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Mouse monoclonal antihuman thrombomodulin antibodies bind to and activate endothelial cells through NF-╨Ю╤ФB activation in vitro.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 5, May 2006, pp 1629–1637
DOI 10.1002/art.21797
© 2006, American College of Rheumatology
Mouse Monoclonal Anti–Human Thrombomodulin Antibodies
Bind to and Activate Endothelial Cells Through
NF-␬B Activation In Vitro
Hiroyuki Nara,1 Hiroshi Okamoto,2 Seiji Minota,1 and Taku Yoshio1
Objective. To clarify whether mouse monoclonal
antibodies (mAb) against human thrombomodulin
(TM), which react with human TM present on the
endothelial cell (EC) surface, have anti–endothelial cell
antibody (AECA) activity and influence antiinflammatory properties of human TM expressed on the EC
surface in vitro.
Methods. Three preparations of mouse mAb
against human TM that react with different sites of the
human TM epidermal growth factor–like domain were
tested for their ability to 1) bind to ECs, 2) modulate
cytokine secretion from ECs, EC adhesion molecule
expression, and neutrophil adhesion to ECs, and 3)
stimulate nuclear translocation of NF-␬B through the
degradation of cytoplasmic I␬B in ECs. Recombinant
human interleukin-1␤ (IL-1␤) was used as a positive
control, and mouse IgG1 and mouse IgG2a were used as
negative controls.
Results. The 3 preparations of mouse mAb
against human TM that bind to unfixed EC monolayers
enhanced IL-6 and IL-8 secretion from ECs, upregulated expression of endothelial leukocyte adhesion
molecule 1, vascular cell adhesion molecule 1, and
intercellular adhesion molecule 1 on EC monolayers,
and enhanced neutrophil adhesion to ECs to a degree
similar to that observed with IL-1␤ stimulation, but
they did not induce the secretion of tumor necrosis
factor ␣ or IL-1␤ from ECs throughout the incubation
period. The 3 preparations stimulated nuclear translocation of NF-␬B through the degradation of cytoplasmic
I␬B. Mouse IgG1 and mouse IgG2a did not exhibit such
effects.
Conclusion. These results suggest the possibility
that AECA can react with antigens such as TM that are
present on the EC surface and activate ECs. Such events
on ECs may lead to vascular inflammation and damage
in patients with connective tissue diseases and vasculitis
in which AECA are present.
Anti–endothelial cell antibodies (AECA) have
been detected in the sera of patients with autoimmune
diseases such as systemic vasculitis, connective tissue
disease, and other rheumatic diseases (1). The presence
of AECA has been reported to be associated with
disease activity in systemic lupus erythematosus (SLE)
and systemic vasculitis (2–8). An idiotypic experimental
model of systemic vasculitis provides evidence supporting the concept that AECA can be pathogenic (9).
AECA have also been demonstrated to bind to endothelial cells (ECs) and to activate them by increasing
their surface expression of adhesion molecules and their
secretion of cytokines as well as by inducing the adhesion of leukocytes to them in vitro (10,11). Monoclonal
AECA from a patient with Takayasu arteritis (12) and
from a patient with SLE (13) have been demonstrated to
activate ECs through NF-␬B activation, although the
exact identity of the antigen recognized by monoclonal
AECA remains unclear, and NF-␬B activation may have
been due to the autocrine effects of cytokines such as
interleukin-1␤ (IL-1␤) produced by ECs that had been
stimulated by monoclonal AECA.
Human thrombomodulin (TM) is an integral
glycoprotein receptor expressed on the EC surface and
the cofactor for thrombin-mediated activation of the
anticoagulant protein C. In addition to its anticoagulant
role, the N-terminal lectin-like domain of TM has
Supported by a grant from the Ministry of Health, Labour and
Welfare of Japan.
1
Hiroyuki Nara, MD, Seiji Minota, MD, PhD, Taku Yoshio,
MD, PhD: Jichi Medical School, Tochigi-ken, Japan; 2Hiroshi Okamoto, MD, PhD: Tokyo Women’s Medical University, Tokyo, Japan.
Address correspondence and reprint requests to Taku Yoshio, MD, PhD, Division of Rheumatology and Clinical Immunology,
Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigiken 329-0498, Japan. E-mail: takuyosh@jichi.ac.jp.
Submitted for publication August 2, 2005; accepted in revised
form January 27, 2006.
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NARA ET AL
recently been demonstrated to have antiinflammatory
properties (14,15). Autoantibodies against human TM
have been found in the sera of patients with SLE (16,17)
and patients with lupus anticoagulant and unexplained
thrombosis (18). On the other hand, Ruiz-Arguelles et al
have reported the possibility that anticardiolipin antibodies (aCL), present in the sera of patients with
primary antiphospholipid syndrome (APS), react with
human TM (19). Furthermore, monoclonal aCL from
(NZB ⫻ NZW)F1 mice have recently been shown to
react with human TM (20). Taken together, these findings indicate that autoantibodies bound to human TM
present in the sera of patients with SLE and APS might
have an immunomodulatory effect on the vascular endothelium in vivo, such as EC activation through the
stimulation of nuclear translocation of NF-␬B and the
increase in neutrophil adhesion to ECs. We therefore
investigated whether mouse monoclonal antibodies
(mAb) against human TM have AECA activity and
influence the antiinflammatory properties of human TM
expressed on the EC surface in vitro.
MATERIALS AND METHODS
Reagents and antibodies. Monoclonal antibodies
against human TM (21-5D2 [IgG1], 21-4G3 [IgG2a], and
21-9H12 [IgG1]) were gifts from Daiichi Fine Chemical Industries (Toyama, Japan). As shown in Figure 1, structurally, the
extracellular portion of human TM is composed of 3 domains:
an N-terminal lectin-like domain, followed by an epidermal
growth factor (EGF)–like domain consisting of 6 EGF-like
repeats, and an O-glycosylation–rich domain (21–23). Protein
C binds to the fourth EGF-like repeat of human TM, and
thrombin binds to the fifth EGF-like repeat (24). The epitope
for mAb 21-5D2 is located in the region composed of the first
through third EGF-like repeats (24). The epitope for mAb
21-4G3 is located in the region composed of the fifth EGF-like
repeat (24). The epitope for mAb 21-9H12 is located in the
region composed of the third through fourth EGF-like repeats
(24).
Mouse IgG1 and mouse IgG2a were purchased from
Zymed (South San Francisco, CA). Recombinant human
IL-1␤ was purchased from Genzyme (Cambridge, MA).
Biotin-conjugated mouse mAb against human endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell
adhesion molecule 1 (VCAM-1) were purchased from
Monosan (Uden, The Netherlands). Biotin-conjugated mouse
mAb against intercellular adhesion molecule 1 (ICAM-1) was
purchased from Exalpha (Boston, MA). We purchased 2⬘,7⬘bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECFAM) from Molecular Probes (Eugene, OR). Goat polyclonal
antibodies against NF-␬B subunit p65 were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA).
Human EC culture. ECs were prepared from a fresh
human umbilical vein and cultured as described (25). ECs were
serially passed by brief exposure to 0.25% trypsin (Difco,
Figure 1. Suggested epitopes in human thrombomodulin (TM) for
mouse monoclonal antibodies (mAb) against human TM (MoaTM;
21-5D2, 21-4G3, and 21-9H12). The epitope for mAb 21-5D2 is
located in the region composed of the first through third epidermal
growth factor (EGF)–like repeats. The epitope for mAb 21-4G3 is
located in the region composed of the fifth EGF-like repeat. The
epitope for mAb 21-9H12 is located in the region composed of the
third through fourth EGF-like repeats. Reprinted, with permission,
from ref. 24.
Detroit, MI) and 0.04% EDTA (Sigma, St. Louis, MO). Only
cells from the second passage were used. The cells were
positive for von Willebrand factor.
Binding of mouse mAb against human TM to ECs.
ECs (1 ⫻ 105/well) were seeded in 96-well flat-bottomed tissue
culture plates (Costar, Cambridge, MA) precoated with 5%
gelatin (Sigma). After 2 days in culture, the cells reached
confluence as a monolayer, and 200 ␮l of mouse mAb against
human TM, mouse IgG1, mouse IgG2a diluted in medium 199
(Nissui, Tokyo, Japan) containing 1% fetal calf serum (FCS;
Medical and Biological Laboratories, Nagoya, Japan) (IgG
concentrations used ranged from 0.31 ␮g/ml to 10 ␮g/ml), or
medium 199 containing 1% FCS was added to each well and
incubated for 1 hour at 37°C.
After 2 gentle washes with 200 ␮l/well of medium 199
containing 1% FCS, ECs were fixed by incubation with 1%
paraformaldehyde (Sigma) in phosphate buffered saline (PBS)
for 10 minutes at room temperature. After 2 washes with PBS
at 200 ␮l/well, the remaining free protein sites were blocked by
adding 300 ␮l/well of blocking buffer (PBS containing 3%
bovine serum albumin [BSA; Sigma]) and incubated for at
least 1 hour at room temperature. After 2 washes with washing
buffer (PBS containing 1% BSA), 100 ␮l of peroxidaseconjugated goat anti-mouse IgG (Dako, Glostrup, Denmark)
diluted 1:1,000 in washing buffer was added to each well. Plates
were incubated for 1 hour at room temperature. After 5 washes
with washing buffer, 100 ␮l of tetramethylbenzidine (TMB)
soluble reagent (ScyTek, Logan, UT) as peroxidase substrate
EC ACTIVATION BY ANTIBODIES AGAINST HUMAN TM
buffer was added to each well and incubated for 15 minutes at
room temperature. Color development was stopped by adding
100 ␮l of TMB stop buffer (ScyTek). The color intensity of the
reaction was read at an optical density (OD) of 450 nm using
an enzyme-linked immunosorbent assay (ELISA) reader (Titertek Multiskan; Flow, McLean, VA). The binding of mouse
mAb against human TM, mouse IgG1, and mouse IgG2a was
expressed as OD.
Inhibition assay. Three preparations of mouse mAb
against human TM (IgG concentrations used ranged from 0.31
␮g/ml to 10 ␮g/ml) were mixed with recombinant human TM
(a gift from Asahi Kasei Pharma Corporation, Tokyo, Japan;
final concentration 1 mg/ml) and incubated at 4°C overnight.
Three preparations of mouse mAb against human TM mixed
with recombinant human TM were used for binding to unfixed
ECs. We then performed the same procedure described above
for binding of mouse mAb against human TM to ECs.
Binding of mouse mAb against human TM to cardiolipin and ␤2-glycoprotein I (␤2GPI). Binding of 3 preparations
of mouse mAb against human TM (IgG concentrations used
ranged from 0.31 ␮g/ml to 10 ␮g/ml) to cardiolipin and ␤2GPI
was performed using the MESACUP cardiolipin test (Medical
and Biological Laboratories) and the anti-CL-␤2GPI EIA kit
(Yamasa, Choshi, Japan), respectively. The procedures were
performed according to the manufacturers’ instructions.
Peroxidase-conjugated goat anti-mouse IgG was used instead
of peroxidase-conjugated anti-human IgG.
Cytokine production by ECs. ECs (1 ⫻ 105/well) were
seeded in 96-well flat-bottomed tissue culture plates precoated
with 5% gelatin. After 2 days in culture, the cells reached
confluence as a monolayer, and 200 ␮l of mouse mAb against
human TM, mouse IgG1, mouse IgG2a diluted in medium 199
containing 1% FCS (IgG concentrations used ranged from 0.31
␮g/ml to 10 ␮g/ml), medium 199 containing 1% FCS, or 5
units/ml (equivalent to 17.9 pg/ml) of IL-1␤ in medium 199
containing 1% FCS was added to each well and incubated for
different periods of time (maximum 24 hours) at 37°C. After
the incubation period, the cell-free supernatant was collected
and frozen at ⫺30°C prior to measurement of cytokine concentrations.
Tumor necrosis factor ␣ (TNF␣), IL-1␤, IL-6, and
IL-8 determinations. TNF␣, IL-1␤, IL-6, and IL-8 concentrations in the cell-free supernatant were evaluated by human
TNF␣, IL-1␤, IL-6, and IL-8 immunoassay (Quantikine; R&D
Systems, Minneapolis, MN), respectively. All assays were
performed according to the manufacturer’s instructions.
ELISA for detection of adhesion molecule expression.
EC monolayers were pretreated exactly as for studies of
cytokine production by ECs and then fixed by incubation with
1% paraformaldehyde in PBS for 10 minutes at room temperature. After 2 washes with PBS at 200 ␮l/well, the remaining
free protein sites were blocked by adding 300 ␮l/well of
blocking buffer and incubated for at least 1 hour at room
temperature. After 2 washes with washing buffer at 200 ␮l/well,
100 ␮l of biotin-conjugated mAb against human ELAM-1,
VCAM-1, and ICAM-1 diluted in washing buffer (final concentration of mAb 1 ␮g/ml) was added to each well and
incubated for 1 hour. After 5 washes with washing buffer, 100
␮l of peroxidase-conjugated streptavidin (Dako) diluted
1:1,000 in washing buffer was added to each well. Plates were
incubated for 1 hour at room temperature. The subsequent
1631
stages were the same as those described above for binding of
mouse mAb against human TM to ECs. Levels of ELAM-1,
ICAM-1, and VCAM-1 were expressed as OD.
Neutrophil adhesion assay. Human neutrophils were
prepared as described by Haslett et al (26), using FicollHypaque (Histopaque-1077; Pharmacia, Uppsala, Sweden)
gradient and erythrocyte lysis. More than 98% of cells prepared by this method were always viable by trypan blue
exclusion, and ⬎95% were neutrophils. Next, neutrophils were
labeled intracellularly with fluorescein using BCECF-AM according to the manufacturer’s instructions. The intracellular
BCECF–labeled neutrophils were washed 3 times with Hanks’
balanced salt solution (HBSS; Life Technologies, San Diego,
CA) and adjusted to a concentration of 2 ⫻ 106/ml in HBSS.
EC monolayers were pretreated exactly as for studies of
cytokine production by ECs. Two hundred microliters of
mouse mAb against human TM, mouse IgG1, and mouse
IgG2a, each diluted in medium 199 containing 1% FCS (IgG
concentrations used ranged from 0.31 ␮g/ml to 10 ␮g/ml),
medium 199 containing 1% FCS, or 5 units/ml of IL-1␤ in
medium 199 containing 1% FCS was added to each well and
incubated for 4 hours at 37°C.
After removing the supernatant from the wells, EC
monolayers were gently washed twice with medium 199 containing 1% FCS, and 2 ⫻ 105 intracellular BCECF–labeled
neutrophils (0.1 ml) were added to each well and coincubated
for 70 minutes at 37°C. After incubation, the nonadherent
intracellular BCECF–labeled neutrophils were removed by
carefully inverting plates for 1 hour, and the plates were gently
washed twice with HBSS. Next, intracellular BCECF–labeled
neutrophils adherent to ECs were solubilized by adding 200 ␮l
of 1% Nonidet P40 (NP40; Sigma) in PBS to each well. After
solubilization of intracellular BCECF–labeled neutrophils adherent to ECs, fluorescence was read with a Titertek Fluoroskan fluorometer (Labsystems, Helsinki, Finland) with excitation at 485 nm and emission at 538 nm. A standard curve was
constructed by serial dilution of the intracellular BCECF–
labeled neutrophil preparation, and the percentage of neutrophils adherent to ECs was calculated from this.
Nuclear translocation of NF-␬B by immunoperoxidase
staining. ECs (4 ⫻ 105/well) were seeded in 8-well type I
collagen–coated chamber slides (Asahi Techno Glass, Tokyo,
Japan). After 2 days in culture, the cells reached confluence as
a monolayer, and 600 ␮l of mouse mAb against human TM,
mouse IgG1, and mouse IgG2a, each diluted in medium 199
containing 1% FCS (IgG concentrations used ranged from 0.31
␮g/ml to 10 ␮g/ml), medium 199 containing 1% FCS, or 5
units/ml of IL-1␤ in medium 199 containing 1% FCS was
added to each well and incubated for 1 hour at 37°C.
After 2 gentle washes with medium 199 containing 1%
FCS, ECs were fixed by incubation with 4.5% paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized with 1% Triton X-100 (Sigma) in PBS for 20 minutes
at room temperature. After 2 washes with PBS, the remaining
free protein sites were blocked by adding 600 ␮l/well of
blocking buffer and incubated for at least 1 hour at room
temperature. After 2 washes with washing buffer, 600 ␮l of
goat polyclonal antibodies against NF-␬B subunit p65 diluted
1:50 in washing buffer was added for 1 hour at 37°C. After 4
washes with washing buffer, the cells were incubated with
second antibodies (biotin-conjugated donkey anti-goat IgG
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NARA ET AL
[Santa Cruz Biotechnology]) for 30 minutes at room temperature. After 4 washes with washing buffer, the cells were
incubated with a solution of peroxidase-conjugated streptavidin (Santa Cruz Biotechnology) for 30 minutes at room
temperature. After 4 washes with washing buffer, immunoperoxidase staining was performed using the ImmunoCruz
Staining System (Santa Cruz Biotechnology) according to the
manufacturer’s instructions. Thirty cells were counted from 10
areas in each well after immunoperoxidase cell staining (300
cells were counted). Percent nuclear translocation of NF-␬B in
ECs was calculated as (number of cells positive for nuclear
translocation of NF-␬B/300 cells) ⫻ 100.
Degradation of I␬B. In order to monitor the degradation of I␬B, ECs were stimulated with IL-1␤ (10 ng/ml), mouse
mAb against human TM, mouse IgG1, mouse IgG2a (IgG
concentrations used ranged from 2.5 ␮g/ml to 20 ␮g/ml), or
medium alone for 30 minutes, and the cells were lysed in 350
␮l of ice-cold lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl,
2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 0.2% NP40, 10 mM sodium fluoride, 10 ␮g/ml
aprotinin, 10 ␮g/ml leupeptin, and 1 ␮g/ml pepstatin A). The
cell lysate was resolved by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and transferred on polyvinylidene difluoride membranes (Millipore, Bedford, MA). The
membranes were incubated with antibodies to I␬B␣ (Santa
Cruz Biotechnology). The immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK). The gels were stained with
colloidal Coomassie (Pierce, Rockford, IL), equilibrated with
5% glycerol, and dried onto filter paper (Whatman, Brentford,
UK).
Statistical analysis. Data were analyzed using Student’s unpaired t-test. P values less than 0.05 were considered
significant.
RESULTS
Binding of mouse mAb against human TM,
mouse IgG1, or mouse IgG2a to unfixed ECs, cardiolipin, and ␤2GPI. Figure 2 shows binding of 3 preparations of mouse mAb against human TM as well as
binding of mouse IgG1 and mouse IgG2a to unfixed
ECs. Mouse IgG1 or mouse IgG2a did not bind to
unfixed ECs. The 3 preparations of mouse mAb against
human TM bound to unfixed ECs in an IgG dose–
dependent manner and in a similar manner, and the
binding of each of the 3 preparations to unfixed ECs was
significantly higher than that of mouse IgG1 or mouse
IgG2a at each IgG concentration (P ⬍ 0.01).
Binding of the 3 preparations of mouse mAb
against human TM to unfixed ECs (IgG concentrations
used ranged from 0.31 ␮g/ml to 10 ␮g/ml) was completely eliminated by preincubation of the preparations
with recombinant human TM (final concentration 1
mg/ml) overnight (not shown). Furthermore, the 3 preparations did not bind to cardiolipin or ␤2GPI (not
Figure 2. Binding of 3 preparations of mouse mAb against human
TM as well as binding of mouse IgG1 and mouse IgG2a to unfixed
endothelial cells (ECs). Binding of mAb 21-5D2 (circles), mAb 21-4G3
(solid squares), and mAb 21-9H12 (solid triangles) and binding of
mouse IgG1 (open squares) and mouse IgG2a (open triangles) to
unfixed ECs was measured using peroxidase-conjugated goat antimouse IgG and by enzyme-linked immunosorbent assay. Results are
expressed as the mean ⫾ SD of triplicate data and are representative
of those from 4 similar experiments. OD ⫽ optical density (see Figure
1 for other definitions).
shown). These results suggest that the 3 preparations of
mouse mAb against human TM bind to ECs through
human TM on the EC surface.
Cytokine secretion from ECs in the presence of
mouse mAb against human TM. To investigate whether
mouse mAb against human TM enhance cytokine secretion from ECs, ECs were incubated with 3 preparations
of mouse mAb against human TM or with mouse IgG1
or mouse IgG2a for 24 hours (IgG concentrations used
ranged from 0.31 ␮g/ml to 10 ␮g/ml). The 3 preparations
produced an IgG dose–dependent increase in IL-6 and
IL-8 secretion from ECs at 24 hours of incubation.
Neither mouse IgG1 nor mouse IgG2a exhibited such
effects (not shown). The 3 preparations did not induce
secretion of TNF␣ or IL-1␤ from ECs at 24 hours of
incubation (IgG concentrations used ranged from 0.31
␮g/ml to 10 ␮g/ml) (not shown).
Figure 3 shows the time course of IL-6 and IL-8
secretion from ECs in the presence of IL-1␤ (5 units/ml),
the 3 preparations of mouse mAb against human TM,
mouse IgG1 or mouse IgG2a (the IgG concentration
used was 10 ␮g/ml), or medium alone. Mouse IgG1 and
mouse IgG2a induced the secretion of IL-6 and IL-8
from ECs in a manner similar to that with medium
alone. In contrast, IL-1␤ (5 units/ml) and the 3 preparations of mouse mAb against human TM produced a
time-dependent increase in IL-6 and IL-8 secretion from
ECs. However, the 3 preparations of mouse mAb against
EC ACTIVATION BY ANTIBODIES AGAINST HUMAN TM
1633
surface after 4 hours of incubation, VCAM-1 expression
on the EC surface after 8 hours of incubation, and
ICAM-1 expression on the EC surface after 24 hours of
incubation in an IgG dose–dependent manner (IgG
concentrations used ranged from 0.31 ␮g/ml to 10
␮g/ml) (not shown). Neither mouse IgG1 nor mouse
IgG2a exhibited such effects.
Figure 3. Time course of interleukin-6 (IL-6) (A) and IL-8 (B)
secretion from endothelial cells in the presence of IL-1␤ (5 units/ml)
(diamonds), 3 preparations of mouse mAb against human TM (mAb
21-5D2 [solid circles], mAb 21-4G3 [solid squares], or mAb 21-9H12
[solid triangles]), mouse IgG1 (open squares) or mouse IgG2a (open
triangles) (the IgG concentration used was 10 ␮g/ml), or medium
alone (open circles). Results are expressed as the mean ⫾ SD of
triplicate data and are representative of those from 4 similar experiments. See Figure 1 for other definitions.
human TM did not induce the secretion of TNF␣ or
IL-1␤ from ECs throughout the 24-hour incubation (not
shown).
Effects of mouse mAb against human TM on EC
adhesion molecule expression. To investigate whether
the 3 preparations of mouse mAb against human TM
up-regulate adhesion molecule expression on the EC
surface, ECs were treated with these 3 preparations. The
3 preparations enhanced ELAM-1 expression on the EC
Figure 4. Time course of expression of endothelial leukocyte adhesion molecule 1 (ELAM-1) (A), vascular cell adhesion molecule 1
(VCAM-1) (B), and intercellular adhesion molecule 1 (ICAM-1) (C)
on the endothelial cell surface in the presence of interleukin-1␤ (5
units/ml) (diamonds), 3 preparations of mouse mAb against human
TM (mAb 21-5D2 [solid circles], mAb 21-4G3 [solid squares], or mAb
21-9H12 [solid triangles]), mouse IgG1 (open squares) or mouse
IgG2a (open triangles) (the IgG concentration used was 10 ␮g/ml), or
medium alone (open circles). Results are expressed as the mean ⫾ SD
of triplicate data and are representative of those from 4 similar
experiments. OD ⫽ optical density (see Figure 1 for other definitions).
1634
Figure 5. Percent neutrophil adhesion to endothelial cells (ECs)
pretreated with interleukin-1␤ (IL-1␤), 3 preparations of mouse mAb
against human TM (mAb 21-5D2, mAb 21-4G3, or mAb 21-9H12),
mouse IgG1 or mouse IgG2a, or medium alone. Results are expressed
as the mean and SD of triplicate data and are representative of those
from 4 similar experiments. See Figure 1 for other definitions.
Figure 4 shows the time course of EC adhesion
molecule expression in the presence of IL-1␤ (5 units/
ml), the 3 preparations of mouse mAb against human
TM, mouse IgG1 or mouse IgG2a (the IgG concentration used was 10 ␮g/ml), or medium alone. Neither
mouse IgG1 nor mouse IgG2a enhanced EC adhesion
molecule expression during the time course. In contrast,
IL-1␤ and the 3 preparations of mouse mAb against
human TM enhanced the expression of ELAM-1,
VCAM-1, and ICAM-1 on the EC surface over time.
The maximal expression of ELAM-1 on the EC surface
induced by IL-1␤ and the 3 preparations was reached
after 4 hours, and ELAM-1 expression on the EC
surface decreased thereafter (Figure 4A). The maximal
expression of VCAM-1 on the EC surface induced by
IL-1␤ and the 3 preparations was reached after 8 hours,
and VCAM-1 expression on the EC surface decreased
thereafter (Figure 4B). ICAM-1 expression on the EC
surface induced by IL-1␤ and the 3 preparations gradually increased in a time-dependent manner (Figure 4C).
Effects of mouse mAb against human TM on
neutrophil adhesion to ECs. To investigate whether ECs
pretreated with mouse mAb against human TM enhance
neutrophil adhesion to ECs, the adherence of intracellular BCECF–labeled neutrophils to ECs pretreated with
IL-1␤ (5 units/ml), the 3 preparations of mouse mAb
against human TM, or mouse IgG1 or mouse IgG2a for
4 hours at 37°C was analyzed. Figure 5 shows neutrophil
adhesion to ECs induced by IL-1␤, the 3 preparations,
or mouse IgG1 or mouse IgG2a (IgG concentrations
used ranged from 0.31 ␮g/ml to 10 ␮g/ml). Neither
mouse IgG1 nor mouse IgG2a enhanced neutrophil
NARA ET AL
adhesion to ECs (P not significant compared with medium alone). In contrast, IL-1␤ enhanced neutrophil
adhesion to ECs. In addition, the 3 preparations enhanced neutrophil adhesion to ECs in an IgG dose–
dependent manner, and the mean percent neutrophil
adhesion induced by each of the 3 preparations was
significantly higher than that induced by mouse IgG1 or
mouse IgG2a at each IgG concentration from 1.25 ␮g/ml
to 10 ␮g/ml (P ⬍ 0.05).
Nuclear translocation of NF-␬B in ECs promoted
by mouse mAb against human TM. To investigate
whether AECA activity of mouse mAb against human
TM depends on the nuclear translocation of NF-␬B, we
analyzed the nuclear translocation of NF-␬B in ECs
pretreated with mouse mAb against human TM. We
repeated this experiment 6 times (see Figure 6A). The
mean ⫾ SD nuclear translocation of NF-␬B was 29.3 ⫾
3.29% when ECs were stimulated with IL-1␤ (5 units/
ml) for 1 hour. Mouse IgG1 or mouse IgG2a (IgG
concentrations used ranged from 0.31 ␮g/ml to 10
␮g/ml) did not stimulate nuclear translocation of NF-␬B
in ECs, and the percent nuclear translocation of NF-␬B
stimulated with mouse IgG1 or mouse IgG2a at each
IgG concentration used did not differ significantly from
that with medium alone. In contrast, the 3 preparations
of mouse mAb against human TM enhanced nuclear
translocation of NF-␬B in ECs in an IgG dose–
dependent manner, and with each, the percent nuclear
translocation of NF-␬B was significantly higher than that
with mouse IgG1 or mouse IgG2a at each IgG concentration used (from 1.25 ␮g/ml to 10 ␮g/ml) (P ⬍ 0.01).
Degradation of I␬B␣ induced by mouse mAb
against human TM. Furthermore, to investigate whether
the enhanced nuclear translocation of NF-␬B in ECs by
mouse mAb against human TM was due to the degradation of cytoplasmic I␬B␣, we analyzed the degradation of cytoplasmic I␬B␣ pretreated with mouse mAb
against human TM. Figure 6B shows the effects of IL-1␤
(10 ng/ml), the 3 preparations of mouse mAb against
human TM (IgG concentrations used ranged from
2.5 ␮g/ml to 20 ␮g/ml), or medium alone on the degradation of cytoplasmic I␬B␣. Coomassie staining revealed that an equal amount and quality of protein
were loaded on the gel. IL-1␤ stimulation completely
depleted cytoplasmic I␬B␣ in ECs. The 3 preparations
of mouse mAb against human TM substantially decreased the levels of cytoplasmic I␬B␣ in ECs in an IgG
dose–dependent manner. However, mouse IgG1 or
mouse IgG2a (not shown) or medium alone did not
affect I␬B␣ expression. These results strongly suggest
that AECA activity of mouse mAb against human TM,
EC ACTIVATION BY ANTIBODIES AGAINST HUMAN TM
1635
nuclear translocation of NF-␬B through the degradation
of cytoplasmic I␬B.
DISCUSSION
Figure 6. Effects of mouse mAb against human TM on NF-␬B
activation. A, Percent nuclear translocation of NF-␬B in endothelial
cells (ECs) treated with interleukin-1␤ (IL-1␤), 3 preparations of
mouse mAb against human TM (mAb 21-5D2, mAb 21-4G3, or mAb
21-9H12), mouse IgG1 or mouse IgG2a, or medium alone. Results are
expressed as the mean and SD of data from 6 independent experiments. B, Levels of I␬B in ECs incubated with IL-1␤, mAb 21-5D2,
mAb 21-4G3, mAb 21-9H12, or medium alone (–) for 30 minutes. See
Figure 1 for other definitions.
such as induction of increases in cytokine secretion from
ECs and up-regulation of adhesion molecule expression on the EC surface, is induced by an increase of
Since NF-␬B is known to play a crucial role in the
induction of IL-6, IL-8, and adhesion molecules as a
positive transcriptional regulator (27), we examined
whether mouse mAb against human TM could stimulate
the degradation of cytoplasmic I␬B and the nuclear
translocation of NF-␬B in ECs, leading to an increase in
cytokine release from ECs and to up-regulation of
adhesion molecule expression on the EC surface. In our
study, mouse mAb against human TM stimulated the
degradation of cytoplasmic I␬B, resulting in the nuclear
translocation of NF-␬B in ECs to a degree comparable
with that observed upon stimulation with IL-1␤ (used as
a positive control). This was concomitant with increased
secretion of IL-6 and IL-8 from ECs, increased expression of adhesion molecules on the EC surface, and
increased neutrophil adhesion to ECs.
NF-␬B activation by monoclonal AECA from a
patient with Takayasu arteritis (12) and from a patient
with SLE (13) has been suggested to result from the
autocrine effects of cytokines such as IL-1␤ produced by
the binding of monoclonal AECA to ECs. In our study,
IL-1␤ or TNF␣ that was released from ECs bound by
mouse mAb against human TM could not be detected
throughout the 24-hour incubation. Three preparations
of mouse mAb against human TM that bind to different
sites at the human TM EGF-like domain (24) showed
similar effects.
Signaling by an interaction between mouse mAb
against human TM and the human TM EGF-like domain on the EC surface, but not between the specific
binding sites of the human TM EGF-like domain and
mouse mAb against human TM, might be important in
stimulating the degradation of cytoplasmic I␬B without
the autocrine effects of cytokines. Three preparations of
mouse mAb against human TM have already been
shown to inhibit protein C activation by thrombin in the
presence of recombinant human TM (24). This binding
of the 3 preparations of mouse mAb against human TM
to the human TM EGF-like domain on the EC surface
diminishes the potent antiinflammatory properties of
activated protein C.
The N-terminal lectin-like domain of human TM
has recently been demonstrated to have antiinflammatory properties (the domain interferes with neutrophil
adhesion to ECs through the suppression of ERK activation and suppresses proinflammatory events by bind-
1636
ing high mobility group box chromosomal protein 1
DNA binding protein, a proinflammatory cytokine)
(14,15). More recently, NF-␬B activation induced by
cytokines such as TNF␣ and IL-1␤ has been demonstrated to effectively inhibit TM gene expression (28).
NF-␬B activation in ECs by the 3 preparations of mouse
mAb against human TM might also inhibit human TM
gene expression and diminish such antiinflammatory
actions by the N-terminal lectin-like domain of human
TM.
Activated neutrophils and their released products
such as elastase and cathepsin G have been reported to
down-regulate human TM activity on the EC surface
(29). In our study, the binding of 3 preparations of
mouse mAb against human TM to the human TM
EGF-like domain on the EC surface increased neutrophil adhesion to ECs. After adhering to ECs, neutrophils
may be activated, and there may be increased release of
products such as elastase and cathepsin G. These activated neutrophils and released products might downregulate human TM activity on the EC surface, leading
to a further increase in inflammation and hypercoagulability in vascular space and perivascular space. Such
antibodies that recognize the identified binding site on
the EC surface have never been reported to activate
NF-␬B in ECs without the autocrine effects of cytokines.
Our study strongly suggests the possibility that AECA
may react with antigens on the EC surface and activate
ECs through NF-␬B activation without the autocrine
effects of cytokines, leading to vascular damage and
inflammation such as vasculitis.
There is controversy as to whether autoantibodies against TM are present in patients with SLE or APS
or in the lupus-prone mouse. Gibson et al (30) found no
autoantibody to human TM in patients with SLE. Mouse
mAb against human cardiolipin from the MRL/l mouse,
which have been reported to show polyreactivity, did not
react with recombinant human TM (16). However,
Ruiz-Arguelles et al (19) reported that the reactivity of
aCL with cardiolipin in sera of patients with primary
APS was eliminated by preincubation with human TM.
Furthermore, Oosting et al (16) reported that autoantibodies against human TM were transiently present in the
sera of 2 antiphospholipid antibody (aPL)–positive patients with SLE and 4 aPL-negative patients with histories of thrombosis. IgG fractions of these sera inhibited
the activation of protein C and bound to a recombinant
version of the human TM EGF-like domain.
Recently, we have reported the presence of antibodies against recombinant human TM in sera of patients with SLE and mixed connective tissue disease,
NARA ET AL
determined by ELISA (17), although whether they react
with cardiolipin or ␤2GPI remains unclear. However, in
our study, the 3 preparations of mouse mAb against
human TM did not bind to cardiolipin or ␤2GPI. VegaOstertag et al have recently reported that IgG aPL from
patients with APS induce significant increases in tissue
factor transcription, function, and expression, in IL-6
and IL-8 up-regulation, and in inducible nitric oxide
synthase expression on human umbilical vein ECs, and
these processes involve phosphorylation of p38 MAPK
and activation of NF-␬B, although the binding sites of
aPL on the EC surface remain unknown (31). Anticardiolipin antibodies/aPL in sera of patients with connective tissue diseases such as SLE and APS might act as
AECA by binding to human TM on the EC surface.
These antibodies might suppress the antiinflammatory
properties and anticoagulant role of human TM in vivo,
leading to inflammation and hypercoagulability in vascular space and perivascular space. Further investigation
is needed to determine whether other AECA in addition
to antibodies against human TM also activate ECs
through activation of NF-␬B or other signal transduction
pathways.
ACKNOWLEDGMENTS
We thank Ms Mamiko Semba and Ms Mika Kasahara
for their technical assistance.
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