Involvement of TL1A and DR3 in induction of
pro-inflammatory cytokines and matrix
metalloproteinase-9 in atherogenesis
Yoon-Joong Kanga, Won-Jung Kima, Hyung-Uk Baea,
Dong-Ik Kimb, Yong Bok Parka, Jeong-Euy Parkb,
Byoung S. Kwonc, Won-Ha Leea,*
aDepartment of Genetic Engineering, Kyungpook National University, Taegu 702-701, Republic of Korea
bSamsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
cImmunomodulation Research Center, University of Ulsan, Republic of Korea
Received 26 April 2004; received in revised form 16 November 2004; accepted 16 December 2004
TL1A (VEGI/TNFSF15) is the ligand for DR3 (TNFRSF12) and is a newly identified member of the tumor necrosis factor
superfamily (TNFSF). Previously, DR3 has been shown to have a role in atherogenesis through stimulation of matrix degrading
enzymes including matrix metalloproteinase (MMP)-9. Immunohistochemical staining of human carotid atherosclerotic plaques
revealed a high-level expression of TL1A in regions rich in macrophage/foam cells. To investigate the role of TL1A and DR3 in the
functioning of macrophage/foam cells in relation to atherogenesis, we have analyzed cellular events mediated by TL1A and DR3 in
a human macrophage-like cell line, THP-1. Treatment of THP-1 cells with immobilized anti-DR3 monoclonal antibody in
combination with IFN-g caused induction of pro-atherogenic cytokines/chemokines such as TNF-a, monocyte chemoattractant
protein (MCP)-1, and interleukin (IL)-8. Treatment of THP-1 cells with recombinant TL1A in combination with IFN-g also caused
induction of MMP-9 and IL-8. Furthermore, the expression of DR3 in peripheral blood monocytes was induced after atherogenic
stimulation. These data suggest that TL1A and DR3 is involved in atherosclerosis via the induction of pro-inflammatory cytokines/
chemokines and decreasing plaque stability by inducing extracellular matrix degrading enzymes.
? 2005 Elsevier Ltd. All rights reserved.
Keywords: Atherosclerosis; Foam cells; Matrix metalloproteinase; Monocyte; Tumor necrosis factor superfamily
TL1A (TNFSF15/VEGI) is a recently identified
member of tumor necrosis factor superfamily (TNFSF)
that was shown to be expressed primarily in endothelial
cells, and it is a ligand for the death domain-containing
receptor DR3 (TNFRSF12) . Early experiments
suggested that DR3 is the receptor for TWEAK ,
but later experiments disapproved the result [3,4].
Currently, the ligand for DR3 is known to be TL1A
, while the receptor for TWEAK is known to be
TweakR/Fn14 . DR3 was reported to contain a death
domain similar to tumor necrosis factor receptor-1
(TNFR-1) and CD95 (also called Fas or APO-1) hence
the name DR3 (death receptor 3). Expression of DR3
appears to be restricted to tissues enriched in lympho-
cytes and signaling through DR3 induces both apoptosis
and activation of NF-kB .
* Corresponding author. Tel.: C82 53 950 5388; fax: C82 53 943
E-mail address: email@example.com (W.-H. Lee).
1043-4666/$ - see front matter ? 2005 Elsevier Ltd. All rights reserved.
Cytokine 29 (2005) 229e235
The interaction between the members of the tumor
necrosis factor superfamily and their receptors (e.g.
TNF-a, CD40/CD40L and LIGHT/TR2) elicit diverse
biological responses, which are known to be involved in
atherosclerosis [7e9]. These responses include the in-
duction of pro-inflammatory cytokines, matrix metal-
loproteinases (MMPs), adhesion molecules, and tissue
factor, and are known to make the atherosclerotic
plaque unstable. The stability of atherosclerotic plaques
depends greatly on the integrity of the fibrous cap,
which in turn depends on its content of extracellular
matrix protein. Dysregulation of the interaction between
the matrix degrading enzymes, matrix metalloprotei-
nases, and their inhibitors, tissue inhibitor of metal-
loproteinases (TIMPs), is believed to be responsible for
the rupture of atherosclerotic plaques .
We reported the expression of DR3 in atherosclerotic
plaques and stimulation of DR3 in human monocytic
cell line THP-1 caused induction of matrix metal-
loproteinases 1, 9 and 13 (MMP-1, 9 and 13) . In
this report, we identified the cell types expressing TL1A
in human carotid atherosclerotic plaques and also
characterized the possible role of TL1A and DR3 in
2.1. Immunohistochemical analysis of TL1A
and DR3 in atherosclerotic plaques
plaques obtained from carotid endoarterectomy tissue
revealed the expression of TL1A and DR3 in regions
rich in foam cells (Fig. 1A). Foam cells, as detected by
CD68 staining, are distributed in the necrotic cores and
shoulder regions of plaques. TL1A is also co-localized
with CD68 indicating that CD68 positive cells express
TL1A (Fig. 1B). As previous reports indicated, expres-
sion of TL1A was detected in endothelial cells as well
(data not shown).
analysis of atherosclerotic
2.2. Stimulation of DR3 cause induction of MMP-9
and cytokines in THP-1 cells
To investigate cellular response mediated by TL1A
and DR3 interaction, we used human macrophage-like
cell line, THP-1. Flow cytometry using anti-TL1A and
anti-DR3 monoclonal antibody (mAb) revealed that the
THP-1 cells expressed high basal level of DR3 (Fig. 2A)
and relatively lower level of TL1A (Fig. 2B).
It has become common use to activate cellular
cytokine receptors by cross-linking them to immobilized
antibodies. Antibodies to type I TNF receptor have
been shown to trigger a variety of TNF like effects upon
cross-linkage of the receptor molecules . Further-
more, cross-linking CD40 using immobilized anti-CD40
can induce B cell proliferation and secretion of IgE
following isotype switching . We investigated the
cellular response after the activation of THP-1 cells with
immobilized anti-DR3 monoclonal antibody. Activation
of THP-1 cells with immobilized anti-DR3 antibody or
IFN-g alone induced only low levels of TNF-a, MCP-1
and IL-8. However, concomitant treatment of THP-1
cells with immobilized anti-DR3 monoclonal antibody
and 100 U/ml IFN-g resulted in synergistic induction of
these cytokines (Fig. 3).
We then investigated TL1A mediated cellular re-
sponse after treating THP-1 cells with recombinant
human TL1A (rhTL1A). We previously reported that
the activation of THP-1 cells with immobilized anti-
DR3 monoclonal antibody induces MMP-1, 9, and 13
. Stimulation of THP-1 cells with rhTL1A induced
expression of MMP-9 in a dose dependent manner as
observed in a gelatin zymogram (Fig. 4A). Treatment of
THP-1 cells with recombinant human TL1A (rhTL1A)
and IFN-g also induced IL-8 (Fig 4B) but failed to
induce TNF-a or MCP-1 (data not shown).
2.3. Expression of DR3 is induced in activated
We then analyzed the expression of DR3 in
peripheral blood monocytes. Cell surface expression
level of DR3 in peripheral blood monocytes was very
low (Fig. 5A). These data indicate that the expression of
DR3 in monocyte/macrophage is not constitutive but
induced after stimulation. Monocyte/macrophage cells
in the atherosclerotic plaques are under constant
stimulation by various atherogenic stimuli from oxida-
tively modified LDL, infection, and pro-inflammatory
cytokines. To find out which of these atherogenic stimuli
are responsible for the activation of DR3 in foam cells,
human peripheral blood monocytes were analyzed after
the activation of total human blood with various
from normal healthy subjects for 2 h in the presence of
TNF-a induced expression of DR3 in monocytic cells
(Fig. 5B). Concomitant increase in the monocyte
activation marker, CD14, was also observed (data not
shown). Treatment of peripheral blood with bacterial
LPS also induced expression of DR3 (Fig. 5C). We then
isolated peripheral blood monocytes and incubated
these cells for a week to induce macrophage differenti-
ation [13,14]. High-level expression of DR3 was also
observed in these cells (Fig. 5D). The expression of
TL1A, however, was not detected in peripheral blood
monocytes before or after the activation indicating that
the TL1A induction requires more specific stimula-
230 Y.-J. Kang et al. / Cytokine 29 (2005) 229e235
Our data provide, for the first time, direct evidence
that TL1A and its receptor DR3 are involved in
atherogenesis. Previous research revealed that TL1A
have role in another inflammatory disease, inflammatory
bowl disease . Increased expression of TL1A and
DR3 was observed in macrophages and lymphocytes in
the intestinal lamina propria. Furthermore, rhTL1A
treatment to cultures of PHA-stimulated lamina propria
mononuclear from patients induced IFN-g production.
These results in combination with our data suggest that
TL1A and DR3 are involved in multiple diseases where
inflammation plays major role.
Flow cytometry data (Fig. 2) showing that TL1A is
expressed on the surface of THP-1 cells suggest that
TL1A, as is the case in TNF-a [16,17], is expressed on cell
indicates that the same cell can express both the ligand,
TL1A, and the receptor, DR3. This is similar to CD40
and CD40L, both of which are known to be expressed in
foam cells in atherosclerotic plaques [18e20].
The expression of TL1A was shown to be induced by
TNF-a and IL-1a . Since TNF-a is expressed and has
CD68 & TL1ATL1A
Fig. 1. Immunohistochemical analyses revealed the expression of TL1A and DR3 in foam cells in atherosclerotic plaques. (A) The shoulder region of
a human carotid endoarterectomy specimen was stained with eosins, monoclonal antibody to foam cell specific marker (CD68), TL1A or DR3, and
then counterstained with autohematoxylin as indicated. Low magnification (1:100) pictures are shown in the upper panels. High magnification
images of the foam cell rich regions are shown in the lower panels (1:400). Boxes indicate the regions illustrated by the high magnification pictures.
(B) Single (TL1A, red staining, left panel) and double staining (TL1A, red/CD68, brown, middle panel) of foam cell rich region are shown. Original
magnification, !400. High magnification images (!1000) of the foam cell rich regions are shown in the right panel. Box in the middle panel indicates
region magnified in the right panel.
231 Y.-J. Kang et al. / Cytokine 29 (2005) 229e235
major role in atherosclerosis , it is likely that the
expression of TL1A in atherosclerotic plaque is induced
by TNF-a. The expression pattern of DR3 in monocyte/
macrophage, however, is not known yet. Our data
(Fig. 5) indicate that the normal circulating monocytes
do not express any detectable level of DR3. Upregula-
tion of the DR3 expression level in peripheral blood
monocytes incubated with TNF-a or LPS indicates that
activation of monocytes results in the upregulation of
DR3. This is further supported by the expression of
DR3 in macrophages derived from peripheral blood
monocytes. Monocytes cultured for 7 days with 10%
autologous serum were reported to be spontaneously
differentiated into macrophage-like cells expressing
macrophage-specific CD68 antigen and high basal levels
of tissue factor activity, a property unique to mature
macrophages [13,14]. Monocytes/foam cells present
within the plaque are under stimulation by various
agents including oxidized-LDL, pro-atherogenic cyto-
kines, and cell-to-cell contact with activated T-lympho-
cytes. These agents could be responsible for the
expression of DR3 in activated monocytes.
The occurrence of acute coronary events heavily
depends on the stability of the atherosclerotic plaques,
which in turn depends on the inflammatory process
within the plaque. Stimulation of foam cells through
interaction between TL1A and DR3 appear to acceler-
ate inflammation via induction of MMP-9 and pro-
atherogenic cytokines/chemokines such as TNF-a,
IL-8, and MCP-1. IL-8, a member of CXC (a-type)
Fig. 2. Flow-cytometric analysis for the expression of DR3 (A) and TL1A (B) in THP-1 cells. Each filled area represents TL1A and DR3 specific
fluorescence and the empty area represents background levels of fluorescence caused by secondary antibody. The analysis was repeated twice with
essentially same results.
TNF-α conc. (pg/ml)
IL-8 conc. (pg/ml)
IL-8 conc. (pg/ml)
Cont. 1000x100x1000x100x LPS
Fig. 3. Activation of DR3 induces expression of pro-atherogenic cytokines in THP-1 cells. THP-1 cells (1 ! 105/well in a 96-well plate) were treated
with 1 mg/ml LPS or immobilized a-DR3 monoclonal antibodies with (solid bar) or without (open bat) the addition of IFN-g (100 U/ml).
Supernatants were collected 24 h after activation and the expression levels of TNF-a (A), IL-8 (B), or MCP-1 (C) were measured by ELISA. The
analysis was performed in triplicate and the error bars represent the standard deviation.
232 Y.-J. Kang et al. / Cytokine 29 (2005) 229e235
chemokines, was reported to activate MMP expression
 and to suppress TIMP expression . MCP-1,
a member of CC (b-type) chemokines, was reported to
induce the expression of tissue factor (TF) in human
SMCs and THP-1 cells  as well as peripheral blood
monocytes . MMP-9, which degrades non-fibrillar
collagen, is expressed in foam cell rich regions in
atherosclerotic plaques .
Our results suggest that TL1A (TNFRSF15) and
DR3 are involved in atherosclerosis via the induction of
pro-atherogenic cytokines and decrease in plaque
stability by inducing extracellular matrix degrading
enzymes. Since these functions have also been sub-
stantively linked to other cytokines, such as TNF-a and
CD40/CD40L [7,12,16,20,26,27], it is reasonable to
expect that TL1A and DR3 are the contributing factors
in atherosclerosis. Further investigations are required to
find out whether blocking the interaction between TL1A
and DR3 could suppress the atherogenic process.
4. Materials and methods
4.1. Histological analysis
For immunohistochemical analysis, carotid endoar-
terectomy specimens were obtained from 13 patients,
aged from 63 to 81, who underwent surgery at Samsung
Seoul Hospital. The study was approved by an in-
stitutional review committee and the subjects gave
informed consent. Atherosclerotic plaque specimens
were washed with saline and embedded in OCT to
make frozen sections. Standard 5 mm sections were
stained using the LSAB kit (DAKO, Copenhagen,
Denmark) according to the manual provided by the
manufacturer. For double staining of CD68 and TL1A,
specimen was sequentially treated with anti-TL1A
IFN- (100 U/ml)
LPS (1 g/ml)
-- 100 250100 250
1.0 21.0 3.8 1.86.7 1.9
IL-8 conc. (pg/ml)
Fig. 4. TL1Ainduces expressionof MMP-9andIL-8 inTHP-1cells.(A)
THP-1 cells (1 ! 105cells/well in 96-well plate) were stimulated with
1 mg/mlLPSor100 and250 ng/mlofrhTL1A inthe presence orabsence
of IFN-g (100 U/ml). Supernatants were collected after 24 h and the
level of MMP-9 was tested using gelatin zymogram. The analysis was
repeated twice with essentially same results and the representative data
are shown in the figure. The relative intensities of MMP-9 bands are
shown below the zymogram. (B) THP-1 cells were treated with 1 mg/ml
of LPS or 100 and 250 ng/ml of rhTL1A in the presence or absence of
IFN-g (100 U/ml), and IL-8 concentrations in the supernatants were
measured by ELISA after 24 h of activation. The experiments were
performed in triplicate and error bars represent the standard deviation.
Fig. 5. Activation of peripheral blood monocytes results in the upregulation of DR3. Diluted human peripheral blood were incubated without
stimulation for 6 h (A), in 2 ng/ml TNF-a for 2 h (B), or in 1 mg/ml LPS for 6 h (C). The expression levels of DR3 in monocytes were tested using
flow cytometry as described in methods. (D) Peripheral blood monocytes were isolated and incubated for 1 week to induce macrophage
differentiation and were tested for the expression of DR3. Each filled area represents the DR3 specific fluorescence and the empty area represents
background levels of fluorescence caused by secondary antibody.
233 Y.-J. Kang et al. / Cytokine 29 (2005) 229e235
monoclonal antibody; biotin-linked secondary anti-
body; streptavidinealkaline phosphatase; fuchsin for
visualization of TL1A staining (red color); anti-CD68
monoclonal antibody which was pre-conjugated with
biotin using an Animal Research Kit (DAKO, Copen-
hagen, Denmark) according to the manual provided by
the manufacturer; streptavidinehorseradish peroxidase;
diaminobenzidine (DAB) for visualization of CD68
(brown color); and finally counterstained with hema-
toxylin (blue color) for visualization of nuclei.
4.2. Cell culture
Human monocytic leukemia THP-1 cells  were
obtained from the American Type Culture Collection
(Rockville, MD). For the analysis of peripheral blood
monocytes, whole blood was collected either in heparin
vacutainer or CTAD Diatubes (Becton Dickinson/
Diagnostica Stago) that contain dipyridamole and
theophylline to prevent in vitro platelet activation.
Peripheral blood monocytes were isolated using gradient
centrifugation with Lymphoprep? (Nycomed Pharma
As, Oslo, Norway) as described previously . For the
generation of macrophage-like cells from peripheral
blood monocytes, isolated monocytes were incubated in
RPMI1640 medium supplemented with 10% heat
inactivated (at 68?C for 1 h) fetal bovine serum for
3 days, after which the medium was changed and
incubation continued for 4 additional days.
4.3. Monoclonal antibodies
Monoclonal antibodies to TL1A and DR3 were
purchased from Immunogenics (Ulsan, Korea). Mono-
clonal antibody to CD68 (KP1) was purchased from
DAKO (Glostrup, Denmark). Phycoerythrin labeled
anti-CD14 monoclonal antibody was purchased from
Caltag Laboratories (Burlingame, CA, USA).
4.4. Flow cytometric analysis
Flow cytometric analysis of human peripheral blood
monocytes and THP-1 cells was performed on FACS-
vantage (BectoneDickinson, Mountain View, CA).
Whole blood was diluted 10-fold with RPMI1640
medium without serum and incubated in CO2incubator
in the presence of 2 ng/ml TNF-a (R&D systems,
Minneapolis, MN, USA) or 1 mg/ml lipopolysaccharide
(LPS) (SigmaeAldrich, St. Louis, Missouri). After
incubation, 1 ml of cell suspension was pelleted and
the cells were sequentially incubated with 0.5 mg of
monoclonal antibody specific for DR3, 0.5 mg of FITC
labeled rat anti-mouse IgG (Caltag Laboratories,
Burlingame, CA, USA), and anti-CD14-PE according
to a previously described method . The fluorescence
profile of 1! 104cells was obtained and CD14 positive
monocyte/macrophage cells were analyzed for the
expression of DR3.
4.5. Activation of cells, enzyme-linked
immunosorbent assay (ELISA), and gelatin
For TL1A mediated activation, 1 ! 105THP-1 cells
in 100 ml of serum-free RPMI1640 medium were added
in 96 well plates. Recombinant human TL1A (rhTL1A,
R&D systems, Minneapolis, MN, USA) was added in
100e250 ng/ml concentrations in each well. The super-
natants were collected 24 h after activation. To activate
the cells with immobilized anti-DR3 monoclonal anti-
body, monoclonal antibodies were diluted in PBS and
added to the wells of a 96-well plate and incubated at
4?C overnight. The wells were washed with PBS
and THP-1 cells (1 ! 105cells in 100 ml of serum-free
RPMI1640 medium) were added in each well. Culture
supernatants were collected after 24 h. Cytokines were
measured bya sandwich ELISA (Endogen Inc., Woburn,
MA, USA). The detection limits were !10 pg/ml for
all the cytokines The MMP activity in the culture
supernatant was determined by performing substrate
gel electrophoresis as described previously .
This work was supported by a grant (No. 2000-1-
20500-001-5) from the Basic Research Program of the
Korea Science & Engineering Foundation and the SRC
Fund to IRC, University of Ulsan, by KOSEF and the
Korean Ministry of Sciences and Technology.
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