© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Glucocorticoid Inhibits Oxidized LDL-Induced Macrophage Growth by Suppressing the Expression of Granulocyte/Macrophage Colony-Stimulating Factor
From the Department of Metabolic Medicine, Kumamoto University School of Medicine (M.S., T.B., T.M., T.T., H.M., Y.A., T.S., M.S.) and the Division of Cardiology, Kumamoto National Hospital (S.K.), Kumamoto, Japan.
Correspondence to Masakazu Sakai, MD, PhD, Department of Metabolic Medicine, Kumamoto University School of Medicine, 1–1-1 Honjo, Kumamoto 860-8556, Japan.
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Abstract—Glucocorticoid, an anti-inflammatory agent, inhibits the development of atherosclerosis in various experimental animal models. This is partially explained by its ability to inhibit smooth muscle cell migration and proliferation in the intima and to reduce chemotaxis of circulating monocytes and leukocytes into the subendothelial spaces. We have recently demonstrated that oxidized LDL (Ox-LDL) has a mitogenic activity for macrophages in vitro in which Ox-LDL–induced granulocyte/macrophage colony-stimulating factor (GM-CSF) production plays an important role. Proliferation of cellular components is one of the characteristic events in the development and progression of atherosclerotic lesions. In the present study, we investigated the effects of glucocorticoids on Ox-LDL–induced macrophage growth. Dexamethasone, prednisolone, and cortisol inhibited Ox-LDL–induced thymidine incorporation into macrophages by 85%, 70%, and 50%, respectively. Ox-LDL induced a significant production of GM-CSF by macrophages, which was effectively inhibited by dexamethasone, prednisolone, and cortisol by 80%, 65%, and 50%, respectively. Dexamethasone-mediated inhibition of Ox-LDL–induced GM-CSF mRNA expression and macrophage growth was significantly abrogated by RU-486, a glucocorticoid receptor antagonist. Our results suggest that the inhibitory effects of glucocorticoids on macrophage growth may be due to the inhibition of Ox-LDL–induced GM-CSF production through transactivation of the glucocorticoid receptor.
Key Words: glucocorticoids • dexamethasone • oxidized LDL • macrophage growth • atherosclerosis
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Introduction |
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Macrophage-derived foam cells are the key cellular elements in the early stages of atherosclerosis and play an important role in the development and progression of atherosclerosis through production of various active molecules, such as cytokines and growth factors.1 Macrophages take up oxidized LDL (Ox-LDL) through the scavenger receptor pathways and become foam cells.2 3 4 Macrophages and macrophage-derived foam cells proliferate in the atherosclerotic lesions, particularly in the early lesions.5 6 7 Because the growth of cellular components is one of the characteristic features in atherosclerotic lesions,1 it seems reasonable to expect that macrophage growth might be linked to atherosclerotic processes, probably by enhancing its progression. Yui et al8 reported that Ox-LDL could induce the growth of starch-induced mouse peritoneal macrophages in vitro. Recent studies have shown that Ox-LDL exhibited a growth-stimulating capacity for other macrophages, such as mouse resident peritoneal macrophages,9 10 rat resident peritoneal macrophages,11 and human monocyte-derived macrophages.12 Moreover, our biochemical studies showed that scavenger receptor-mediated endocytic internalization of lysophosphatidylcholine (lyso-PC), a major modified lipid moiety of Ox-LDL, is important for Ox-LDL–induced macrophage growth13 14 and that Ox-LDL–induced granulocyte/macrophage colony-stimulating factor (GM-CSF) production plays an important role in macrophage growth.15
Atherosclerosis is viewed as a chronic inflammatory process because one of its features is accumulation of inflammatory cells and cytokines.1 Moreover, administration of an anti-inflammatory agent, dexamethasone, suppresses the development of atherosclerosis in various experimental animal models, including Watanabe heritable hyperlipidemic rabbits,16 cholesterol-fed rabbits,17 cuff-induced intimal thickening of the rabbit carotid artery,18 and rat balloon angioplasty model.19 The inhibitory mechanisms of dexamethasone for the development of atherosclerosis are partially explained by inhibition of smooth muscle cell migration20 and proliferation21 and by a reduction in chemotaxis of circulating monocytes22 and leukocytes23 into the subendothelial spaces. In addition, Asai et al24 reported that dexamethasone inhibited the growth of U937 cells, a monocyte/macrophage-like cell line. However, the exact inhibitory mechanism of dexamethasone for the development of atherosclerosis remains to be elucidated.
Because macrophage-derived foam cells play an essential role in the development of the early stages of atherosclerosis, we investigated the effect of glucocorticoids on Ox-LDL–induced macrophage growth in the present study. Our results demonstrated that glucocorticoids suppressed Ox-LDL–induced macrophage growth through inhibition of Ox-LDL–induced production of GM-CSF, which might be one of the underlying mechanisms of the inhibitory effect of dexamethasone on the development of experimental atherosclerosis in animal models.
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Methods |
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Chemicals
[methyl-3H]Thymidine and [14C]palmitoyl lyso-PC were purchased from DuPont-NEN. Dexamethasone, prednisolone, cortisol, aldosterone, progesterone, estrone, estragiol, estriol, dehydroepiandrosterone (DHEA), testosterone, and mifepristone (RU-486) were purchased from Sigma Chemical Co. All other chemicals were the highest grade available from commercial sources.
Lipoproteins
Human LDL (d=1.019 to 1.063 g/mL) was isolated by sequential
ultracentrifugation from the plasma of normolipidemic
subjects after overnight fasting. LDL was dialyzed against 0.15 mol/L
NaCl and 1 mmol/L ethylenediamine-tetraacetic acid (EDTA),
pH 7.4. Ox-LDL was prepared by incubation of LDL with 5 µmol/L
CuSO4 for 20 hours at 37°C followed by the addition of 1 mmol/L
EDTA and cooling.25 The concentrations of proteins were
determined by bicinchonic acid protein assay reagent (Pierce)
using bovine serum albumin as a standard.26 Ox-LDL
was labeled with [14C]lyso-PC as described
previously.13 Specific radioactivity of
[14C]lyso-PC–labeled Ox-LDL was 16 000
cpm/µg protein. The level of endotoxin associated with these
lipoproteins was <1 pg/µg protein, as measured by a commercially
available kit (Toxicolor system; Seikagaku Corp). Moreover,
macrophage growth was not influenced by endotoxin at a
concentration of <1 ng/mL in our experimental system.
Cell Culture
Peritoneal macrophages were collected from nonstimulated
male C3H/He mice (25 to 30 g) (Japan SLC, Inc, Hamamatsu, Japan)
and suspended in RPMI 1640 medium (Nissui Seiyaku Co)
supplemented with 10% heat-inactivated FBS (Life
Technologies, Inc), streptomycin (0.1 mg/mL), and penicillin (100 U/mL)
(medium A).
Human peripheral blood monocytes were isolated by the method of Fogelman et al27 using Ficoll/Hypaque gradient centrifugation. The mononuclear cells thus obtained were resuspended in RPMI 1640 supplemented with 20% autologous serum, 0.1 mg/mL of streptomycin, and 100 U/mL of penicillin, plated on serum-treated 10 cm dishes (Falcon), and incubated for 2 hours. Nonadherent cells were removed by washing 3x with PBS, then the adherent cells were detached by incubation in PBS/5% autologous serum containing 0.02% EDTA at 4°C for 30 minutes. The cells were then washed extensively and resuspended in RPMI 1640 supplemented with 5% autologous serum, 0.1 mg/mL of streptomycin, and 100 U/mL of penicillin (medium B). The cells were plated on 10 cm dishes and incubated for 9 days to differentiate into macrophages. The medium was aspirated and replaced every 3 days with fresh medium B. After 9-day incubation, differentiation of monocytes into macrophages was identified by 4 criteria, including (1) adherence to culture plates, (2) morphologic features resembling mononuclear cells after Giemsa staining, (3) the capacity to take up carbon particles, and (4) positive immunohistochemistry with antibody for CD 68. The cells contained >95% macrophages and were >95% viable as determined by trypan blue staining and lactic dehydrogenase release.
Tritiated Thymidine Incorporation Assay
The peritoneal cells were adjusted to
4x105 cells/mL for the
[3H]thymidine incorporation
assay.10 Cell suspensions (100 µL) were dispersed in
each well of 96-well tissue culture plates (6.4 mm in diameter,
Falcon) and incubated for 90 minutes at 37°C. Nonadherent cells were
removed by washing 3x with 100 µL of prewarmed medium A. More than
98% of adherent cells were confirmed to be macrophages by both
Giemsa staining and carbon particle uptake.10 These
macrophages were cultured at 37°C in 0.1 mL of medium A in
the presence of the test lipoproteins without a medium change. Eighteen
hours before the termination of the experiments, 10 µL of 10 µCi/mL
[3H]thymidine was added to each well and
incubated for 18 hours at 37°C. The medium was discarded, and the
cells were dissolved in 0.1 mL of 0.5% sodium dodecyl sulfate
and subsequently precipitated with 0.1 mL of ice-cold 10%
trichloroacetic acid (TCA). The resulting TCA–insoluble material was
collected on filters with Labomash LM-101 (Labo Science). The filters
were dried, and their radioactivity was counted in a liquid
scintillation spectrophotometer.
Cell-Counting Assay
Peritoneal cells or human monocyte-derived macrophages
were adjusted to 2x104 cells/mL, and 1 mL of
cell suspension was dispersed in each well of 24-well tissue culture
plates (16 mm in diameter, Falcon) and incubated for 90 minutes at
37°C. Nonadherent cells were removed by washing 3x with 1 mL of
prewarmed medium A or B, respectively. These macrophages
were cultured at 37°C in 1 mL of medium A or B, respectively, with or
without the test lipoproteins. After incubation for 7 days without
medium change, adherent cells in triplicate wells were lysed in 1%
(wt/vol) Triton X-100, and the number of naphthol blue-black–stained
nuclei was counted in a hemocytometer, as described
previously.10
Endocytic Degradation and Cell-Association of
[125I]Ox-LDL
Mouse peritoneal macrophages
(2x106 cells) in 1.0 mL of medium A were seeded
onto each plastic culture dish (22 mm in diameter, Falcon) and
incubated for 90 minutes at 37°C. The monolayers thus formed were
washed 3x with 1.0 mL of medium A. Each well was incubated with
[125I]Ox-LDL for 6 hours at 37°C in the
absence or presence of the indicated concentrations of
dexamethasone. Endocytic degradation was determined by
TCA-soluble radioactivity in the medium after precipitating free
iodine with AgNO3, as described
previously.28 Cells were solubilized with 1.0 mL of 0.1
mol/L NaOH and the cell-associated radioactivity was determined
as described previously.29
Mouse macrophage monolayers were incubated at 37°C for 3 hours with 10 µg/mL [14C]lyso-PC–labeled Ox-LDL in the presence or absence of the indicated concentrations of dexamethasone. After washing 3x with PBS, cellular lipids were extracted by hexan:isopropanol (2:1, vol:vol), and [14C]lyso-PC was counted in a liquid scintillation spectrophotometer.13
ELISA for GM-CSF
Mouse peritoneal macrophages were adjusted to
5x105 cells/mL, and 10 mL of the cell suspension
was dispersed in 10 cm plates (10 cm in diameter, Falcon) then
incubated for 90 minutes at 37°C. Nonadherent cells were removed by
washing 3x with 5 mL of PBS, and adherent cells were cultured at
37°C in 15 mL of medium A with or without the test lipoproteins.
During incubation for 24 hours, 300 µL of the medium were collected
at the indicated time intervals (the actual incubation times were 0, 3,
6, 9, 12, and 24 hours) and immediately centrifuged at
10 000g for 1 minute to remove any particulate material.
The supernatant was stored at -80°C immediately. Then, the frozen
culture supernatants were quickly thawed to determine GM-CSF levels in
the medium. The concentration of GM-CSF was determined by a
commercially available GM-CSF–specific ELISA kit (sensitivity, 5
pg/mL, Amersham) using recombinant murine GM-CSF as a
standard.15
Reverse Transciptase Polymerase Chain Reaction (RT-PCR)
Analysis for GM-CSF
Standard molecular biological techniques were
used.30 After incubation of murine peritoneal
macrophage monolayers (2x106 cells/well
in 6-well plate, 3.5 cm in diameter, Nunc) with 20 µg/mL Ox-LDL for 1
hour, total RNA was extracted with TRIzol (Life Technologies, Inc). The
first strand cDNA synthesis containing 1 µg of total RNA was primed
with oligo dT. Primers used for PCR amplification of GM-CSF and
ß-actin were designed on the basis of murine GM-CSF
cDNA31 and murine ß-actin cDNA32 sequences
as follows: for GM-CSF: forward primer, TGT GGT CTA CAG CCT CTC AGC AC
(nucleotide 64 to 87 of murine GM-CSF coding sequence);
reverse primer, CAA AGG GGA TAT CAG TCA GAA AGG T
(nucleotide 343 to 368 of murine GM-CSF coding
sequence,31 the size of RT-PCR products of GM-CSF was
expected to be 305 base pairs); for ß-actin: forward primer;
GTG GGC CGC TCT AGG CAC CAA (nucleotide 25 to 45 of murine
ß-actin coding sequence); reverse primer, CTC TTT GAT GTC ACG CAC GAT
TTC (nucleotide 541 to 564 of murine ß-actin coding
sequence32 the size of RT-PCR products of ß-actin
was expected to be 540 base pairs). The cycling conditions in
the GeneAmp 9600 System consisted of a first step of 94°C
denaturation for 10 minutes, followed by 30 cycles of annealing at
54°C for 60 seconds, extension at 75°C for 90 seconds, and
denaturation at 94°C for 30 seconds, with a final elongation step at
75°C for 10 minutes. Amplification products were analyzed
by 1.5% agarose gel electrophoresis. To verify that the amplification
products were consistent with the reported sequences of
murine GM-CSF and ß-actin, they were ligated into pGEM-T (Promega),
transfected into Escherichia coli XL1-Blue and sequenced by
using 373A DNA sequencer (Applied Biosystems).
Miscellaneous
Data were expressed as mean±SD. Differences between groups were
examined for statistical significance using the Student's t
test. A probability value <5% was considered significant. The
experimental protocol was approved by the Human Ethics Review Committee
and the Ethics Review Committee for Animal Experimentation of the
Kumamoto University School of Medicine.
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Results |
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Glucocorticoids Inhibit Ox-LDL–Induced Macrophage Growth
As shown in Figure 1
, Ox-LDL–induced [3H]thymidine incorporation
into mouse peritoneal resident macrophages was significantly
inhibited by dexamethasone in a dose-dependent manner,
whereas dexamethasone alone did not affect
[3H]thymidine incorporation into
macrophages. The cell counting assay also showed that the
Ox-LDL–induced increase in cell number from
1.7x104 cells/well to
3.2x104 cells/well was inhibited by 75% when 10
nmol/L of dexamethasone was added (the Table). Under these
conditions, dexamethasone had no cytotoxic effects on these
cells; more than 95% of the cells were viable as confirmed by
microscopic examination after trypan blue staining and lactic
dehydrogenase release (data not shown). These results demonstrated that
dexamethasone inhibits Ox-LDL–induced macrophage
growth.
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) or the
presence (•) of 20 µg/mL of Ox-LDL. During the last 18 hours of
incubation, cells in each well were chased with
[3H]thymidine, harvested, and the radioactivity was
determined as described in Methods. Data represent the mean±SD
of 4 separate experiments.
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We next examined the effect of several steroids on Ox-LDL–induced
thymidine incorporation into macrophages. As shown in Figure 2, dexamethasone,
prednisolone, and cortisol significantly inhibited Ox-LDL–induced
[3H]thymidine incorporation by 85%, 70%, and
50%, respectively. In contrast, aldosterone did not
inhibit [3H]thymidine incorporation by Ox-LDL
(Figure 2). Moreover, other steroids, including progesterone,
estrone, estradiol, estriole, dehydroepiandrosterone, and testosterone,
did not have any effect on [3H]thymidine
incorporation by Ox-LDL (data not shown). These results demonstrated
that glucocorticoids inhibited Ox-LDL–induced macrophage
growth.
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To investigate whether transactivation of glucocorticoid receptor by
dexamethasone causes inhibition of Ox-LDL–induced
macrophage growth, we examined the effect of RU-486, a
glucocorticoid receptor antagonist,33 on this
phenomenon. As shown in Figure 3, [3H]thymidine incorporation into
macrophages was not affected by RU-486 in the presence or
absence of Ox-LDL. However, RU-486 significantly abrogated
dexamethasone-mediated inhibition of Ox-LDL–induced
[3H]thymidine incorporation (Figure 3).
These results suggested that the inhibitory effect of
dexamethasone on Ox-LDL–induced macrophage growth
might be mediated by transactivation of glucocorticoid receptor.
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Effect of Dexamethasone on the Uptake of Ox-LDL
and Lyso-PC
Dexamethasone increases acetyl-LDL degradation by
human monocyte-derived macrophages34 or suppresses
the expression of acetyl-LDL receptor on a human macrophage
cell line, THP-1 cell.35 Moreover, we previously
demonstrated that the internalization of lyso-PC concomitant with
Ox-LDL into macrophages through the scavenger receptor A-I/A-II
played an important role in macrophage
growth.13 14 Thus, to elucidate whether
dexamethasone modulates the uptake of Ox-LDL or lyso-PC by
macrophages, we examined the effects of
dexamethasone on cell-association and degradation of Ox-LDL
and on the uptake of lyso-PC by macrophages. Figure 4A shows that cell-association of
[125I]Ox-LDL to macrophages was
inhibited by dexamethasone in a dose-dependent fashion with
maximal inhibition by 20%. Moreover, degradation of
[125I]Ox-LDL by cells was also inhibited by
dexamethasone by 30% (Figure 4B). Under these
conditions, the transfer of lyso-PC from Ox-LDL to macrophages
was examined using [14C]lyso-PC–labeled
Ox-LDL.9 Dexamethasone inhibited the amount of
[14C]lyso-PC transferred from Ox-LDL to cells
by 20% (Figure 4C). Thus, the inhibition of macrophage
growth by dexamethasone could be explained at least in part
by the inhibition of lyso-PC uptake, although several other
inhibitory mechanisms remain unknown.
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) or cold Ox-LDL (
). Cell-associated radioactivity
(A) and TCA-soluble radioactivity in the medium (B) were determined as
described in Methods. The raw values of cell-association (A) and
endocytic degradation (B) equivalent to 100% were 1.28 and 2.53
µg · 6 h-1 · mg-1 cell
protein, respectively. (C) Macrophages (2x106)
were incubated for 3 hours with 10 µg/mL of
[14C]lyso-PC–labeled Ox-LDL in the absence or presence
of the indicated concentrations of dexamethasone (•).
After incubation, cellular radioactivity of [14C]lyso-PC
was measured as described in Methods. Specific radioactivity of
[14C]lyso-PC–labeled Ox-LDL was 16000 cpm/µg protein.
[14C]Lyso-PC contents of
[14C]lyso-PC–Ox-LDL was 112 nmol/mg protein. Data
represent the mean±SD of 3 separate experiments.
Glucocorticoids Inhibit Ox-LDL–Induced GM-CSF Production
We have recently demonstrated that Ox-LDL–induced
production of GM-CSF plays an important role in Ox-LDL–induced
macrophage growth as a growth priming factor.14
Thus, we next examined the effect of glucocorticoids on Ox-LDL–induced
GM-CSF production into the medium using ELISA method. As shown
in Figure 5, addition of 20 µg/mL of
Ox-LDL significantly induced GM-CSF release into the medium, and the
maximal release was observed at 6 hours, although it decreased to the
basal level at 24 hours. When macrophages were incubated with
Ox-LDL together with different concentrations of
dexamethasone, Ox-LDL–induced GM-CSF release was inhibited
in a dose-dependent manner (Figure 5). Figure 6 shows that prednisolone and cortisol
also significantly inhibited GM-CSF release, whereas estradiol and
testosterone had no effect. Among the glucocorticoids tested in the
present study, dexamethasone was the most potent
inhibitor of GM-CSF release, a finding consistent
with its inhibitory effect on Ox-LDL–induced
[3H]thymidine incorporation into
macrophages (Figure 2
). Figure 6 also shows that
RU-486 abrogated dexamethasone-mediated inhibition of
Ox-LDL–induced GM-CSF release.
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), or Ox-LDL with 10 nmol/L of
dexamethasone (
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To elucidate whether dexamethasone inhibits GM-CSF release
from macrophages at the mRNA level, we determined the level of
GM-CSF mRNA in macrophages using RT-PCR analysis with
parallel determination of ß-actin mRNA as a control. As shown in
Figure 7, the levels of ß-actin mRNA
were not affected by Ox-LDL, dexamethasone, and/or RU-486.
In contrast, Ox-LDL caused a significant induction of GM-CSF mRNA,
which was significantly inhibited by dexamethasone.
Consistent with the effect of RU-486 on thymidine incorporation
(Figure 3), dexamethasone-mediated inhibition of
Ox-LDL–induced GM-CSF mRNA expression was significantly abrogated by
RU-486 (Figure 7).
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Effects of Dexamethasone or RU-486 on Ox-LDL–Induced
Growth of Human Monocyte-Derived Macrophages
Finally, to elucidate the effect of dexamethasone on
Ox-LDL–induced human macrophage growth, human monocyte-derived
macrophages (2x104 cells/well) were
incubated with 40 µg/mL of Ox-LDL in the presence or absence of 10
nmol/L of dexamethasone or 1 µmol/L of RU-486 for 7
days, and then cell numbers were counted. Ox-LDL–induced increase in
the number of human monocyte-derived macrophages from
1.9x104 cells/well to
3.1x104 cells/well was significantly inhibited
by dexamethasone to 2.2x104
cells/well. Dexamethasone-mediated inhibition of
macrophage growth was abrogated by RU-486 to
2.9x104 cells/well. These results suggested that
dexamethasone-mediated inhibition of Ox-LDL–induced human
macrophage growth might be also mediated by activation of the
glucocorticoid receptor.
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Discussion |
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Previous studies have demonstrated that dexamethasone inhibits atherosclerosis in various experimental models.16 17 18 19 Because macrophage-derived foam cells play an important role in the development and progression of atherosclerosis1 and were reported to proliferate in the atherosclerotic lesions,5 6 7 we investigated in the present study the effect of dexamethasone on the Ox-LDL–induced macrophage growth. Our results showed that glucocorticoids, including dexamethasone, prednisolone, and cortisol, significantly inhibited Ox-LDL–induced macrophage growth (Figures 1
and 2, and the Table). Furthermore, these glucocorticoids
significantly inhibited Ox-LDL–induced GM-CSF production
(Figures 5 to 7), an effect prevented by a
glucocorticoid receptor antagonist, RU-486 (Figures 3 and 7). These results suggested that the
inhibitory effect of glucocorticoids on Ox-LDL–induced
macrophage growth might be mediated by inhibition of
Ox-LDL–induced GM-CSF production through the activation of
glucocorticoid receptor.
In cholesterol-fed rabbits, administration of
glucocorticoid suppresses the development of atherosclerotic
lesions17 and reduces macrophage cell counts in
atherosclerotic lesions.24 These effects were partially
explained by a reduction in chemotaxis of circulating
monocytes.22 In the present study, we demonstrated a
clear inhibitory effect of dexamethasone on
Ox-LDL–induced macrophage growth (Figure 1 and the
Table). Moreover, Hamilton36 demonstrated that
dexamethasone inhibited M-CSF–induced growth of
mouse peritoneal macrophages. Becuause Ox-LDL and M-CSF are
present in atherosclerotic lesions,1 2 3 4 in which
macrophage growth is also reported,5 6 7 inhibition
of macrophage growth by dexamethasone might be a
possible mechanism that reduces macrophage cell counts in
atherosclerotic lesions. It is possible that such an effect may lead to
the prevention of development and/or progression of
atherosclerosis in cholesterol-fed
rabbits.
Based on our recent study that internalization of lyso-PC into
macrophages plays an important role in Ox-LDL–induced
macrophage growth,13 14 we believe that the
inhibitory mechanism of dexamethasone on
Ox-LDL–induced macrophage growth is partially explained by the
inhibition of uptake of Ox-LDL and lyso-PC (Figure 4). However,
when macrophages were incubated with 20 µg/mL of Ox-LDL
together with 10 nmol/L of dexamethasone, inhibition of
macrophage growth and uptake of lyso-PC were 80% and 20%,
respectively (Figures 2 and 4). Based on these results,
we postulate the presence of other major inhibitory
mechanism(s) of Ox-LDL–induced macrophage growth.
Glucocorticoids, such as dexamethasone, prednisolone, and
cortisol, significantly inhibited Ox-LDL–induced GM-CSF
production (Figures 5 and 6), which was
consistent with their inhibitory effect on
macrophage growth (Figure 2 and the Table). These
results suggested that inhibition of GM-CSF production by
glucocorticoids might cause inhibition of Ox-LDL–induced
macrophage growth. Our previous report15 and the
present study demonstrated that Ox-LDL could induce an increase in
GM-CSF mRNA level and protein release. Moreover, our subsequent
preliminary experiments using luciferase assay in which we examined the
promoter activity of GM-CSF gene showed that Ox-LDL activated
reporter gene expression, suggesting that Ox-LDL could enhance GM-CSF
mRNA expression at transcription level. However, post-transcriptional
regulation of GM-CSF in macrophages is still unknown at
present; the mechanisms of the inhibitory effect of
dexamethasone are also poorly understood. Several
mechanisms might explain the inhibitory effect of
glucocorticoids on GM-CSF expression. Dexamethasone
inhibits phorbol 12-myristate 13-acetate–induced phospholipase
A2 expression in mouse macrophages via
suppression of several kinase activities in the signal
chain,37 suggesting that dexamethasone might
suppress Ox-LDL–induced signal transduction for GM-CSF expression.
Moreover, glucocorticoid receptor itself is a transcriptional factor
and the binding of glucocorticoid receptor to glucocorticoid regulating
element in promoter lesion of various genes regulates their expression
positively or negatively.38 39 Therefore, it is possible
that glucocorticoids may inhibit Ox-LDL–induced macrophage
growth through inhibition of certain gene expression required for
Ox-LDL–induced macrophage growth, including GM-CSF.
Furthermore, glucocorticoid receptor is known to bind to
activator protein-1 (AP-1) and then inhibit AP-1 action as
a transcription factor.40 In T-lymphocytes, AP-1 plays an
important role in GM-CSF expression induced by activation of protein
kinase C (PKC).41 42 43 44 45 46 In this regard, we recently
demonstrated that Ox-LDL initiated an increase in intracellular
Ca2+ with subsequent activation of
PKC.47 In a series of preliminary experiments, we also
showed that AP-1 and AP-2 are important transcription factors in GM-CSF
expression in mouse macrophages using gel retardation and
luciferase assays (Matsumura et al, unpublished data, 1998).
Moreover, Ares et al48 demonstrated that Ox-LDL induced
AP-1 activation in human vascular smooth muscle cells. Thus, we
speculate that activation of the glucocorticoid receptor by
dexamethasone may inhibit AP-1 activation, thereby
inhibiting the expression of GM-CSF in macrophages.
Dexamethasone inhibits the growth of several cell types, such as the growth of interleukin-6–induced myeloma cells,49 thrombin-induced growth of human smooth muscle cells,50 serum-induced growth of mouse fibroblasts,51 and mouse uterine epithelial cells.52 Recently, dexamethasone has also been shown to arrest G1 cell cycle in rat hepatoma cells53 through the expression of CCAAT/enhancer-binding protein alpha.54 Moreover, dexamethasone enhanced an expression of p21 waf1/cip1, a cyclin-dependent kinase inhibitor, which induced G1 arrest in rat hepatoma cell, lung alveolar cells, and mouse fibroblasts.55 56 57 Thus, it is possible that the cell cycle of macrophages might also be arrested in G1 phase by dexamethasone in Ox-LDL–induced macrophage growth.
In the present study, we demonstrated that glucocorticoids inhibited macrophage growth. Moreover, several other anti-atherogenic properties of glucocorticoids have been reported in vitro20 21 22 23 24 and in vivo studies.16 17 18 19 However, it is still not clear whether glucocorticoid is a useful agent against atherosclerosis in humans. Because glucocorticoids have various side effects that might worsen atherosclerosis, such as increase in blood pressure, induction of hypercholesterolemia and hypertriglyceridemia, impairment of glucose tolerance, and imbalance of thrombosis and fibrinolysis,58 59 long-term clinical trials have not been performed to elucidate the effect of glucocorticoid in normal human subjects. Interestingly, treatment with glucocorticoid in certain patients with rheumatoid arthritis60 and systemic lupus erythematosus61 is associated with increased mortality due to cardiovascular diseases. The exact reasons for the discrepancy between the effect of glucocorticoids on such patients and animal models remains to be resolved. Further epidemiological and pathological studies might solve this issue if a safe local delivery system of glucocorticoid into the atherosclerotic lesions or a glucocorticoid-derivative without these side effects is developed in the future.
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Acknowledgments |
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We are grateful to Drs Akira Miyazaki and Hideki Hakamata and Prof Seikoh Horiuchi from the Department of Biochemistry, Kumamoto University School of Medicine, for their helpful discussion. This work was supported in part by a grant from Ono Medical Research Foundation.
Received October 6, 1998; accepted December 22, 1998.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modification of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]
3.
Palinski W, Rosenfeld ME., Ylä-Herttuala S,
Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg
D, Witztum JL. Low density lipoprotein undergoes oxidative modification
in vivo. Proc Natl Acad Sci U S A. 1989;86:1372–1376.
4. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086–1095.
5.
Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell
proliferation inhuman coronary arteries. Proc Natl Acad
Sci U S A. 1990;87:4600–4604.
6.
Rosenfeld ME, Ross R. Macrophage and smooth
muscle cell proliferation in atherosclerotic lesions of WHHL and
comparably hypercholesterolemic fat-fed rabbits.
Arteriosclerosis. 1990;10:680–687.
7. Spagnoli LG, Orlandi A, Santeusanio G. Foam cells of the rabbit atherosclerotic plaque arrested in metaphase by colchicine show a macrophage phenotype. Atherosclerosis. 1991;88:87–92.[Medline] [Order article via Infotrieve]
8.
Yui S, Sasaki T, Miyzaki A, Horiuchi S, Yamazaki M.
Induction of murine macrophage growth by modified LDLs.
Arterioscler Thromb. 1993;13:331–337.
9.
Martens JS, Reiner NE, Herrena-Velit P, Steinbrecher
UP. Phosphatidylinositol 3-kinase is involved in the induction of
macrophage growth by oxidized low density lipoprotein.
J Biol Chem. 1998;273:4915–4920.
10.
Sakai M, Miyazaki A, Hakamata H, Sasaki T, Yui S,
Yamazaki M, Shichiri M, Horiuchi S. Lysophosphatidylcholine plays an
essential role in the mitogenic effect of oxidized low
density lipoprotein on murine macrophages. J Biol
Chem. 1994;269:31430–31435.
11. Sato Y, Kobori S, Sakai M, Yano T, Higashi T, Matsumura T, Morikawa W, Terano T, Miyazaki A, Horiuchi S, Shitiri M. Lipoprotein(a) induces cell growth in rat peritoneal macrophages through inhibition of transforming growth factor-ß activation. Atherosclerosis. 1996;125:15–26.[Medline] [Order article via Infotrieve]
12.
Sakai M, Miyazaki A, Hakamata H, Sasaki T, Yui S,
Yamazaki Y, Shichiri M, Horiuchi S. Lysophosphatidylcholine potentiates
the mitogenic activity of modified LDL for human
monocyte-derived macrophages. Arterioscler Thromb Vasc
Biol. 1996;16:600–605.
13.
Sakai M, Miyazaki A, Hakamata H, Kodama T, Suzuki H,
Kobori S, Shichiri M, Horiuchi S. The scavenger receptor serves as a
route for internalization of lysophosphatidylcholine in oxidized low
density lipoprotein-induced macrophage proliferation.
J Biol Chem. 1996;271:27346–27352.
14. Sakai M, Shichiri M, Hakamata H, Horiuchi S. Endocytosed lysophosphatidylcholine, through the scavenger receptor, play an essential role in oxidized low density lipoprotein- induced macrophage proliferation. Trends Cardiovasc Med. 1998;8:119–124.
15.
Biwa T, Hakamata H, Sakai M, Miyazaki A, Suzuki H,
Kodama T, Shichiri M, Horiuchi S. Induction of murine
macrophage growth by oxidized low density lipoprotein is
mediated by granulocyte macrophage colony-stimulating factor.
J Biol Chem. 1998;273:28305–28313.
16. Makheja AN, Bloom S, Muesing R, Simon T, Bailey JM. Anti-inflammatory drugs in experimental atherosclerosis in WHHL rabbits and inhibition by cortisone acetate. Atherosclerosis. 1989;76:155–161.[Medline] [Order article via Infotrieve]
17. Naito M, Yasue M, Asai K, Yamada K, Hayashi T, Kuzuya M, Funaki C, Yoshinine N, Kuzuya F. Effects of dexamethasone on experimental atherosclerosis in cholesterol-fed rabbits. J Nutr Sci Vitamin. 1992;38:255–264.
18. Hayashi H, Nomoto A, Mutoh S, Yamaguchi I, Ono T. Role of inflammatory response in initiation of atherosclerosis: effects of anti-inflammatory drugs on cuff-induced leukocyte accumulation and intimal thickening of rabbit carotid artery. Atherosclerosis. 1991;91:107–116.[Medline] [Order article via Infotrieve]
19. Villa AE, Guzman LA, Chen W, Golomb G, Levy RJ, Topol EJ. Local delivery of dexamethasone for prevention of neointimal proliferation in a rat model of balloon angioplasty. J Clin Invest. 1994;93:1243–1249.
20. Van Put DJ, Van Hove CE, De Meyer GR, Wuyts F, Herman AG, Bult H. Dexamethasone influences intimal thickening and vascular reactivity in the rabbit collared carotid artery. Eur J Pharmacol. 1995;294:753–761.[Medline] [Order article via Infotrieve]
21. Voisard R, Seitzer U, Baur R, Dartsch PC, Osterhues H, Hoher M, Hombach V. Corticosteroid agents inhibit proliferation of smooth muscle cells from human atherosclerotic arteries in vitro. Int J Cardiol. 1994;43:257–267.[Medline] [Order article via Infotrieve]
22. Yamada K, Naito M, Hayashi T, Asai K, Yoshimine N, Iguchi A. Effects of dexamethasone on migration of human monocytes in response to oxidized beta-very low density lipoprotein. Artery. 1993;20:253–267.[Medline] [Order article via Infotrieve]
23. Prescott MF, McBride CK, Court M. Development of intimal lesions after leukocyte migration into the vascular wall. Am J Pathol. 1989;135:835–846.[Abstract]
24.
Asai K, Funaki C, Hayashi T, Yamada K, Naito M, Kuzuya
M, Yoshida F, Yoshimine N, Kuzuya F.
Dexamethasone-induced suppression of aortic
atherosclerosis in cholesterol-fed rabbits:
possible mechanisms. Arterioscler Thromb Vasc Biol. 1993;13:892–899.
25. Sakai M, Miyazaki A, Sakamoto Y, Horiuchi S. Cross-linking of apolipoprotein is involved in a loss of the ligand activity of high density lipoprotein upon Cu2+-mediated oxidation. FEBS Lett. 1992;314:199–202.[Medline] [Order article via Infotrieve]
26. Miyazaki A, Rahim ATMA, Araki S, Morino Y, Horiuchi S. Chemical cross-linking alters high-density lipoprotein to be recognized by a scavenger receptor in rat peritoneal macrophages. Biochim Biophys Acta. 1991;1082:143–151.[Medline] [Order article via Infotrieve]
27. Fogelman AM, Haberland ME, Seager J, Hokom M, Edwards. Factors regulating the activities of the low density lipoprotein receptor and the scavenger receptor on human monocyte-macrophages. J Lipid Res. 1981;22:1131–1141.[Abstract]
28. Hakamata H, Miyazaki A, Sakai M, Sakamoto Y, Matsuda H, Kihara K, Horiuchi S. Differential effects of an acyl-coenzyme A: cholesterol acyltransferase inhibitor on HDL- induced cholesterol efflux from rat macrophage foam cells. FEBS Lett. 1995;363:29–32.[Medline] [Order article via Infotrieve]
29.
Miyazaki A, Sakai M, Suginohara Y, Hakamata H, Sakamoto
Y, Horiuchi S. Acetylated low density lipoprotein reduces its
ligand activity for the scavenger receptor after interaction with
reconstituted high density lipoprotein. J Biol Chem. 1994;269:5264–5269.
30. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1989.
31. Miyatake S, Otsuka T, Yokota T, Lee F, Arai K. Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes. EMBO J. 1985;4:2561–2568.[Medline] [Order article via Infotrieve]
32. Alonso S, Minty A, Bourlet Y. Comparison of three actin-coding sequences in the mouse: evolutionary relationships between the actin genes of warm-blooded vertebrates. J Mol Evol. 1986;23:11–22.[Medline] [Order article via Infotrieve]
33. Agarwai MK. The antiglucocorticoid action of mifepristone. Pharmacol Therapeut. 1996;70:183–213.[Medline] [Order article via Infotrieve]
34. Hirsch LJ, Mazzone T. Dexamethasone modulates lipoprotein metabolism in cultured human monocyte-derived macrophages: stimulation of scavenger receptor. J Clin Invest. 1986;77:485–490.
35.
Moulton KS, Wu H, Barnett J, Parthasarathy S, Glass CK.
Regulated expression of the human acetylated low density
lipoprotein receptor gene and isolation of promoter sequences.
Proc Natl Acad Sci U S A. 1992;89:8102–8106.
36. Hamilton JA. Glucocorticoids and prostaglandin inhibit the induction of macrophage DNA synthesis by macrophage growth factor and phorbol ester. J Cell Physiol. 1983;115:67–74.[Medline] [Order article via Infotrieve]
37. Gewert K, Sundler R. Dexamethasone down-regulates the 85 kDa phospholipase A2 in mouse macrophages and suppresses its activation. Biochem J. 1995;307:499–504.
38. Green S, Chambon P. Nuclear receptors enhance our understanding of transcription regulation. Trend Genet. 1988;4:309–314.[Medline] [Order article via Infotrieve]
39.
Evans RM. The steroid and thyroid hormone receptor
family. Science. 1988;240:889–895.
40. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–157.[Medline] [Order article via Infotrieve]
41. Brorson KA, Beverly B, Kang S-M, Lenaldo M, Schwartz RH. Transcriptional regulation of cytokine genes in nontransformed T Cells. J Immunol. 1991;147:3601–3609.[Abstract]
42.
Gasson JC. Molecular Physiology of
Granulocyte-Macrophage Colony-Stimulating Factor.
Blood. 1991;77:1131–1145.
43.
Sugimoto K, Tuboi A, Miyatake S, Arai K, Arai N.
Inducible and non-inducible factors co-operatively activate the
GM-CSF promoter by interacting with two adjacent DNA motifs.
Inter Immunol. 1990;2:787–794.
44.
Masuda ES, Tokumitu H, Tsuboi A, Shilomai J, Hung P,
Arai K-I, Arai N. The granulocyte-macrophage colony-stimulating
factor promoter cis-acting element CLE0 mediates induction signals in T
cells and is recognized by factors related to AP-1 and NFAT. Mol
Cell Biol. 1993;13:7399–7407.
45.
Wang C-Y, Bassuk AG, Boise LH, Thompson CB. Bravo R,
Leiden JM. Activation of the granulocyte-macrophage
colony-stimulating factor promoter in T cells requires cooperative
binding of Elf-1 and AP-1 transcription factors. Mol Cell
Biol. 1994;14:1153–1159.
46.
Tuboi A, Muramatsu M, Tsutsumi A, Arai K, Arai N.
Calcineurin activates transcription from the GM-CSF promoter in
synergy with either protein kinase C or NF-
B/AP-1 in T cells.
Biochem Biophys Res Commun. 1994;199:1064–1072.[Medline]
[Order article via Infotrieve]
47.
Matsumura T, Sakai M, Kobori S, Biwa T, Takemura T,
Matsuda H, Hakamata H, Horiuchi S. Shichiri M. Two intracellular
signaling pathways for activation of protein kinase C are involved in
oxidized low density lipoprotein-induced macrophage growth.
Arterioscler Thromb Vasc Biol. 1997;17:3013–3020.
48.
Ares MPS, Kallin B, Eriksson J, Nelson J. Oxidized LDL
induces transcription factor activator protein-1 but
inhibits activation of nuclear factor-B in human vascular
smooth muscle cells. Arterioscler Thromb Vasc Biol. 1995;15:1584–1590.
49.
Chen YH, Desai P, Shiao RT, Lavelle D, Haleem A, Chen
J. Inhibition of myeloma cell growth by dexamethasone
and all-trans retinoic acid: synergy through modulation of
interleukin-6 autocrine loop at multiple sites. Blood. 1996;87:314–323.
50.
Nakano T, Raines EW, Abraham JA, Wenzel FG 4th,
Higashiyama S, Klagburn M, Ross R. Glucocorticoid inhibits
thrombin-induced expression of platelet-derived growth factor
A-chain and heparin-binding epidermal growth factor in human aortic
smooth muscle cells. J Biol Chem. 1993;268:22941–22947.
51.
Fagot D, Buquet-Fagot C, Mester J.
Antimitogenic effects of dexamethasone in
chemically transformed mouse fibroblasts. Endocrinology. 1991;129:1033–1041.
52. Bigsby RM, Cunha GR. Progesterone and dexamethasone inhibition of uterine epithelial proliferation in two models of estrogen-independent growth. Am J Obstet Gynecol. 1988;158:646–650.[Medline] [Order article via Infotrieve]
53. Sanchez I, Goya L, Vallerga AK, Firestone GL. Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing an early G1 block in cell cycle progression. Cell Growth Differ. 1993;4:215–225.[Abstract]
54. Ramos RA, Nishiono Y, Maiyar AC, Simon KE, Ridder CC, Ge Y, Firestone GL. Glucocorticoid-stimulate CCAAT/enhancer binding protein alpha expression is required for steroid-induced G1 cell cycle arrest of minimal-deviation rat hepatoma cells. Molec Cell Biol. 1996;16:5288–5301.[Abstract]
55.
Cha HH, Cram EJ, Wang EC, Huang AJ, Kasler HG,
Firestone GL. Glucocorticoids stimulate p21 gene expression by
targeting multiple transcriptional elements within a steroid responsive
region of the p21 waf1/cip1 promoter in rat hepatoma cells.
J Biol Chem. 1998;273:1998–2007.
56.
Corroyer S, Nabeyrat E, Clement A. Involvement of the
cell cycle inhibitor CIP1/WAF1 in lung alveolar epithelial
cell growth arrest induced by glucocorticoids.
Endocrinology. 1997;138:3677–3685.
57.
Ramalingam A, Hirai A, Thimpson EA. Glucocorticoid
inhibition of fibroblast proliferation and regulation of the cyclin
kinase inhibitor p21 Cip. Mol Endocrinol. 1997;11:577–586.
58. Nashel DJ. Is atherosclerosis a complication of long-term corticosteroid treatment? Am J Med. 1986;80:925–929.[Medline] [Order article via Infotrieve]
59.
Maxwell SR, Moots RJ, Kendall MJ.
Corticosteroids: do they damage the
cardiovascular system? Postgrad Med J.. 1994;70:863–870.
60.
Kalbak K. Incidence of atherosclerosis
in patients with rheumatoid arthritis received long-term
corticosteroid therapy. Ann Rheum Dis. 1972;31:196–201.
61. Bulkley BH, Roberts WC. The heart in systematic lupus erythematosus and the changes induced in it by corticosteroid therapy. Am J Med. 1975;58:246–264.
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