The Journal of Immunology

Expression of Cytokine, Chemokine, and Adhesion Molecules

during Endothelial Cell Activation Induced by Antibodies
against Dengue Virus Nonstructural Protein 11

Chiou-Feng Lin,* Shu-Chen Chiu,* Yu-Ling Hsiao,* Shu-Wen Wan,* Huan-Yao Lei,*
Ai-Li Shiau,* Hsiao-Sheng Liu,* Trai-Ming Yeh,† Shun-Hua Chen,* Ching-Chuan Liu,‡ and
Yee-Shin Lin2*
Vascular dysfunction is a hallmark associated with disease onset in dengue hemorrhagic fever and dengue shock syndrome. In
addition to direct viral damage, immune responses to dengue virus (DV) infection may also underlie the pathogenesis of disease.
We have proposed a mechanism of molecular mimicry in which Abs directed against DV nonstructural protein 1 (NS1) cross-react
with endothelial cells and induce damage. In this study, we demonstrated the inflammatory endothelial cell activation induced by
anti-DV NS1 via the transcription factor NF-␬B-regulated pathway. Protein phosphorylation and NF-␬B activation were observed
after anti-DV NS1 stimulation in a human microvascular endothelial cell line-1. The cytokine and chemokine production, including
IL-6, IL-8, and MCP-1, but not RANTES, in endothelial cells increased after treatment with anti-DV NS1 Abs. The expression of
IL-6, IL-8, and MCP-1 was blocked by the preabsorption of anti-DV NS1 with DV NS1 or by the inhibition of NF-␬B activation.
Furthermore, the increases in both ICAM-1 expression and the ability of human PBMC to adhere to endothelial cells were also
observed, and these effects were inhibited by pretreatment with anti-ICAM-1 or anti-MCP-1 Abs. Therefore, in addition to
endothelial cell apoptosis, as previously reported, inflammatory activation occurs in endothelial cells after stimulation by anti-DV
NS1 Abs. These results suggest the involvement of anti-DV NS1 Abs in the vasculopathy of DV infection. The Journal of
Immunology, 2005, 174: 395– 403.

P atients with dengue virus (DV)3 infection present a wide
range of diseases from mild dengue fever to life-threaten-
ing dengue hemorrhagic fever and dengue shock syn-
drome (DHF/DSS) (1, 2). There is no vaccine against DHF/DSS
because its pathogenic mechanisms are still not fully understood
(3– 6). In dengue pathogenesis, the involvement of viral and im-
mune responses has been hypothesized (3–9). Virus variation and
virus load involving Ab-dependent enhancement of infection have
been suggested to be responsible for the progression and severity
of dengue disease (10 –17). Cell activation, impaired proliferation,
cytokine and chemokine production, and apoptosis are caused after
DV infection by a direct (18 –32) or indirect route (33, 34). It is
likely that different mechanisms are involved in the pathogenesis
of DV infection.
DHF/DSS represents a pathological complication caused by
multiple symptoms, including thrombocytopenia, coagulopathy,
Departments of *Microbiology and Immunology, †Medical Technology, and ‡Pedi-
atrics, National Cheng Kung University Medical College, Tainan, Taiwan
Received for publication March 22, 2004. Accepted for publication October 19, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1 have been observed in patients with DV infections (8, 27, 45–52).
This work was supported by Grant NSC 91-3112-B006-002 from the National Sci-
ence Council, Taiwan.
2
Address correspondence and reprint requests to Dr. Yee-Shin Lin, Department of
Microbiology and Immunology, National Cheng Kung University Medical College, 1
University Road, Tainan 701, Taiwan. E-mail address: yslin1@mail.ncku.edu.tw
3
Abbreviations used in this paper: DV, dengue virus; AEC, 3-amino-9-ethylcarba-
zole; AECA, anti-endothelial cell Ab; DHF, dengue hemorrhagic fever; DSS, dengue
shock syndrome; EGM, endothelial cell growth medium; HMEC-1, human micro-
vascular endothelial cell line-1; IKK, I-K␤ kinase; JEV, Japanese encephalitis virus;
␻
L-NAME, N -nitro-L-arginine methyl ester; NS1, nonstructural protein 1; PDTC, pyr-
rolidine dithiocarbamate; zVAD-fmk, benzyloxycarbonylvalylalanylaspartic acid flu-
oromethyl ketone.
and vasculopathy (7, 35–37). Plasma leakage, also called the hem-
orrhagic syndrome, occurs once vessel endothelium is disrupted
and is followed by the loss of its barrier function. The pathogenesis
of endothelial dysfunction resulting in vascular leakage remains
unclear, however. In addition to direct viral damage, such as the
induction of apoptosis in endothelial cells as observed in vitro (27),
complement activation and cytokine and chemokine production
induced by DV infection appear to be involved in the induction of
endothelial cell damage (26, 27, 38, 39). DV can also indirectly
modulate endothelial cell function through Ab-enhanced infection
of DV in peripheral blood monocytes (33). Furthermore, the in-
volvement of anti-endothelial cell autoantibodies in DV pathogen-
esis has been proposed (40 – 42). Endothelial cell apoptosis caused
by autoantibodies may be related to the transient leakage syndrome
in dengue vasculopathy.
Inflammatory immune responses facilitate the progression of
disease severity in DHF/DSS (7–9). Lymphocytes, monocytes and
macrophages, mast cells, endothelial cells, liver cells, and den-
dritic cells are the targets of DV, and these cells could produce
cytokines or chemokines, or both, after DV infection (20 –22, 24 –
27, 33, 38, 39, 43, 44). Increased levels of cytokines, including
IL-2, IL-6, IL-8, IL-10, IL-13, IL-18, IFN-␥, TNF-␣, and MCP-1,
The roles of various cytokines and chemokines in the pathogenesis
of DHF/DSS need to be clarified, especially on endothelial cell
activation, which is related to vasculopathy.
Previous studies in our laboratory showed the pathogenic role of
Abs against nonstructural protein 1 (NS1) present in dengue pa-
tients or generated in mice (40, 41). Anti-DV NS1 Abs cross-
reacted with endothelial cells and induced apoptosis via an NO-
mediated pathway (40, 42). Pathogenic anti-DV NS1 may
contribute to the progression of DHF/DSS (41). In the present

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

396 ANTI-DENGUE NS1 Ab-INDUCED ENDOTHELIAL CELL ACTIVATION
study, we demonstrated protein tyrosine phosphorylation and
NF-␬B activation after endothelial cells had been bound by anti-
DV NS1 Abs. We also demonstrated that the increased levels of
cytokine and chemokine expression were regulated by NF-␬B, and
that anti-DV NS1 stimulation caused adhesion molecule expres-
sion and PBMC adhesion to endothelial cells.
Materials and Methods
Mice
BALB/cByJ breeder mice were obtained from The Jackson Laboratory and
maintained on standard laboratory food and water ad libitum in our medical
college laboratory animal center. Their 8-wk-old progeny were used for the
generation of Abs in the present study.
Abs and reagents
Both purified and FITC-conjugated mAbs against phosphotyrosine (clone
PY20) were purchased from Oncogene Research Products. Rabbit Abs
specific for NF-␬B p65 were purchased from Chemicon International.
Anti-human IL-6, IL-8, MCP-1, RANTES, and ICAM-1 mAbs were from
BD Pharmingen. Neutralizing anti-human MCP-1 and ICAM-1 mAbs were
also from BD Pharmingen, and mouse mAb to ␤-actin was from Sigma-
Aldrich. FITC- or HRP-conjugated goat anti-mouse or anti-rabbit IgG,
NF-␬B inhibitor pyrrolidine dithiocarbamate (PDTC), protein synthesis in-
hibitor cycloheximide, caspase inhibitor benzyloxycarbonylvalylalany-
laspartic acid fluoromethyl ketone (zVAD-fmk), and NO synthase inhibitor
N␻-nitro-L-arginine methyl ester (L-NAME) were obtained from
Sigma-Aldrich.
Polyclonal Abs against DV or Japanese encephalitis virus (JEV) NS1
were obtained from BALB/c mice immunized i.p. with purified recombi-
nant DV-2 (New Guinea C strain) or JEV (NT109 strain) NS1 proteins, as
described previously (40). The IgG fractions from hyperimmunized mouse
sera were purified with a protein G-Sepharose affinity chromatography col-
umn (Amersham Biosciences) and recovered with HCl-glycine. The reac-
tivity of purified IgG against NS1 was confirmed by SDS-PAGE and West-
ern blot. The control IgG was eluted from a protein G column loaded with
normal mouse sera. The endotoxin concentration of each of these prepa-
rations was ⬍0.03 EU/␮g, as determined by a Limulus amebocyte lysate
assay (Associates of Cape Cod).
Patient sera
Dengue patient sera were obtained from N. Hung (Department of Dengue
Hemorrhagic Fever, Children’s Hospital No. 1. Seventeen serum samples
were collected from patients with DHF disease severity grades I-III. Di-
agnosis of DHF was based on the clinical criteria established by the World
Health Organization. Sera from five healthy volunteers were used as the
normal controls.
Cell and virus culture
Human microvascular endothelial cell line-1 (HMEC-1) was passed in cul-
ture plates containing endothelial cell growth medium (EGM; Cambrex)
composed of 2% FBS, 1 ␮g/ml hydrocortisone, 10 ng/ml epidermal growth
factor, and antibiotics (40). Cells were detached using 1000 U/ml trypsin
and 0.5 mM EDTA. Only those cultures from three to five passages with
a viability of ⬎95% by eosin-Y staining were used for experiments. Baby
hamster kidney cell line and C6/36 cells were cultured in DMEM medium
containing 10% FBS and antibiotics. PBMC were isolated from normal
volunteer blood using Ficoll-Paque isolation (Amersham Biosciences), ac-
cording to the standard procedures, and were used for adhesion assay.
Dengue-2 virus (PL046, Taiwan isolated) was maintained in the C6/36
cells. Briefly, monolayers of C6/36 were incubated with DV at a multi-
plicity of infection of 0.01 and incubated at 26°C in 5% CO2 for 5 days.
The cultured medium was harvested, and cell debris was removed by cen-
trifugation at 900 ⫻ g for 10 min. After further centrifugation at 16,000 ⫻
g for 10 min, the virus supernatant was collected and stored at ⫺70°C until
use. Virus titer was determined by plaque assay using the BHK-21 cell line,
as described previously (26).
Western blotting and flow cytometry for detection of protein
tyrosine phosphorylation
HMEC-1 cells underwent starvation in serum-free EGM for 1 h before the
experiment. Next, the cells were treated with mouse anti-DV NS1, anti-
JEV NS1, or control IgG for various time intervals. Cells were then har-
vested and lysed with a buffer containing 1% Triton X-100, 50 mM Tris
(pH 7.5), 10 mM EDTA, 0.02% NaN3, and a protease inhibitor mixture
(Boehringer Mannheim). After being freeze thawed once, cell lysates were
centrifuged at 12,000 rpm for 20 min at 4°C. The supernatants were col-
lected and boiled in sample buffer for 5 min. Following SDS-PAGE, pro-
teins were transferred to polyvinylidene difluoride membrane (Millipore),
blocked overnight at 4°C in PBS-T (PBS plus 0.1% Tween 20) containing
5% skim milk, and probed with Abs against phosphotyrosine at 4°C over-
night. After being washed with PBS-T, blots were incubated with a 1/5000
dilution of HRP-conjugated goat anti-rabbit IgG for 1 h at 4°C. The protein
bands were developed with a 3-amino-9-ethylcarbazole (AEC) substrate kit
(Zymed Laboratories).
For immunostaining followed by flow cytometric analysis, cells were
washed in PBS, fixed with 1% paraformaldehyde in PBS at room temper-
ature for 10 min, and permeabilized with 70% ethanol. After being washed
with PBS three times, cells were incubated with FITC-conjugated mAbs
against phosphotyrosine for 1 h at 4°C and analyzed by flow cytometry
(FACSCalibur; BD Biosciences) with excitation set at 488 nm.
Western blotting and EMSA for detection of NF-␬B activation
Nuclear translocation of NF-␬B was detected by Western blotting. Har-
vested cells were lysed with buffer A (10 mM HEPES (pH 7.8), 5 mM
MgCl2, 10 mM KCl, 1 mM ZnCl2, 0.2 mM EDTA, 1 mM Na3VO4, 10 mM
NaF, 0.5 mM DTT, and 0.5 mM PMSF), incubated on ice for 10 min, and
centrifuged at 12,000 rpm for 20 min at 4°C. The supernatants were col-
lected as the cytosolic lysates, and the nuclear extracts were obtained from
pellets lysed in buffer B (20 mM HEPES (pH 7.8), 5 mM MgCl2, 300 mM
NaCl, 1 mM ZnCl2, 0.2 mM EDTA, 25% glycerol, 1 mM Na3VO4, 10 mM
NaF, 0.5 mM DTT, and 0.5 mM PMSF) and incubated for 15 min on ice
with occasional mixing. Following SDS-PAGE, proteins were transferred
to polyvinylidene difluoride membrane, blocked overnight at 4°C in PBS-T
containing 5% skim milk, and probed with Abs against NF-␬B p65 subunit
at 4°C overnight. After being washed with PBS-T, blots were incubated
with a 1/5000 dilution of HRP-conjugated goat anti-rabbit IgG for 1 h at
4°C. The protein bands were developed with an AEC substrate kit.
For EMSA, nuclear extracts were collected, as described above, and
separated by 6% acrylamide gel. The DIG Gel Shift kit (Roche Diagnos-
tics) was used according to the manufacturer’s instructions. The detection
probe specific for NF-␬B consisted of a digoxigenin-labeled double-strand
oligonucleotide (5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘). Specificity
of protein-DNA complexes was detected by immunoreactivity with sheep
anti-digoxigenin conjugated with alkaline phosphatase and developed us-
ing chemiluminescent substrate disodium 3-(4-methoxyspiro{1,2-dioxet-
ane-3-,2⬘-(5⬘-chloro)tricyclo[3.3.1.13.7]decan]-4-yl) phenyl phosphate
(Roche). The generated chemiluminescent signals were recorded on x-ray
film by autoradiography.
Immunohistochemistry and flow cytometry for detection of
cytokine, chemokine, and adhesion molecule
Monolayers of HMEC-1 cells were cultured on sterile glass slides, fol-
lowed by treatment with mouse anti-DV NS1, anti-JEV NS1, or control
IgG for various time intervals. For flow cytometric analysis, cells were
detached using 1000 U/ml trypsin and 0.5 mM EDTA. Cells were fixed and
permeabilized with Cytofix/Cytoperm kit (BD Pharmingen). For immuno-
histochemical staining, fixed cells were incubated with 0.3% H2O2 in PBS
for 5 min to quench endogenous peroxidase activity and then washed again
with PBS. Abs specific for IL-6, IL-8, RANTES, MCP-1, and ICAM-1
were added to cells and incubated for 1 h at 4°C. After being washed with
PBS, cells were incubated with HRP- or FITC-conjugated anti-primary Ab
for 1 h at 4°C. After being washed with PBS, cells were developed using
the AEC substrate kit (Zymed Laboratories), counterstained with hema-
toxylin (Sigma-Aldrich), and viewed with light microscopy or analyzed by
flow cytometry with excitation set at 488 nm.
ELISA
The concentrations of cytokines and chemokines, including IL-6, IL-8, and
MCP-1, in culture supernatants and DHF patient sera were determined
using ELISA kits (R&D Systems). The manufacturer’s instructions were
followed, and the concentrations were determined by spectrophotometry at
450 nm (Molecular Devices).
RT-PCR
The expression of MCP-1 and RANTES mRNA was determined using
RT-PCR analysis. Total cellular RNA from endothelial cells was extracted
using TRIzol reagent (Invitrogen Life Technologies), according to the
manufacturer’s instructions. The concentration of RNA was quantified by
spectrophotometry at 260 nm (U-2000; Hitachi). The cDNA was prepared
The Journal of Immunology 397

by reverse transcription, as previously described (40), and PCR was per-

formed using a PCR controller (GeneAmp PCR System 2400;
PerkinElmer). PCR was conducted in 50 ␮l of the reaction mixture (1.5
mM MgCl2 and 0.2 mM each of dATP, dGTP, dCTP, and dTTP) contain-
ing primers at 1.5 ␮M each, 0.2 ␮g/ml RNase A (Sigma-Aldrich), and 1 U
of TaqDNA polymerase (Promega), as follows: 94°C for 1 min; 30 thermal
cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min; and a final
cycle at 72°C for 5 min. The oligonucleotide primers for human MCP-1
(sense, 5⬘-CAAACTGAAGCTCGCACTCTCGCC-3⬘ and antisense, 5⬘-AT
TCTTGGGTTGTGGAGTGAGTGTTCA-3⬘), RANTES (sense, 5⬘-TGCC
TCCCCATATTCCTCGG-3⬘ and antisense, 5⬘-TCATGTTTGCCAGTAA
GC-3⬘), and ␤-actin (sense, 5⬘-AGCGGGAAATCGTGCGTG-3⬘ and anti-
sense, 5⬘-CAGGGTACATGGTGGTGGTGCC-3⬘) were used according to
previously published sequences (27, 40). DV primer set (sense, 5⬘-GATATG
GGTTATTGGATAGA-3⬘ and antisense, 5⬘-CTGATTTCCATCCCGTA-3⬘)
was used as an infected control (26). The PCR products were analyzed by
1.5% agarose gel electrophoresis, stained with ethidium bromide, and viewed
with UV light.
Transient transfection
In experiments using dominant-negative mutant, HMEC-1 cells were co-
transfected with the dominant-negative IKK-␤ (the kind gift of C. Chen,
Department of Pharmacology, National Taiwan University) or the empty
vector pcDNA3.1 (the kind gift of M. Lai, Department of Biochemistry,
National Cheng Kung University). Briefly, HMEC-1 cells were grown to FIGURE 1. Tyrosine phosphorylation of cellular proteins after treat-
50% confluent in six-well plate, then transfected with the dominant-nega- ment with anti-DV NS1 Abs in endothelial cells. After serum-free starva-
tive IKK-␤ (0.5 ␮g) or the empty vector (1.0 ␮g) using Lipofectamine tion for 1 h, HMEC-1 cells were treated with 5 ␮g of anti-DV NS1, anti-
2000 reagent (Invitrogen Life Technologies). After 24 h of transfection, the
JEV NS1, or control IgG, or replaced with EGM (used as positive controls
cells were used for experiments.
for cell signaling) composed of 2% FBS for 30 min. The tyrosine-phos-
Adhesion assay phorylated protein levels were analyzed by Western blotting (A, upper
panel) and quantified by measuring OD (A, lower panel). The cells were
HMEC-1 cells (5 ⫻ 104 cells/well) were plated into eight-well glass cham-
stained with FITC-conjugated phosphorylated tyrosine-specific IgG and
ber slides (Nalge Nunc International). When monolayers were confluent,
the cells were stimulated with anti-DV NS1, anti-JEV NS1, or control IgG analyzed using flow cytometry. A histogram and the percentage of positive
in serum-free culture medium. After 24 h of incubation, the cells were cells are shown (B, upper panel). Time-kinetic changes of protein tyrosine
washed once with medium and incubated for 2 h at 37°C with isolated phosphorylation induced by control IgG (F), anti-DV NS1 (Œ), and EGM
PBMC (1 ⫻ 105 cells/well) in a total volume of 250 ␮l/well. At the end of (f) are shown, respectively. The untreated cells (E) were used as negative
the incubation period, the nonadherent cells were removed by washing controls (B, lower panel).
twice with 0.1% BSA in PBS. Adherent cells were stained with Liu’s stain
(TONYAR Biotech) and viewed with light microscopy. The adherent cells cells after they had been treated with DHF patient sera (data not
were counted on three consecutive microscopic fields (53, 54).
shown).
Statistical analysis We next investigated the involvement of NF-␬B in the anti-DV
Comparisons between various treatments were performed by Student’s t NS1-triggered signaling pathways. After Ab treatment for 3 or
test with SigmaPlot version 4.0 for Windows (Cytel Software). Values 24 h, endothelial cell extracts were separated for cytoplasmic and
were considered statistically significant at p ⬍ 0.05. nuclear fractions, and then followed by immunoblotting for NF-
␬B. The expression of NF-␬B decreased in the cytoplasmic frac-
Results tion and increased in the nuclear fraction in cells with anti-DV
Protein tyrosine phosphorylation and NF-␬B activation induced NS1 treatment compared with those with anti-JEV NS1 or control
by anti-DV NS1 Abs in human endothelial cells IgG treatment (Fig. 2A). Activation of NF-␬B as indicated by its
In a previous study (40), we showed that anti-DV NS1 cross-re- translocation from cytoplasm to nucleus was also observed by im-
acted with endothelial cells. To assess the signal transduction in- munohistochemical staining (data not shown). The DNA-binding
duced by anti-DV NS1 in endothelial cells, we investigated protein ability of NF-␬B was determined by EMSA, which showed pos-
phosphorylation and NF-␬B activation in the present study. We itive signals in the anti-DV NS1-treated cells compared with the
used Western blotting to assay the levels of protein tyrosine phos- untreated, anti-JEV NS1, or control IgG group (Fig. 2B). The
phorylation from Ab-treated HMEC-1 cells (Fig. 1A, upper panel). DNA-binding ability of NF-␬B was inhibited when anti-DV NS1
We also determined the relative ODs of bands (Fig. 1A, lower was preabsorbed with rDV NS1 proteins. The specificity of the
panel). Results showed tyrosine phosphorylation of proteins in cross-reactivity of anti-NS1 Abs was thus confirmed. To further
HMEC-1 after binding with anti-DV NS1, but not with anti-JEV demonstrate whether NF-␬B activation is requisite for its DNA-
NS1 or control IgG. EGM-treated cells were used as the positive binding ability mediated by anti-DV NS1, the effect of NF-␬B
control for protein phosphorylation induced by growth factors. inhibitor PDTC was examined. Results showed that the addition of
Protein tyrosine phosphorylation was further analyzed by flow cy- PDTC to cell cultures caused an inhibition of the DNA-binding
tometry as presented by the percentages of fluorescence-positive activity of NF-␬B in anti-DV NS1-treated cells (data not shown).
cells (Fig. 1B, upper panel). The time-kinetic changes of phos-
phorylated cells after Ab stimulation are shown in Fig. 1B (lower Production of cytokines and chemokines in endothelial cells
panel). Results indicated that protein tyrosine phosphorylation after anti-DV NS1 stimulation
reached its highest level 30 min after anti-DV NS1 treatment. The Immune activation of endothelial cells along with the induction of
specificity of Ab stimulation was demonstrated by the blockage of proinflammatory responses underlie dengue vascular disorders (7,
protein tyrosine phosphorylation when anti-DV NS1 Abs were 9). We therefore examined whether anti-DV NS1 Abs could in-
pretreated with rDV NS1 proteins (data not shown). Tyrosine duce inflammatory factors in endothelial cells. Results from im-
phosphorylation of cellular proteins was also observed in HMEC-1 munohistochemical staining revealed the expression of IL-6, IL-8,
398 ANTI-DENGUE NS1 Ab-INDUCED ENDOTHELIAL CELL ACTIVATION
FIGURE 2. NF-␬B activation induced by anti-DV NS1 Abs in endo-
thelial cells. A, HMEC-1 cells were untreated or treated with 5 ␮g of
anti-DV NS1, anti-JEV NS1, or control IgG for 3 or 24 h, and the expres-
sion of NF-␬B in cytoplasm and nucleus was determined using Western
blotting. ␤-actin was used as an internal control of total cell lysate before
cytoplasmic and nuclear extraction. B, The DNA-binding ability of NF-␬B
in HMEC-1 cells after treatment with anti-DV NS1 (5 or 25 ␮g), anti-JEV
NS1 (25 ␮g), or control IgG (25 ␮g) for 3 h was detected by EMSA using
NF-␬B-specific probes. Cells treated with anti-DV NS1 preabsorbed with
25 ␮g of rDV NS1 protein were tested for the specificity of Ab stimulation.
NF-␬B p65 fragment was used as a positive control. Free probe was used
as a loading control.
and MCP-1 by HMEC-1 cells after they had been treated with
anti-DV NS1 for 24 h (Fig. 3A). The expression of RANTES,
however, could not be detected in anti-DV NS1-treated cells. Ex-
pression of IL-6, IL-8, and MCP-1 was further quantified using
both flow cytometric analysis (Fig. 3B) and ELISA (Table I).
Treatment with anti-JEV NS1 Abs showed only basal levels of
IL-6, IL-8, MCP-1, and RANTES, similar to those with control
IgG. The ELISA (Table I) and immunohistochemical staining
(data not shown) showed IL-6, IL-8, and MCP-1 production after
anti-DV NS1 treatment for as short as 3– 6 h. The specificity of the
cross-reactivity of anti-NS1 Abs was further confirmed by the in-
hibitory effect of preabsorption of anti-DV NS1 with recombinant
DV NS1, but not JEV NS1 proteins (Fig. 3B). Further studies
showed that the mRNA expression of MCP-1, but not RANTES,
was elevated after anti-DV NS1 stimulation in HMEC-1 cells, as
detected via RT-PCR (Fig. 4). By contrast, cells infected with DV
showed increased mRNA expression in RANTES, but not in
MCP-1. The ELISA quantification demonstrated high levels of
MCP-1 in DHF patient sera at different disease grades compared
with healthy controls (Table II), similar to results in a study of DSS
patients (27).
FIGURE 3. Production of IL-6, IL-8, and MCP-1, but not RANTES, by
endothelial cells after treatment with anti-DV NS1 Abs. HMEC-1 cells
were treated with 5 ␮g of anti-DV NS1, anti-JEV NS1, or control IgG for
24 h. The expression of IL-6, IL-8, MCP-1, and RANTES was detected
with immunohistochemical staining using specific Abs, as described in
Materials and Methods (A, the positive cells are indicated by arrowheads),
and with flow cytometry analysis (B). For flow cytometric analysis, three
individual cultures were performed, and the averages of the percentages of
positive cells are shown (mean ⫾ SD). Cells treated with anti-DV NS1
preabsorbed with 5 ␮g of recombinant DV or JEV NS1 protein were tested
for the specificity of Ab stimulation.
Requirement for NF-␬B in anti-DV NS1-induced cytokine and
chemokine production
Anti-DV NS1 induced NF-␬B activation (Fig. 2). We further ex-
amined the involvement of NF-␬B modulation on the expression
of IL-6, IL-8, and MCP-1. After treatment with anti-DV NS1 in
HMEC-1 cultures for 24 h, the protein levels of IL-6, IL-8, and
MCP-1 increased. NF-␬B inhibitor PDTC, but not NO synthase
inhibitor L-NAME or caspase inhibitor zVAD-fmk, was able to
inhibit the anti-DV NS1-mediated elevations of IL-6, IL-8, and
MCP-1 expression (Fig. 5, left). Further confirming the require-
ment for NF-␬B activation, HMEC-1 cells transfected with dom-
inant-negative IKK-␤ showed a blockage in IL-6, IL-8, and
MCP-1 production compared with the vector control (Fig. 5,
right). These results revealed that NF-␬B in anti-DV NS1-induced
endothelial cell activation up-regulates cytokine and chemokine
expression.
MCP-1 facilitates the expression of ICAM-1 induced by anti-DV
NS1 in endothelial cells
The activation of endothelial cells involves not only the induction
of cytokines and chemokines; our data showed that the expression
of adhesion molecules was also augmented. After anti-DV NS1
The Journal of Immunology 399

Table I. Production of IL-6, IL-8, and MCP-1 by endothelial cells after

treatment with anti-DV NS1 Abs for various time periodsa

Ab
Time Dose
(h) (␮g) Control IgG Anti-JEV NS1 Anti-DV NS1

IL-6 (pg/cell)
0 0.12 ⫾ 0.02
3 5 0.30 ⫾ 0.11 0.14 ⫾ 0.10 0.22 ⫾ 0.10
6 5 0.31 ⫾ 0.10 0.22 ⫾ 0.08 1.05 ⫾ 0.04ⴱ
12 5 0.32 ⫾ 0.15 0.35 ⫾ 0.15 4.03 ⫾ 0.55ⴱⴱ
24 5 0.42 ⫾ 0.14 0.39 ⫾ 0.11 5.75 ⫾ 1.20ⴱⴱ
24 25 0.45 ⫾ 0.11 0.41 ⫾ 0.12 4.15 ⫾ 1.07ⴱⴱ
IL-8 (pg/cell)
0 0.07 ⫾ 0.01
3 5 0.10 ⫾ 0.01 0.12 ⫾ 0.05 0.52 ⫾ 0.12ⴱ FIGURE 4. The expression of MCP-1, but not RANTES mRNA by
6 5 0.12 ⫾ 0.03 0.20 ⫾ 0.01 2.75 ⫾ 1.07ⴱ endothelial cells after anti-DV NS1 Ab treatment. Total cellular mRNA
12 5 0.23 ⫾ 0.09 0.22 ⫾ 0.11 5.33 ⫾ 1.34ⴱⴱ was extracted at 24 h from HMEC-1 cells after treatment with 5 ␮g of
24 5 0.21 ⫾ 0.11 0.31 ⫾ 0.15 7.65 ⫾ 2.52ⴱⴱ anti-DV NS1, anti-JEV NS1, or control IgG or with 100 multiplicity of
24 25 0.29 ⫾ 0.08 0.61 ⫾ 0.20 5.97 ⫾ 1.30ⴱⴱ infection of dengue-2 virus, and detected using RT-PCR. ␤-actin was used
MCP-1 (fg/cell) as an internal control.
0 0.29 ⫾ 0.09
3 5 0.30 ⫾ 0.01 0.29 ⫾ 0.11 0.52 ⫾ 0.01
6 5 0.43 ⫾ 0.01 0.41 ⫾ 0.08 1.75 ⫾ 0.09ⴱ
12 5 0.46 ⫾ 0.07 0.58 ⫾ 0.05 4.33 ⫾ 0.50ⴱⴱ
24 5 0.45 ⫾ 0.03 0.71 ⫾ 0.01 4.65 ⫾ 0.23ⴱⴱ expressed on endothelial cells was involved in this binding effect.
24 25 0.54 ⫾ 0.14 0.87 ⫾ 0.14 4.03 ⫾ 1.07ⴱⴱ We also tested the potential regulatory role of MCP-1 on ICAM-
a
ELISA kits were used for the assay. Three individual cultures were performed in 1-mediated PBMC adherence to endothelial cells. A reduction in
each group, and the averages of values are expressed as mean ⫾ SD; ⴱ, p ⬍ 0.05 vs
control IgG; ⴱⴱ, p ⬍ 0.01 vs control IgG.
PBMC adherence was demonstrated when endothelial cells were
pretreated with anti-MCP-1 Abs (Fig. 7). These results thus sug-
gested that the ICAM-1-mediated ability of PBMC to adhere to
endothelial cells was at least partially regulated by MCP-1. To
stimulation, an increase in the expression of ICAM-1 was detected
further investigate whether the up-regulation of cell adherence was
with immunohistochemical staining (Fig. 6A). The increase in
dependent on new protein synthesis, HMEC-1 cells were pre-
ICAM-1 expression was confirmed using flow cytometric analysis
treated with protein synthesis inhibitor cycloheximide. Results
(see Fig. 6B for time kinetics and dose-response data). To further
showed that cycloheximide treatment inhibited PBMC adhesion,
investigate the regulation of ICAM-1 expression by NF-␬B in anti-
suggesting a requirement for protein synthesis of ICAM-1 or
DV NS1-treated cells, PDTC was used. Results showed a reduc-
MCP-1 to increase PBMC adherence to HMEC-1 cells (Fig. 7B).
tion of ICAM-1 expression once cells were pretreated with PDTC
(data not shown). MCP-1 elevates ICAM-1 expression (55–57).
We therefore investigated the relationship between MCP-1 and Discussion
ICAM-1 expression after anti-DV NS1 stimulation. Pretreatment Dengue disease is an emerging public health problem, and an in-
with MCP-1-neutralizing Abs reduced the expression of ICAM-1, creasing number of cases are being reported (5, 6, 58). Vascular
suggesting that the ICAM-1 expressed was regulated, at least in leakage, liver abnormality, and hemorrhagic diathesis are the life-
part, by MCP-1 in anti-DV NS1-treated endothelial cells. Similar threatening complications that occur in dengue patients with DHF/
results were observed using both immunohistochemical staining DSS. Their pathogenic mechanisms, however, are not well under-
(Fig. 6A) and flow cytometric analysis (Fig. 6B). Further studies stood. We have proposed a mechanism of molecular mimicry in
using anti-DV NS1 F(ab⬘)2 confirmed that the activation of endo- which Abs directed against DV NS1 would cross-react with en-
thelial cells, as detected by ICAM-1 expression, was not via an dothelial cells and cause damage. Our studies showed anti-
Fc-mediated signaling pathway (data not shown). endothelial cell Abs (AECA) in dengue patient sera (41). More-
over, endothelial cell damage induced by anti-DV NS1 by the
Regulation of ICAM-1 and MCP-1 on the adherence of PBMC production of NO may play a role in the disruption of vessel en-
to anti-DV NS1-treated endothelial cells dothelium and contribute to the AECA-induced pathogenesis of
vasculopathy (40, 42). When taken together with previous findings
The ability of immune cells to adhere to DV-infected endothelial
that anti-DV NS1 Abs also cross-reacted with platelets (59) and
cells was investigated next. Based on the findings shown in Fig. 6,
anti-DV NS1 Ab-induced expression of ICAM-1 was down-regu-
lated by anti-MCP-1. To explore the effects of ICAM-1 up-regu-
lation on endothelial cell and immune cell interaction, the adhesion
Table II. MCP-1 levels in DHF patient seraa
of PBMC to anti-DV NS1-treated HMEC-1 cells was assessed.
Higher levels of PBMC adhering to endothelial cells could be ob-
Group MCP-1 (pg/ml)
served in cells treated with anti-DV NS1 for 24 h, compared with
those with anti-JEV NS1 or control IgG (Fig. 7A). The increase in Controls (n ⫽ 5) 72.9 ⫾ 18.9
PBMC adherence was quantified by counting the numbers of ad- DHF patients (n ⫽ 17) 2695.6 ⫾ 444.0ⴱⴱⴱ
Grade I (n ⫽ 1) 3009.5 ⫾ 351.4ⴱⴱⴱ
herent cells under a microscope (see Fig. 7B for time kinetics and
Grade II (n ⫽ 13) 2610.8 ⫾ 383.2ⴱⴱⴱ
dose-response data). We further investigated whether the increase Grade III (n ⫽ 3) 2958.5 ⫾ 618.6ⴱⴱⴱ
in ICAM-1 expression was related to the ability of PBMC to ad- a
ELISA kits were used for the assay. Three individual cultures were performed
here to endothelial cells. Pretreatment with ICAM-1-neutralizing for each person, and the averages of values are expressed as mean ⫾ SD; ⴱⴱⴱ, p ⬍
Abs inhibited the adherence of PBMC, suggesting that ICAM-1 0.001 vs controls.
400 ANTI-DENGUE NS1 Ab-INDUCED ENDOTHELIAL CELL ACTIVATION
FIGURE 5. The involvement of NF-␬B in the expression of IL-6, IL-8,
and MCP-1 in endothelial cells after anti-DV NS1 stimulation. In the pres-
ence or absence of PDTC (100 ␮M), L-NAME (10 ␮M), or zVAD-fmk (10
␮M) for 1-h pretreatment or transfected with dominant-negative IKK-␤
(⌬IKK-␤) or empty vector, HMEC-1 cells were treated with 5 ␮g of anti-
DV NS1, anti-JEV NS1, or control IgG for 24 h. The expression of IL-6,
IL-8, and MCP-1 was detected using ELISA, as described in Materials and
Methods. Three individual cultures were performed in ELISA, and the
averages are shown (mean ⫾ SD).
that transient thrombocytopenia in DV-infected mice was associ-
ated with the generation of anti-platelet Abs (60), our results sug-
gest that the onset of autoimmune responses in DV infection may
have implications in DHF immunopathogenesis (9). Several other
reports have also shown the autoimmune responses in DV infec-
tion (61, 62).
In addition to cell apoptosis (40), we showed in this study that
immune activation in endothelial cells also occurred after anti-DV
NS1 stimulation. Protein tyrosine phosphorylation and NF-␬B ac-
tivation in HMEC-1 cells were observed. There was also an in-
crease in the expression of cytokines and chemokines, including
IL-6, IL-8, and MCP-1, but not RANTES. The involvement of
NF-␬B in the expression of IL-6, IL-8, and MCP-1 was also dem-
onstrated. Up-regulation of ICAM-1 expression and ICAM-1-me-
diated adherence of PBMC to endothelial cells were observed after
anti-DV NS1 stimulation. The expression of ICAM-1 was modu-
lated, at least in part, by MCP-1 and by NF-␬B. Taken together,
these facts indicate that endothelial cell activation that involves
NF-␬B-regulated cytokine and chemokine production and adhe-
sion molecule expression, as well as adherence of immune cells to
endothelial cells, may represent the inflammatory mechanisms me-
diated by anti-DV NS1 Abs. The signaling pathways induced by
anti-DV NS1 in endothelial cells leading to immune activation and
inflammation are shown in Fig. 8. Although our previous study
showed an NO-mediated mechanism of apoptosis induced by anti-
DV NS1, immune activation is NF-␬B dependent, but NO and
apoptosis independent.
Several in vitro studies have shown that endothelial cells are the
targets of DV and that induction of proinflammatory responses and
apoptotic cell death are directly caused by DV (26, 27, 63). We
have shown somewhat similar effects mediated by anti-DV NS1
Abs, specifically that endothelial cells underwent apoptosis (40 –
42) and, in the present study, induced proinflammatory effects. The
combined effects of DV and anti-NS1 Abs on endothelial cells
FIGURE 6. MCP-1 regulates the expression of ICAM-1 in endothelial
cells after anti-DV NS1 Ab stimulation. A, HMEC-1 cells were treated with
5 ␮g of anti-DV NS1, anti-JEV NS1, or control IgG for 24 h. Cells were
labeled with mouse anti-human ICAM-1 Abs, followed by HRP- or FITC-
conjugated goat anti-mouse IgG. Cells treated with anti-DV NS1 in the
presence of 5 ␮g of neutralizing anti-MCP-1 were also tested. The ICAM-1
expression was detected using immunohistochemical staining. B, HMEC-1
cells were treated with 1, 5, or 25 ␮g of anti-DV NS1, anti-JEV NS1, or
control IgG for 6, 12, or 24 h. The ICAM-1 expression was detected using
flow cytometry analysis. Three individual cultures were performed, and the
percentages of positive cells are shown (mean ⫾ SD).
related to dengue pathogenesis remain to be investigated. Re-
cently, DV NS1 has been reported to express in a GPI-linked form
that results in signal transduction, as evidenced by tyrosine phos-
phorylation of cellular proteins (64). In this study, we showed
tyrosine phosphorylation and NF-␬B activation after anti-DV NS1
stimulation in endothelial cells without DV infection. The role of
protein tyrosine phosphorylation in the NF-␬B-mediated immune
activation requires further clarification. To explore the signaling
pathways mediated by anti-DV NS1, the surface molecules of en-
dothelial cells, GPI linked or not, recognized by these autoanti-
bodies need to be identified. Studies on potential autoantigens us-
ing anti-DV NS1 Abs and DHF/DSS patient sera are in progress.
Various autoimmune diseases associated with vascular dysfunc-
tion may be related to the generation of AECA (42). Vasculopathy
in autoimmune diseases involves both cytotoxicity and inflamma-
tion facilitated by AECA via a direct or an indirect route. The
elevated expression of inflammatory mediators, including cyto-
kines (IL-1 and IL-6), chemokines (IL-8 and MCP-1), and adhe-
sion molecules (ICAM-1, VCAM-1, and E-selectin), has been
shown in AECA-stimulated endothelial cells (65– 68). However,
the molecular mechanisms of AECA-mediated endothelial cell ac-
tivation remain poorly defined. Studies on the mechanisms of en-
dothelial activation have shown that degradation of I-␬B followed
by NF-␬B activation might contribute to the induction of proin-
flammatory responses (67). Activation of NF-␬B was also detected
when anti-DV NS1 cross-reacted with uninfected endothelial cells.
A recent study provided a possible molecular mechanism of AE-
CA’s regulation of ICAM-1 expression through ERK1/2 activation
in Behcet’s disease (69).
In dengue pathogenesis, elevated levels of inflammatory IL-6
and IL-8 were correlated with clinical presentation of DHF (46 –
48, 52). Also, endothelial cells infected with DV induced IL-6 and
The Journal of Immunology
FIGURE 7. The ability of PBMC to adhere to anti-DV NS1-treated en-
dothelial cells is blocked by pretreatment with anti-ICAM-1 and anti-
MCP-1. A, HMEC-1 cells were cultured in eight-well glass chamber slides
and treated with 5 ␮g of anti-DV NS1, anti-JEV NS1, or control IgG for
24 h. In anti-DV NS1-treated groups, cells cultured with 5 ␮g of neutral-
izing anti-ICAM-1 or anti-MCP-1 were also tested. Cells were washed with
PBS, and then cocultured with fresh human PBMC for 2 h. Cell adhesion
was observed using Liu’s stain. B, Time kinetics and dose response of
anti-DV NS1 on the PBMC adhesion to endothelial cells in the presence or
absence of 100 ␮M cycloheximide or 5 ␮g of anti-ICAM-1 or anti-MCP-1.
The numbers of adherent cells were quantified in each tested culture.
IL-8 production (26, 27). In the present study, we showed that
anti-DV NS1 promoted proinflammatory endothelial cell activa-
tion causing increases in IL-6 and IL-8 protein expression and
secretion. Furthermore, anti-DV NS1 induces the generation of
MCP-1 in endothelial cells. A previous study showed MCP-1 in
DSS patient plasma and pleural fluid, but not in DV-infected en-
dothelial cells (27). We found, in the present study, MCP-1 pro-
duction by endothelial cells after anti-DV NS1 stimulation; MCP-1
in DHF patient sera was also confirmed. In addition to the two
chemokines IL-8 and MCP-1, RANTES was also checked in this
study and was found not induced by anti-DV NS1 stimulation,
although it was produced in endothelial cells after DV infection
(27). MCP-1-mediated elevation of ICAM-1 expression and facil-
itation of leukocyte transmigration have been suggested (53–55).
Results in this study showed that MCP-1 up-regulated ICAM-1
expression in anti-DV NS1-treated HMEC-1 cells. Endothelial cell
activation followed by elevated expression of ICAM-1 may con-
tribute to the adherence of immune cells to endothelial cells in the
inflammatory responses associated with dengue disease pathogen-
esis. The involvement of other cytokines, chemokines, and adhe-
sion molecules is currently under investigation.
In the present study, we showed the inflammatory initiator role
of anti-DV NS1 resulting in endothelial cell activation. In addition
to the increased expression of cytokines and chemokines, a pos-
sible chemotactic effect on PBMC adherence may contribute to the
401
FIGURE 8. The signaling pathways mediated by anti-DV NS1 Abs that
lead to endothelial cell activation. Signal transduction in endothelial cells
after anti-NS1 stimulation involves protein tyrosine phosphorylation and
NF-␬B activation. NF-␬B is required for the expression of cytokines (e.g.,
IL-6) and chemokines (e.g., IL-8 and MCP-1). Expression of ICAM-1
induced by anti-NS1 is, at least in part, regulated by NF-␬B and by MCP-1.
Furthermore, the ability of PBMC to adhere to endothelial cells is mediated
by ICAM-1. The endothelial cell activation induced by anti-DV NS1 Abs
leading to inflammatory responses may be related to DHF pathogenesis.
indirect damage in endothelial cells. Activation of endothelial cells
via DV-infected peripheral blood monocytes has been reported
(33). Endothelial cell activation and apoptosis might play a role in
the fulminant, but short-lived vascular leakage of DHF pathogen-
esis (27). The clinical features indicate that vascular endothelia in
serosal tissues are preferentially affected by dengue pathogenetic
mechanisms, resulting in ascites and pleural effusion for most of
the plasma leakage in dengue shock syndrome (70). Tissue culture
fluid from DV-infected monocytes has activated endothelial cells
(33). Serum inflammatory cytokines may relate to the development
of plasma leakage and disease severity (8, 52). Given all of these
data, endothelial vascular dysfunction in dengue pathogenesis in-
volves multiple factors, including the dengue virus itself and anti-
DV NS1, direct and indirect effects on endothelial cells, and im-
mune activation and apoptosis.
Acknowledgment
We acknowledge the editorial assistance of Bill Franke.
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