nunc) and incubated for
the indicated time periods in a 37°C humid cell incubator.
Subsequently, 1 µCi of 3H-thymidine was added to each
well. After 6 more hours of incubation, DNA was precipitated onto glass
fiber filters by means of an automatic cell harvester, and the amount
of incorporated 3H was determined in a liquid scintillation
counter and expressed as counts per minute. Parallel cultures were made
in all experiments.
Preparation of LDL
Venous blood from healthy donors was drawn after overnight
fasting into precooled vacuum tubes containing Na2EDTA (1
mg/mL). Plasma was recovered by means of low-speed
centrifugation (1400g, 20 minutes) at 1°C
and kept at this temperature throughout the separation procedures. LDL
was isolated from plasma in the density interval 1.025 to 1.050 kg/L by
sequential preparative
ultracentrifugation19 in a 50.3 Ti
Beckman fixed-angle rotor (Beckman L8-80
ultracentrifuge) for 20 hours. The total protein content of
the LDL preparation was determined by the Lowry
technique.20
Oxidation of LDL by Copper
Isolated LDL was dialyzed in 0.02 mol/L phosphate/0.16 mol/L
NaCl buffer, pH 7.4 for 15 hours at 4°C to remove EDTA.
Copper-mediated oxidation of LDL was performed by incubating 0.2
mg/mL of EDTA-free LDL in medium F-10 containing 10-5
mol/L CuSO4 overnight at 37°C. Each preparation of oxLDL
was used within 1 week of the oxidation. The presence of endotoxins in
the lipoprotein preparations was analyzed with the
Limulus assay (Kabi-Pharmacia AB). All endotoxin levels were
below 0.5 ng/mL in the stock solutions and below 5 pg/mL in the test
samples. There was no difference in endotoxin levels between native and
oxLDL. The lipid peroxide contents of oxidized and native LDL were
determined by analyzing TBAR substances and were expressed as MDA
equivalents.21
Detection of IFN-– and IL-4–Producing Cells
The ELISPOT technique was used to determine the frequency
of cells producing IFN-.22 For IL-4
ELISPOTs,23 the method was used as adopted in our
laboratory.
For detection of IFN-–producing cells, 96-well nitrocellulose
plates (Millititre HA, Millipore Co) were used. The plates were prewet
with 200 µL/well fresh sterile PBS 30 minutes before being coated.
Then a Millititer vacuum filtration holder (Millipore) was used to
remove the solution. After three cycles of washing and vacuum suction
using PBS, the plates were coated with 15 µg/mL, 50 µL/well
anti-human IFN- catcher-mAb (1-DIK; Mabtech AB) diluted with
fresh PBS. Plates were then left for coating in humid chambers at 4°C
at least overnight.
PBMCs were prepared from healthy laboratory personnel and blood donors, and then oxLDL, native LDL, or PHA (Wellcome Diagnostics) at the indicated concentrations was added to the respective cell suspensions. Subsequently, the cells were cultured in round-bottomed cell culture plates (Nunc) at 37°C in 5% CO2 incubator for 72 hours.
The coated plates were then washed three times with sterile PBS with the vacuum device and 100 µL cell suspension was transferred to the wells of the precoated detection plates. The cells were then again incubated at 37°C, 5% CO2 overnight.
For the detection of IFN-, the nitrocellulose plates were washed two
times and the PBS flicked off, followed by washing and vacuum suction
as indicated above. Fifty microliters of a detector-mAb
(7-B6-1-Biotin; Mabtech AB) at a concentration of 1 µL/mL was then
added to each well in PBS. Thereafter, the plates were kept at +4°C
overnight. After washing as above, we added 50 µL of
avidin-alkaline phosphatase (Sigma Chemical Co) at 0.6 µg/mL in
PBS, which was left for 2 hours in room temperature. After three more
cycles of washing and vacuum suction, 50 µL of a phosphatase
substrate solution producing an insoluble product (BCIP; Sigma) was
added for 1 hour at room temperature. Thereafter, the plates were
washed three times with distilled water and left to dry at room
temperature. Spots were enumerated using a low-magnification (x20)
dissection microscope, each spot representing one
IFN-–producing cell.
For detection of IL-4, conventional ELISA plates (Immulon II, Dynatech) were used. The plates were coated with 15 µg/mL, 50 µL/well anti-human IL-4 mAb (Mabtech AB). These coated plates were kept at +4°C at least overnight in a moist chamber before use.
The precoated ELISA plates were then washed three times with sterile
PBS. Thereafter, cell suspensions prepared as described above were
transferred to wells (100 µL/well). The plates were then kept at
37°C in 5% CO2 overnight. Thereafter, the plates were
washed three times with sterile PBS, and 50 µL/well of a
detector-mAb (IL4-II-Biotin; Mabtech AB) at a concentration of 1
µL/mL was added. The plates were then kept in moist chambers at 4°C
overnight. Reactions were developed as for IFN- ELISPOTs, except for
the manual washing steps without vacuum device and the fact that BCIP
had to be left in the wells for 4 to 5 hours to make the spots readily
visible. Spots corresponding to cells that had secreted IL-4 were
enumerated under low magnification (x20) in an inverted microscope,
each spot representing one Il-4–producing cell.
Anti-MHC Class II Antibodies
For inhibition of MHC class II–dependent reactions, a pool of
three different mAb reacting against human MHC class II was used at a
maximal total final concentration of 1 µg/mL. This concentration had
in preliminary experiments been shown to block PBMC proliferation and
IFN- ELISPOT production to background levels using a tetanus
toxoid as model antigen (data not shown). The pool consisted of equal
amounts of 2.06 (mouse IgG1, anti-HLA DR; American Type Culture
Collection [ATCC]), IVA 12 (IgG1, anti-HLA DR, DP, DQ; ATCC), and
9,3F10 (IgG2a, anti-HLA DR, DQ; ATCC).
As control antibodies for the MHC class II–blocking experiments, two monoclonals directed against keyhole limpet hemocyanin were used, clone H5 (IgG1) and clone 7-B4 (IgG2a).24 Control antibodies were used in the same concentrations and ratios of IgG subclasses as for the blocking antibodies in each experiment.
As another control, mAb directed toward an irrelevant antigen (human von Willebrand molecule) were used (IgG1, Immunotech).
Detection of Antibody Production
PBMCs from healthy donors were prepared and suspended in
complete medium at a concentration of 2x105 cells/mL.
OxLDL or LDL was added at the indicated concentrations, and cell
suspensions were incubated at 37°C. After 16 hours, cells were washed
three times in PBS, and 200 µL cell suspension at a concentration of
105/well or lower was transferred to the precoated
ELISPOT wells. The frequency of cells producing antibodies was
determined by a modified version of the ELISPOT technique exactly as
recently described from our laboratory.25 26 Spots were
counted under low magnification (x20) with an inverted microscope.
Analysis of Cellular Composition of PBMCs
Standard flow cytometry was used for the quantification of
lymphocyte subsets in a FACScan from Becton Dickinson & Co. The
following directly fluorochrome–conjugated antibodies were used:
anti-CD3 (Leu-4)-fluorescein isothiocyanate
(FITC)-conjugated, anti-CD4 (Leu-3a) phycoerythrin (PE)-conjugated,
anti-CD8 (Leu-2a)-PE all purchased from Becton Dickinson, and anti-CD19
(HB37), A/S anti-HLe-1 (anti-CD45), Leu-M3 (anti-CD14) purchased from
DAKO. For the automatic lymphocyte gating, the compound reagent
Simultest Leucogate consisting of anti-CD45 (anti-HLe-1)-FITC and
anti-CD14 (Leu-M3)-PE was used, and as negative control the compound
reagent Simultest Control g1/g2a was used (both from Becton Dickinson).
Cells, 200 000/tube in PBS, were stained with antibodies for 15
minutes in room temperature. After staining, the cell suspension was
washed once in PBS and then directly analyzed on a FACSort flow
cytometer, using the SIMULSET software for automatic
lymphocyte gating. T lymphocytes were defined as CD3+
positive cells, T helper lymphocytes as
CD3+CD4+ double positive cells, T
suppressor/killer lymphocytes as CD3+CD8+
double positive cells, and B lymphocytes as
CD19+CD3- single positive cells.
Statistics
Conventional methods were used for calculation of means and
standard deviations. Coefficients of skewness and kurtosis were
calculated to test deviations from a normal distribution. Differences
in individual experiments between control samples and samples
stimulated with oxidized or native LDL were analyzed by
Student's t test. Effects of native and oxLDL in the whole
group of individuals tested were analyzed by Wilcoxon
signed-rank test.
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Results |
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LDL Oxidation
Exposure of native LDL to 10 µmol/L of Cu2+ for 16 hours at 37°C resulted in an increase in the amount of TBAR material present in the LDL preparation from 1.5±0.4 to 46.0±1.8 MDA equivalents/mg protein and in an increased mobility during agarose gel electrophoresis (data not shown).
PBMC Proliferation
In accordance with our previous results, the proliferation
of PBMCs as determined by DNA synthesis was significantly enhanced
after stimulation with copper-oxidized LDL. The optimal
concentration of oxLDL for T-cell activation varied between individuals
and different experiments, but in general, maximal stimulation was
obtained at oxLDL concentrations between 1 and 10 µg/mL. Also, native
LDL induced proliferation of PBMCs, but in general, higher
concentrations (10 µg/mL) of native LDL were needed for maximal
stimulation (data not shown).
To determine if oxLDL-induced T-cell stimulation is dependent on
MHC class II, we tested the effect of mAb directed against MHC class II
on oxLDL-induced PBMC proliferation. We found that a total
concentration of 1 µg/mL of three pooled anti-MHC class II mAb added
to the cell cultures abolished the T-cell response to 5 µg/mL oxLDL
(Fig 1
). A similar effect was obtained when tetanus
toxoid or native LDL was used as antigen (data not shown). The control
antibody had no inhibitory effect (data not shown).
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or without (
) oxLDL for the indicated time periods in culture
medium in 96-well plates. Mab to MHC class II were added to the cell
cultures with (
) or without (
) oxLDL 30 minutes before
addition of oxLDL. During the last 6 hours of incubation, 1 µCi/mL of
3H-thymidine was added. DNA was precipitated in glass fiber
filters, and the amount of incorporated 3H-thymidine was
determined in a liquid scintillation counter. Each value
represents the mean±SD of six determinations.
***P<.005.
In contrast, antibodies to MHC class II at the same concentration that
completely inhibited the response to oxLDL and tetanus toxoid had no
effect on the T-cell response to the mitogen PHA, which has a strong
stimulatory effect on T cells (Fig 2).
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Effect of OxLDL and Native LDL on Cytokine Pattern of
PBMCs
To allow a quantitative determination of the induction of
cytokines at the single-cell level, the ELISPOT assay was
used. In 27 healthy blood donors tested, oxLDL at 5 µg/mL induced a
167% (SEM±32%; P<.001) increase. LDL at 10 µg/mL
induced a 52% (SEM±42%) increase, but this difference was not
significant. For comparison, incubation of a strong antigen, tetanus
toxoid, at 5 U/mL with PBMCs from 6 healthy blood donors gave a 269%
(SEM±51%; P<.01) increase in the number of
IFN-–producing cells. In most individuals tested, a maximal
effect was obtained at 5 µg/mL oxLDL and 10 µg/mL LDL. Only in 1
individual was a decrease in IFN- secretion noted after stimulation
with oxLDL. A representative experiment demonstrating
the effect of native and oxidized LDL on IFN- secretion is shown in
Fig 3. OxLDL had no effect on secretion of IL-4 in 16 of
18 healthy individuals tested. In 2 individuals, an enhanced IL-4
secretion was detected after stimulation with oxLDL (data not shown).
LDL had no effect on the production of IL-4. A
representative experiment showing IL-4–producing cells
after stimulation with PHA or oxLDL is demonstrated in Fig 4.
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Effect of Antibodies to MHC Class II on OxLDL-Induced IFN-
Secretion at the Single- Cell Level
To determine whether oxLDL-induced IFN- secretion is dependent
on MHC class II, we tested the effect of mAb to MHC class II on
oxLDL-induced IFN- secretion. We found that pooled anti-MHC class II
antibodies added to the cell cultures completely abolished the
oxLDL-induced IFN- secretion, while the control antibodies at the
same concentrations and the same Ig isotypes had no effect (Fig 5). A similar effect was obtained when tetanus toxoid
was used as antigen (data not shown). We then determined if a
monoclonal control antibody to another irrelevant antigen (von
Willebrand factor) had any effect. In a
representative experiment, 5 µg/mL oxLDL induced an
increase from 22±2.0 to 34±1.1 of IFN-–producing
cells/105. When 1 µg/mL control antibody was present,
oxLDL induced an increase from 23.5±4.0 to 37±6.1
IFN-–producing cells/105. The irrelevant antibody
thus had no effect on oxLDL-induced IFN- production. Trace
amounts of copper are present in the cell cultures, but copper had
no effect on production of IFN- (data not shown).
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Effect of OxLDL on Antibody Secretion at the Single-Cell
Level
To determine whether oxLDL-induced T-cell activation also involved
a B-cell response, we tested the production of antibodies in
PBMCs after treatment with oxLDL. We found that oxLDL induced enhanced
antibody production of IgG, IgA, and IgM class, as determined
by the ELISPOT technique (Fig 6). Antibody specificity
was not investigated in these experiments.
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Cellular Composition of PBMCs
The cellular composition of PBMCs was analyzed by FACScan
(Fig 7). Our data indicate that the proportion of
CD4+ and CD8+ T cells and B cells did not
change after 3 days of culture and transfer to nitrocellular plates for
determination of cytokine secretion by the ELISPOT technique.
Furthermore, no difference between cells cultured in 5 µg/mL oxLDL
and cells cultured in medium was detected.
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Discussion |
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The present study demonstrates that oxLDL-induced T-cell activation in healthy individuals is characterized by enhanced secretion of IFN-
. Antibodies to oxLDL have been detected in both
healthy individuals and patients with clinical manifestations of
atherosclerosis,27 and we here demonstrate
that oxLDL induces B-cell activation, as determined by enhanced
secretion of immunoglobulins of IgG, IgA, and IgM classes.
Cytokine and antibody formation was determined by use of the
highly sensitive ELISPOT technique. The immune reaction initiated was
dependent on MHC class II, since antibodies to MHC class II prevented
both oxLDL-induced PBMC proliferation and IFN- secretion. An
irrelevant control antibody had no effect. We found that
incubation with anti-MHC class II antibodies had slight
inhibitory effects on spontaneous IFN- secretion. This
finding is in accordance with earlier reports and depends on T cells
reactive with autologous MHC class II molecules.28
Both the basal IFN- secretion and the response to oxLDL varied
between different individuals but was highly significant in the whole
group (n=27) of healthy donors tested, indicating that an immune
response to oxLDL is common in the population. In accordance with
earlier findings, native LDL also induced proliferation and IFN-
secretion in PBMCs, although not as strongly as oxLDL.
Oxidation of LDL is believed to be a key factor in the development of atherosclerosis, and during recent years the immune mechanisms in the development of atherosclerosis have gained considerable interest.1 2 3 The mechanisms by which oxLDL may activate T cells are largely unknown. In principle, oxLDL may induce T-cell stimulation by unspecific mechanisms, but it is also possible that activation by means of a conventional antigen or a superantigen is involved.
The possibility that the effect of oxLDL on PBMCs is due to an
endotoxin contamination in the lipoprotein preparations cannot be
completely excluded, since both LDL and oxLDL contain trace amounts of
endotoxin (<5 pg/mL in the test samples). However, native LDL
containing amounts of endotoxin similar to those in oxLDL had no
significant effect on IFN- expression in the group tested. It is
therefore not likely that endotoxin is responsible for oxLDL-induced
enhanced expression of IFN-.
IFN- may be produced by T cells and natural killer cells. However,
the finding that antibodies to MHC class II inhibit IFN- formation
indicates that T cells are the main source of IFN-.
Oxidation of LDL is initiated by abstraction of a hydrogen atom from a polyunsaturated fatty acid in a cell membrane or lipoprotein. This leads to a chain reaction with formation of lipid hydroperoxides and aldehydes, fragmentation of the carrier protein apo B-100 and exposure of novel epitopes, and profound changes in the properties of the LDL molecule including increased negative charge, density, and electrophoretic mobility and decreased content of polyunsaturated fatty acids and vitamin E.29 OxLDL but not native LDL is taken up in macrophages after binding by the scavenger receptor, leading to foam cell formation.30
One possibility is that novel epitopes on oxLDL are presented on monocytes in the context of MHC class II and recognized as foreign by T cells, leading to a specific immune response.
We have recently found that oxLDL induces HSP 60 in monocytic cells (J.F. et al, unpublished observation, 1995). Immunity to HSP 60 has been implicated in autoimmune diseases such as rheumatoid arthritis and recently also atherosclerosis.31 32 HSP 60–reactive T cells are present also in healthy control individuals.33 Another possibility is thus that oxLDL activates T cells indirectly, by inducing HSP 60, which in its turn activates T cells in an MHC-dependent manner.
OxLDL-induced T-cell activation may also be dependent on a superantigen. Superantigens can activate T cells by crosslinking V regions of the T-cell receptor with MHC molecules on accessory cells.34 T-cell activation induced by superantigens is in general strong, and oxLDL-induced T-cell stimulation is comparatively weak, which argues against this possibility.
The most likely interpretation of our data is that oxLDL induces a
cell-mediated immune reaction with a preferential induction of
IFN-, a Th1 cytokine. Cells are activated by an MHC
class II–dependent mechanism and thus require the presence of
antigen-presenting cells. Signs of B-cell activation as
determined by induction of immunoglobulin production were seen
after incubation of PBMCs with oxLDL. The mechanisms behind this B-cell
activation and the specificity of antibodies produced are not known,
but this finding is compatible with recent data indicating the presence
of antibodies to oxLDL in both normal control subjects and patients
with clinical manifestations of
atherosclerosis.26
T cells may in principle both inhibit and promote further development
of atherosclerosis. IFN-, induced by oxLDL from T
cells, may exert dual effects on the development of the disease.
IFN- may inhibit smooth muscle cell growth and also suppress
atherosclerosis in experimental animal
models.35 On the other hand, IFN- and other
T-cell–produced cytokines activate
macrophages to secrete inflammatory mediators as well as growth
factors for smooth muscle cells, which may lead to enhanced plaque
growth and also a perpetuation of the inflammatory disease
process.35 36 Another cytokine, TGF-ß, has been
proposed to play an important role in the atherosclerotic lesion, by
effects possibly acting both to suppress and promote disease. TGF-ß
may both inhibit and stimulate smooth muscle cell growth, depending on
the concentration used.37 38 The outcome of a human
inflammatory disease may depend on the subpopulation of T cells that
predominates at the site of inflammation. An intriguing possibility is
thus that the Th1/Th2 balance in the atherosclerotic lesion may
influence the disease progress, and it is possible that the balance
between the various reactions initiated in the early inflammatory
atherosclerotic lesion may influence the final outcome.
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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 (B95-19X-11244-01), the Foundation for Old Servants, King Gustaf V 80th Birthday Fund, the Swedish Society of Medicine, Ostermans Fund, the Swedish Association Against Rheumatism, and the Nanna Svartz Fund. The authors thank Dr Kerstin Carlson for preparation of LDL.
Received November 14, 1994; accepted June 20, 1995.
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