© 2001 American Heart Association, Inc.
Vascular Biology |
Induction of Endothelial-Leukocyte Interaction by Interferon-
Requires Coactivation of Nuclear Factor-
B
From the Vascular Medicine Unit (T.B., U.L., V.L.F., P.L., J.K.L.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass; the CNR Institute of Clinical Physiology (R.D.C., G.L.), Pisa, and the Cardiovascular Division (R.D.C., G.L.), "G. D’Annunzio" University, Chieti, Italy; and the Department of Pathology (A.S.N.), Emory University Hospital, Atlanta, Ga.
Correspondence to James K. Liao, MD, Vascular Medicine and Atherosclerosis Unit, Brigham and Women’s Hospital, 221 Longwood Ave, LMRC-322, Boston, MA 02115. E-mail jliao{at}rics.bwh.harvard.edu
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Abstract—To determine whether nuclear factor (NF)-
B is necessary to confer
endothelial cell responsiveness to interferon (INF)-
in terms of vascular cell adhesion molecule (VCAM)-1 expression and
leukocyte adhesion, human endothelial cells were
treated with IFN- in the presence of low concentrations (LCs) of
interleukin (IL)-1
(
100 pg/mL), which activates NF-B
but does not induce VCAM-1 expression. Although IFN- induced major
histocompatibility complex class II antigen expression and although a
high concentration of IL-1 (10 ng/mL) induced leukocyte adhesion and
VCAM-1 expression, neither IFN- nor LC IL-1 was able to induce
VCAM-1 expression or leukocyte adhesion. However, the combination of
IFN- and LC IL-1 induced VCAM-1 expression and increased
leukocyte adhesion (67% and 49% of high-concentration IL-1,
respectively). Electrophoretic mobility shift assays and
immunoblotting of nuclear extracts showed that IFN-
activated signal transducers and activators of
transcription (STAT)-1 and interferon regulatory factor (IRF)-1 but
not NF-B, whereas LC IL-1 activated NF-B but not
STAT-1 or IRF-1. Nuclear run-on studies showed that LC IL-1 is
necessary but not sufficient for inducing VCAM-1 gene transcription and
that the combination of IFN- and LC IL-1 is required for full
VCAM-1 gene transcription. These findings suggest that factors that
activate NF-B can synergize with IFN- in promoting
endothelial-leukocyte interaction.
Key Words: endothelium • adhesion molecules • cytokines • inflammation
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Introduction |
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The adhesion of leukocytes to vascular endothelium importantly contributes to many inflammatory and immunologic disorders, including early atherogenesis, in which monocyte but not granulocyte accumulation characterizes the formation of fatty streaks.1 Multiple protein families, including chemoattractants and cellular adhesion molecules, provide "traffic signals" for monocyte attachment to the vascular wall. These mediators include molecules overexpressed in atherosclerotic lesions, such as vascular cell adhesion molecule (VCAM)-1. The relatively monocyte-selective "inflammatory" state of the vascular endothelium during early atherogenesis is possibly explained by the fact that the VCAM-1 ligand, the integrin very late antigen-4, is expressed on monocytes but not on neutrophils.2
Cytokines such as interleukin (IL)-1 and tumor
necrosis factor (TNF)-, induce endothelial VCAM-1
expression and the adhesion of monocytes to the
endothelial
surface.3 The ability of these
cytokines to promote VCAM-1 gene expression relates, in part,
to their ability to activate the transcription factor nuclear
factor (NF)-B.4 However,
recent analyses of the VCAM-1 promoter suggest that NF-B
activation alone may not suffice to induce VCAM-1 expression and that
other transcription factors, such as interferon regulatory factor
(IRF)-1, are required for the full induction of VCAM-1 gene
transcription.5 Indeed, IRF-1
has been found to synergize with NF-B in transactivating
cytokine-inducible genes, such as inducible type II NO
synthase6 and
VCAM-1.7
A major product of activated T
lymphocytes, interferon (IFN)-, induces functional changes in the
vascular endothelium, including the expression of major
histocompatibility complex (MHC) class II
antigens.8 9 10
Activated T lymphocytes and MHC-II–positive vascular smooth
muscle cells (SMCs) are colocalized within atherosclerotic
lesions,11 12 and
recent evidence suggests that T lymphocytes contribute to the
development and rupture of atherosclerotic
plaques.13 Interestingly,
IFN-, which is an inducer of IRF-1, cannot alone induce
endothelial VCAM-1 expression or substantial monocyte
adhesion to endothelial cells. In contrast, IFN-
potently stimulates VCAM-1 expression in
SMCs.14 Because SMCs, but not
endothelial cells, in serum-containing media display
basal NF-B
activity,15 16 we
hypothesized that NF-B activation may permit IFN-–induced VCAM-1
expression in SMCs but not endothelial cells.
Therefore, the aim of the present study was to determine whether
minimal activation of NF-B, which in itself is insufficient to
induce VCAM-1 expression, is required for the induction of VCAM-1
expression by IFN-.
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Methods |
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Materials
Human recombinant IL-1
was obtained from
Hoffmann-La Roche. IL-1ß, TNF-, and IFN- were purchased from
Genzyme. The monoclonal antibodies to VCAM-1 (Ab E1/6), E-selectin (Ab
H18/7), ICAM-1 (Ab HU 5/3), and MHC class I (Ab W6/32) were kindly
provided by M.A. Gimbrone (Brigham & Women’s Hospital, Boston, Mass).
The monoclonal antibody to MHC class II (HLA-DR, Ab I-2/IA antigen)
was kindly provided by Arnold Freedman (Dana Farber Cancer Institute,
Boston, Mass). Unless otherwise specified, all other reagents were
obtained from Sigma Chemical Co.
Cell Cultures
Human saphenous vein endothelial
cells were harvested enzymatically with type II collagenase
at 0.1% as
described17 18 and
were maintained in medium 199 (GIBCO-BRL) containing HEPES (25
mmol/L), heparin (50 U/mL), endothelial cell growth
factor (50 µg/mL),
L-glutamine (5
mmol/L), antibiotics, and 5% FCS (Hyclone). Once they were grown to
confluence, the cells were replated on low pyrogen fibronectin (1.5
µg/cm2) at 20 000
cells/cm2. Human saphenous vein
endothelial cells isolated by these techniques form a
confluent monolayer of polygonal cells and express von
Willebrand factor as determined by their content of specific
mRNA and immunoreactive
protein.17 Cell number was
assessed after trypsinization in a Neubauer hemocytometer (VWR
Scientifics). Cellular viability was assessed by trypan blue exclusion.
Bovine aortic endothelial cells
(106 per 100-mm2
culture dish, grown in DME medium with 5% heat-inactivated
FCS) were used within passage 3 for transfection experiments. Cell
number was assessed by direct cell counting of adherent cells, after
trypsin detachment, in a Neubauer hemocytometer and staining by trypan
blue. The percentage of cells excluding trypan blue was taken as a
measure of cell viability.
Cell-Surface Enzyme Immunoassay
The expression of cell-surface adhesion molecules was
determined by enzyme immunoassays. The assays were performed by
incubating endothelial monolayers with specific
monoclonal antibodies (1:10 to 1:1000 dilutions), followed by the
addition of biotinylated goat anti-mouse IgG (Vector Labs, Inc), and
then with streptavidin–alkaline phosphatase (Zymed).
Endothelial monolayers were washed 3 times with PBS
between each incubation step, and the integrity of the monolayers was
monitored by phase-contrast microscopy. The surface expression of each
protein was determined spectrophotometrically at an absorbance of 450
nm after the addition of the chromogenic substrate
3,3',5,5'-tetramethylbenzidine.
Leukocyte Adhesion Assay
Monocytoid U937 cells were obtained from American
Tissue Culture Collection and grown in RPMI medium 1640 (GIBCO-BRL)
containing 10% FCS. The U937 cells were concentrated by
centrifugation to 1x106
cells/mL. For the adhesion assays, endothelial cells
were grown to confluence in 6-well tissue culture plates, after which
IFN- (10 to 1000 U/mL) or IL-1 (0.01 to 10 ng/mL) or both were
added for an additional 24 or 48 hours to allow for the induction of
VCAM-1 and MHC-II antigen, respectively. For control, some monolayers
were pretreated with a monoclonal antibody against VCAM-1 (E1/6). The
adhesion assay was performed by adding 1 mL of the concentrated U937
cell suspension to each monolayer under rotating conditions (63 rpm) at
21°C.18 After 10 minutes,
nonadhering cells were removed by gentle washing with medium 199, and
the monolayers were fixed with 1% paraformaldehyde.
The number of adherent cells was determined by counting 6 different
fields with use of an ocular grid and a x20 objective (0.16
mm2 per field). Microscopic fields chosen
for counting adherent leukocytes were randomly selected at half-radius
distance from the center of the monolayers.
Nuclear Run-On Assay
Nuclei from 3 to 5x107
endothelial cells were prepared 16 hours after
stimulation with IL-1 (low and high concentrations) and IFN-,
either alone or in combination, and in vitro transcription with
[-32P]UTP (800 Ci/mmol) was performed
as described.19 Linearized
plasmids (1 µg) were immobilized on nylon membranes,
hybridized to radiolabeled transcripts (
5 to
8x107 cpm/mL) at 45°C for 48 hours in
hybridization buffer containing 50% formamide, 5x SSC, 2.5x
Denhardt’s solution, 25 mmol/L sodium phosphate buffer (pH 6.5),
0.1% SDS, and 250 µg/mL salmon sperm DNA, and washed in 1x
SSC/0.1% SDS at 65°C before autoradiography.
Densitometric analyses of autoradiographic bands
for Northern hybridization were performed with the aid of Image
software (National Institutes of
Health).20
Electrophoretic Mobility Shift Assay
Endothelial cells were grown to
confluence (5x105 cells) in 10-cm Petri
dishes in serum-containing medium. After stimulation with appropriate
cytokines for the indicated time intervals, the cells were
scraped and collected into prechilled microfuge tubes. Nuclear and
cytosolic extracts were prepared according to Dignam et
al,21 with the additional
step of washing nuclear pellets in low-salt buffer before high-salt
extraction of nuclear proteins to remove any residual cytosolic
contaminants. Aliquots were assayed for protein concentration by the
BCA method (Pierce). Dithiothreitol was added to a final concentration
of 1 mmol/L, and extracts were stored at -80°C.
The oligonucleotide
5'-AGTTGAGGGGACTTTCCCAGGC-3',
corresponding to the tandem B binding site in the human VCAM-1
promoter, and a mutant oligonucleotide with a G
C
substitution in the third nucleotide of the consensus motif
were used to assess NF-B activation. The
oligonucleotide
5'-CATGTTATGCATATTCCTGTAAGTG-3',
containing the consensus binding site for signal transducers and
activators of transcription (STAT)-1 (p91), and a mutant
oligonucleotide with a CCTGGA substitution were used
to assess STAT-1 activation. The oligonucleotide
5'-GGAGTGAAATAGAAAGTCTG-3', corresponding to the IFN-stimulatory
response element (ISRE) site in the human VCAM-1 promoter was used to
assess IRF-1 binding. All oligonucleotides were either
synthesized or obtained from Santa Cruz Biotechnology. The
oligonucleotides were end-labeled by T4
polynucleotide kinase (New England Biolabs) and
[-32P]ATP (3000 Ci/mmol) and purified
by Sephadex G-50 columns (Pharmacia).
Nuclear extract (10 µg) was added to
32P-labeled oligonucleotides
(20 000 cpm) in a buffer containing 2 µg poly[dI·dC]
(Boehringer-Mannheim), 10 µg BSA, 10 mmol/L Tris-HCl (pH
7.5), 50 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L
EDTA, and 5% glycerol. After a 30-minute incubation, DNA-protein
complexes were resolved on 4% nondenaturing polyacrylamide
gels electrophoresed at 12 V/cm in 0.5x Tris-borate EDTA buffer.
Specificity was determined by the addition of RelA (p65), p50, or
STAT-1 antibodies (2 µg IgG per reaction, Santa Cruz
Biotechnology) or excess unlabeled (cold) or mutant
oligonucleotides (20 ng) to the nuclear extracts for 10
minutes before the addition of radiolabeled
probe.
Immunoblotting
Whole-cell lysates were prepared in 2x SDS lysis
buffer (250 mmol/L Tris-HCl [pH 6.8], 20% glycerol, 4% SDS,
and 5% 2-mercaptoethanol), as
described.22 Equivalent
amounts of whole-cell lysates (30 to 40 µg of protein) or nuclear or
cytosolic fractions (prepared as described below) were resolved on 10%
or 12% SDS-polyacrylamide gels, followed by electrophoretic
transfer to polyvinylidene difluoride membranes
(Millipore).
Membranes were incubated in PBS containing 0.1% Tween 20 (PBS-T) with 5% nonfat dry milk for 1 hour at 37°C and then incubated for 1 hour with primary antibodies used at 0.4 µg/mL. Membranes were washed with PBS-T and incubated with horseradish peroxidase–conjugated donkey anti-rabbit IgG as a secondary antibody (Jackson Laboratories), which was diluted 1:15 000 in PBS-T and 5% dry milk. Protein bands were visualized with the use of Renaissance chemiluminescence reagents (DuPont NEN).
Statistical Analysis
Multiple comparisons were performed by 1-way ANOVA,
and individual differences were tested by the Fisher protected least
significance difference test after the demonstration of significant
intergroup differences by
ANOVA.
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Results |
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Cytokine-Induced Expression of VCAM-1 and MHC-II
There was little or no basal VCAM-1 or MHC-II expression in vascular endothelial cells. After stimulation with IL-1
(10 ng/mL), VCAM-1 expression peaked at 14
hours and remained steady for up to 24 to 28 hours before declining to
baseline at 48 hours. Similar VCAM-1 expression was observed with
IL-1ß (data not shown). The expression of MHC-II antigen after
IFN- (1000 U/mL) stimulation was much slower, appearing after 24
hours and peaking at 48 hours. Stimulation with low concentrations of
IL-1 (<0.2 ng/mL) did not induce endothelial VCAM-1
expression
(Figure 1
). However, there was a steep
concentration-dependent increase in VCAM-1 expression with higher
concentrations of IL-1 (1 to 10 ng/mL), with maximal VCAM-1
expression occurring at an IL-1 concentration of 10 ng/mL. However,
high concentrations of IL-1 (1 to 10 ng/mL) were unable to induce
MHC-II expression. Treatment with increasing concentrations of IFN-
(1 to 1000 U/mL) alone produced a gradual increase in MHC-II, but not
VCAM-1, expression. However, the combination of IFN- (1000 U/mL) and
a low concentration of IL-1 (0.1 ng/mL) significantly induced VCAM-1
expression (ie, 67% of high concentration of IL-1). Similar
synergistic effects of IFN- were obtained when low concentrations
(ie, subthreshold for VCAM-1) of TNF- (<0.2 ng/mL) were used
instead of IL-1.
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Leukocyte Adhesion to
Endothelial Cells
The effects of IL-1 (low and high concentrations)
and IFN-, either alone or in combination, on monocytoid cell (U937)
adhesion to endothelial cells were determined in a
rotational adhesion assay (please see Figure I, which can be accessed
online at http://atvb.ahajournals.org). Treatment of
endothelial cells with IFN- (1000 U/mL) alone had no
significant effect on U937 cell adhesion compared with control or
unstimulated endothelial cells (140±30 versus 150±40
cells/mm2,
P>0.05). Stimulation of
endothelial cells with a low concentration of IL-1
(0.1 ng/mL) modestly increased U937 cell adhesion (420±50
cells/mm2), whereas stimulation of
endothelial cells with a high concentration of IL-1
(10 ng/mL) substantially increased monocytoid cell adhesion
(24 700±3700 cells/mm2). However,
stimulation of endothelial cells with IFN- (1000
U/mL) and a low concentration of IL-1 (0.1 ng/mL) produced a 23-fold
increase in U937 cell adhesion (9840±1020
cells/mm2,
P<0.001). The induction of
VCAM-1 expression by IFN- and a low concentration of IL-1
accounted for 70% of U937 cell attachment, inasmuch as pretreatment of
endothelial cells with the blocking anti–VCAM-1
monoclonal antibody E1/6, at saturating concentrations, for 30 minutes
before the adhesion assay decreased the adhesion of these cells by 70%
(2950±320 cells/mm2,
P<0.01).
Endothelial VCAM-1 and
MHC-II Gene Transcription
To determine whether the regulation of VCAM-1
expression occurred at the level of gene transcription, we performed
nuclear run-on assays with nuclei isolated from
endothelial cells stimulated with IFN- and IL-1
(Figure 2). Treatment with IFN- (1000 U/mL) induced
MHC-II, but not VCAM-1, gene transcription, whereas the high
concentration of IL-1 (10 ng/mL) induced VCAM-1, but not MHC-II,
gene transcription. The low concentration of IL-1 (0.1 ng/mL) mildly
induced VCAM-1 gene transcription, but in combination with IFN-, it
substantially induced VCAM-1 gene transcription (ie, 80% of high
concentration of IL-1). Specificity was determined by the lack of
hybridization to the prokaryotic vector DNA, pGEM. Equivalent
ß-tubulin gene transcription confirmed comparable in vitro loading
conditions among the different treatment
conditions.
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Activation of NF-B and IRF-1
Because IL-1 is known to activate NF-B
and because IFN- is known to activate STAT-1 and IRF-1,
we performed electrophoretic mobility shift assays (EMSAs) and
immunoblotting on nuclear extracts isolated from
endothelial cells stimulated with IFN- and IL-1
to determine the potential roles of these transactivating factors in
VCAM-1 gene transcription. Stimulation with IFN- (1000 U/mL) alone
did not activate NF-B
(Figure 3). However, the low concentration of IL-1 (0.1
ng/mL) and, to a greater extent, the high concentration of IL-1 (10
ng/mL) induced NF-B activation. There was no further increase in
NF-B activation when endothelial cells were
stimulated with the combination of IFN- (1000 U/mL) and a low
concentration of IL-1 (0.1 ng/mL) compared with a low concentration
of IL-1 alone. Specificity of the NF-B band was evidenced by the
decreased band intensity in the presence of excess unlabeled B but
not mutated B oligonucleotides.
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Stimulation of the type II IFN receptor by IFN- leads to
the activation of Janus kinases and the subsequent tyrosine
phosphorylation of STATs and the transcriptional
induction of
IRF-1.22 23 By
EMSA, stimulation with IFN- (1000 U/mL), but not IL-1 (10 ng/mL),
leads to the activation of STAT-1 (p91)
(Figure 4). Specificity of the band corresponding to
STAT-1 decreased in the presence of excess unlabeled
-activated sequence (GAS) probe but not mutated GAS
oligonucleotides.
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The IRF-1 gene contains functional GAS
cis-acting elements for
transcriptional induction. In a time-dependent manner, IFN- (1000
U/mL) induced IRF-1 activation after 2 hours
(Figure 5). Specificity of the IRF-1 band is evidenced by the
obliteration of the IRF-1 band in the presence of excess cold or
unlabeled ISRE oligonucleotide. Furthermore,
immunoblotting of nuclear extracts with anti–IRF-1
antibody demonstrated that stimulation with IFN- (1000 U/mL), but
not IL-1 (0.1 ng/mL), induced the expression of IRF-1
(Figure 6). However, the combination of IFN- (1000 U/mL)
and IL-1 (0.1 ng/mL) did not increase IRF-1 activation compared with
that of IFN- alone. These findings indicate that IL-1 (0.1 ng/mL)
is unable to activate STAT-1 or
IRF-1.
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Discussion |
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Our findings indicate that IFN-
can induce
endothelial VCAM-1 expression in conjunction with
"subthreshold" concentrations of IL-1. The increase in VCAM-1
expression is due mainly to the increase in VCAM-1 gene transcription.
Our results also indicate that there is no overlapping activation of
transcription factors such as NF-B, STAT-1, and IRF-1 by these
cytokines at the concentrations used, with NF-B being
activated selectively by low concentrations of IL-1 and with
STAT-1 and IRF-1 being selectively activated by IFN-.
Thus, our findings suggest that the mechanism by which IFN- and low
concentrations of IL-1 induce VCAM-1 expression occurs via the
combined action of NF-B and IRF-1.
Although interaction between IL-1 and IFN- is a new
finding in the present study, such interactions between TNF- and
IFN- have been previously
documented.24 25 26
For example, the combination of TNF- and IFN- activates
the human class I MHC promoter synergistically, and this induction
requires the binding of both NF-B and IRF-1 to the class I MHC
promoter.25 Furthermore, a
synergistic interaction between transcription factors NF-B and IRF-1
has been recently reported for the inducible type II NO synthase
expression, whereby NF-B and IRF-1 cooperate in inducing inducible
type II NO synthase gene
transcription.6 Our findings
confirm this cooperative interaction in the VCAM-1 promoter,
particularly at subthreshold concentrations of IL-1. The potential
clinical significance of these results is the demonstration that the
lymphokine, IFN-, can modulate endothelial-leukocyte
interaction, which is relevant in atherosclerosis and
vascular inflammation.
The tandem NF-B sites are necessary for the induction of
VCAM-1 gene transcription by TNF- or
IL-1.27 However, recent
analysis of the VCAM-1 promoter has identified an additional
transcription factor, IRF-1, which is required for full induction of
VCAM-1 gene transcription.5
IRF-1 is a transcription factor involved in the IFN- signal
transduction
pathway.23 24 For
example, overexpression of IRF-1, although unable to
transactivate the VCAM-1 promoter, synergized with
overexpressed NF-B in an ISRE-dependent
manner.5 Furthermore,
recombinant IRF-1 specifically interacted with the VCAM-1 ISRE, acting
to increase the affinity of NF-B to its cognate binding
sites.5 During the preparation
of the present article, a similar synergistic interaction has been
reported for IFN- and TNF.7
However, the present study demonstrates that low concentrations of
IL-1 are unable to induce VCAM-1 gene transcription without the added
effects of IFN-, which alone also cannot induce VCAM-1
expression.
Most of the stimuli that are able to trigger adhesion
molecule expression in endothelial cells, with the
possible notable exception of
IL-4,28 act by increasing the
rate of gene transcription.4
The expression of VCAM-1 is dependent upon the activation of
NF-B.29 30 The
induction of VCAM-1 by the combined treatment with IFN- and
IL-1 is caused by an increased rate of VCAM-1 gene transcription, as
demonstrated by nuclear run-on experiments. These experiments indicate
that neither IFN- nor low concentrations of IL-1 could by
themselves trigger VCAM-1 gene transcription. However, their
combination leads to such an induction. The synergistic activation of
VCAM-1 by IFN- and TNF/IL-1, albeit lower than that obtained with a
maximal concentration of IL-1 or TNF-, was substantial compared
with the null effects of IFN- on the one hand and of barely
detectable effects of the low IL-1 or TNF- concentrations on the
other. Augmented VCAM-1 protein corresponded to an increased adhesion
of leukocytes to endothelial cells, largely
attributable (on the basis of experiments with blocking antibodies) to
VCAM-1 expression. On the basis of such a magnitude of gene activation,
we propose a pathophysiological relevance for these
observations in the setting of atherosclerosis and
vascular inflammation.
These findings support a pathogenetic role of
activated Th1 lymphocytes, the best known source of the
production of IFN-, within the atherosclerotic plaque.
Lymphocytes have been described in early as well as in advanced and
especially "unstable" atherosclerotic
plaques.11 12 Their
production of IFN- could be one important pathogenetic link
between their presence and monocyte recruitment from the circulation,
in conjunction with local production of possibly low
concentrations of IL-1 and TNF- by activated
macrophages and foam cells. Furthermore, inasmuch as other
known triggers of atherogenesis, such as the advanced glycation end
products31 and possibly
minimally modified LDLs,32
also appear to act through endothelial NF-B
activation, likely overriding the tonic inhibition of such activation
by NO, we anticipate a synergistic effect of IFN- on these other
stimuli. The recent demonstration (in a murine cardiac transplant model
of graft atherosclerosis) that arterial
VCAM-1 expression in vascular endothelial and SMCs was
markedly attenuated in IFN-–null mice supports the hypothesis that
IFN-–induced factors, such as IRF-1, are required for maximal
VCAM-1 gene expression.33
Therefore, the present results imply that factors or conditions
such as hypercholesterolemia or diabetes
mellitus, which minimally activate NF-B, would make
endothelial cells much more responsive to IFN- in
terms of VCAM-1
expression.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-52233, HL-48743, and HL-09483, the Italian National Research Council, and the Deutsche Forschungsgemeinshaft. J.K.L. is an Established Investigator of the American Heart Association. We thank Michael A. Gimbrone, Jr, and Arnold Freedman for VCAM-1 and MHC-II antibodies, respectively.
Received February 4, 2000; accepted July 31, 2000.
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