登革病毒感染不同抗原呈現細胞對其免疫及凝血功能的影響(III)

行政院國家科學委員會專題研究計畫 成果報告

登革病毒感染不同抗原呈現細胞對其免疫及凝血功能的影

響(3/3)

中 華 民 國 93 年 10 月 5 日

Dengue Virus Induces Human Neutrophil Activation and Necrosis

Mu-Jie Suna, Lien-Cheng Chenb, Huan-Yao Leic,Hsiao-Sheng Liuc, Yee-Shin Linc,

Shun-Hua Chenc, Tsuey-Yu Chang d,and Trai-Ming Yeha

a _{Department of Medical Technology, National Cheng Kung University,} b _{Institute of}

Basic Medical Sciences, National Cheng Kung University, c Department of

Microbiology and Immunology, National Cheng Kung University, d _{Department of}

Parasitology, National Cheng Kung University, Tainan, Taiwan, ROC

Short title: Dengue virus and neutrophil Full address:

Dr. Trai-Ming Yeh

Department of Medical Technology, College of Medicine, National Cheng Kung

University, Tainan 701, Taiwan (ROC)

Tel. +886 6 2353535 ext. 5778, Fax +886 6 236 3956, E-Mail:

[email protected].

Abstract

Neutrophils are known to play important role in host defense against pathogens.

Even though neutropenia is common during dengue virus (DV) infection, the role of

neutrophils during DV infection is unclear. In this study, freshly isolated human

neutrophils were incubated with DV to understand the effects of DV and neutrophils

on each other. DV induced the secretion of chemokines such as interleukin 8,

macrophage inflammatory protein (MIP -1α, MIP -1β) and the release of granular enzyme, myeloperoxidase 18 h after incubation. DV-induced neutrophil activation

was further supported by the increased expression of CD11b/CD18 (Mac-1) and

Toll-like receptor 4 on the surface of DV-stimulated neutrophils. In addition, the

phagocytic ability to FITC-carboxylate microspheres (FITC-beads) was increased in

DV-stimulated neutrophils . Co-localization of DV E-antigen with FITC -beads was

found in these neutrophils , indicating that DV may enter neutrophils through

phagocytosis. However, no viral progeny was detected in the supernatants and cell

lysate of neutrophils 18 h after incubation. In addition, the viabilities and apoptosis

of neutrophils were decreased after incubation with DV for 18 h. On the other hand,

DV-stimulated neutrophils was increased in a dose-dependent manner. Therefore,

our results suggest that phagocytosis of DV by neutrophils may help to clear DV

infection. However, DV-induced neutrophil activation and necrosis may also

Introduction

Dengue viruses are mosquito -borne flaviviruses which are widely spread in

tropical and subtropical countries [11]. DV causes a spectrum of disease, from a

relatively benign febrile disease called dengue fever (DF) to a life-threatening illness

called dengue hemorrhage fever and shock syndrome (DHF/DSS) [8]. DF is

characterized by biphasic fever, headache, muscle pain, and rash. DHF/DSS

occurs in a small percentage of dengue patients which is characterized by

hemorrhage and vascular leakage accompanied by severe thrombocytopenia and

hemoconcentration. The pathogenesis of DHF/DSS has not been fully elucidated.

However, abnormal immune responses may be responsible for the progress of DF

to DHF/DSS [16].

Even though DV can infect many different cell types in vitro such as monocytes,

hepatoma, and endothelial cells [17, 12], its primary host target cell is thought to be

monocytes and macrophages. These cells are responsible for the dissemination of

the virus in blood after its initial entry via the mosquito vector [10]. However, the role

not been fully elucidated. Since neutrophils bear an arsenal of weapons against

infection, neutrophils may contribute to the clearance of viral infection as well as to

the pathogenesis of tissue damage [29]. Early clinical and laboratory indicators

suggest neutropenia is a common phenomenon during dengue infection [2, 15].

Furthermore, neutrophil granule enzyme elastase has been found to be significantly

elevated in the sera of dengue patients with shock than in patients without shock

[14]. Therefore, neutrophil activation and degranulation may involve in dengue virus

induced immunopathological process. In this study, the effects of DV and

neutrophils on each other were studied using freshly isolated human neutrophils

incubated with DV in vitro. Results from these studies suggest neutrophils may play

an important role in the clearance of DV as well as in the pathogenesis of DV

Materials and Methods

Reagents

The following reagents were from the indicated sources: fetal calf serum (FCS),

Dulbecco’s modification of Eagle’s minimum essential medium (DMEM),

penicillin-streptomycin-glutamine, and Hank’s balanced salt solution (HBSS) (Gibco

Life Technologies, Grand Island, NY), FITC-carboxylate microspheres (FITC-beads)

(Polysciences, Inc., Warrington, PA), lipopolysaccharide (LPS) of E. coli 0111: B4

(Difco Laboratories, Detroit, MI) , RBC lysis solution (Genera, Minneapolis, MN),

Histopaque-1.077 lymphocyte separation medium and FITC -labeled mouse

monoclonal anti-human CD11b antibody and goat-polyclonal anti-human TLR4

antibody (Santa Cruz Biotechnology, CA), FITC-conjugated mouse-polyclonal

anti-goat IgG antibody (Jackson ImmunoResearch, West Grove, PA), biotin labeled

mouse-monoclonal anti-DV envelop protein (E), non structural protein 1 (NS-1)

antibodies (Dr. Lei’s laboratory), and PE-conjugated streptoavidin (Sigma, St. Louis,

MO).

Dengue type 2 virus strain (PL0146) was propagated in C6/36 cells from

American Type Culture Collection with DMEM supplemented with 2% FCS and

antibiotics. The virus culture medium was harvested after incubation for 5 days, cell

debris was removed by centrifugation, and the virus supernatant was aliquoted and

stored at -70 °C until used. DV titers were determined on BHK-21 cultures under

nutrient agarose and expressed as PFU per milliliter. The UV inactivation of DV

(UV-DV) was conducted in a Stratagene UV-stratalinker apparatus using 1800 mJ

of UV radiation. The heat inactivation of DV (H -DV) was carried out by heating at

56°C for 30 min. No plaque was found when UV-DV or H-DV was used to infect

BHK cells. A mock DV stock was prepared in exactly the same manner as the DV

preparation, with the exception that the C6/36 cells were not infected with DV

(C6/36).

Neutrophil Isolation

Neutrophils were isolated from the peripheral blood of healthy donors. The

blood was collected in citrated tubes and mixed with 5% dextran i n a ratio of 10:1 to

containing the buffy coat was layered over an equal volume of Histopaque-1.077

lymphocyte separation media and centrifuged (x 650g) at 25 °C for 25 min. The

erythrocytes in the resulting pellet were lysed with RBC lysis buffer and removed by

centrifugation (x 450g) at 25°C for 10 min. Purified neutrophils were washed 3 times

with HBSS and re-suspended in RPMI 1640 plus 10% FCS. The cell preparation

contained 98% neutrophils, with less than 1 platelet/100 neutrophils as determined

by cytospin and Liu’s stain after microscopic counting.

Stimulation of Neutrophils

Neutrophils (2x106) were stimulated with either live DV at multiplicity of

infection (MOI) as indicated, UV-DV (with the same MOI as live DV), or 200 ng/ml

LPS and incubated in 1 ml of RPMI 1640 supplemented with 10% FCS and 2 mM

L-glutamine at 37°C in a 5% CO2 incubator. In addition, cells treated with medium

alone or C6/36 supernatant were also included as controls. After 18 h, the cell

suspensions were centrifuged, and the supernatants were analyzed by ELISA for

chemokines and myeloperoxidase (MPO). In some experiments, the cells were

electron microscopy examination.

Enzyme-Linked Immunosorbent Assay (ELISA)

IL -8, MIP-1α, MIP-1β, and MPO were assessed by commercially available ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s

instructions.

CD11b/CD18 (Mac-1) and TLR4 Staining

Human neutrophils (2 x 104) with or without DV (MOI of 1) or LPS (200 ng/ml)

stimulation were cytospined onto the slides and stained with FITC-labeled mouse

anti-human CD11b monoclonal antibody in a final concentration of 20 µg/ml for 1 h

at RT, and the cells were washed with Tris-buffer and observed by a confocal

spectral microscope. The slides stained with goat anti-human TLR4 antibody (20

µg/ml) for 1 h at 4℃ , were then washed twice and incubated with FITC conjugated

mouse anti-goat IgG antibody (4 µg/ml). After incubation for 30 min at 4 °C in the

dark, the cells were washed with Tris-buffer and observed by a confocal fluorescent

(FACSCalibur, Becton Deckinson, USA).

Electron Microscopy

Neutrophils after incubation with DV were fixed at room temperature for 1 h

using a freshly prepared 3% solution of glutaraldehyde in warm cacodylate buffer.

Fixed cells were resuspended in warm liquid agarose and chilled immediately on ice.

After cutting the agarose into small cubes, cells were postfixed in 2% osmium

tetroxide, dehydrated in a graded series of ethanol and embedded in resin (EMS,

SPUR’S Kit, Washington) according to standard protocols. Ultrathin sections were

cut with ultramicrotome (Reichert-Jung) and contrasted with saturated aqueous

uranyl acetate and lead citrate at room temperature. Specimens were then

observed under a transmission electron microscope (JEOL JEM-1200EX, Tokyo,

Japan) at 100 kV.

Phagocytosis of Neutrophils

The uptake of FITC -carboxylate microspheres (FITC-beads) by neutrophils

MOI of 1 or 5 for 1 h. After pre-incubation, 2 µl of FITC-beads (0.75 µm, 7x106 beads/µl) were added and incubated for 30 min at 37°C. Neutrophils were washed with ice cold 1.6% BSA in PBS to remove free FITC -beads and fixed with 100 µl 4% paraformaldehyde in PBS for 20 min at 4℃ . Some cells were permeabilized with

0.1% saponin in PBS and incubated with biotin labeled monoclonal antibody against

dengue virus E protein at a final dilution of 1:60 for 1 h. The cells were then washed

twice with permeabilization solution and incubated with PE-conjugated streptoavidin

at a final dilution of 1:100 for 30 min at RT in the dark. Fluorescence pattern of

neutrophils was observed by confocal microscopy (Leica TCS SP2, Germany) and

analyzed by dual-laser FACS Calibur (BD Biosciences, Mountain View, CA) using

CellQuest software (BD Biosciences).

Assessment of Neutrophils Viability

Assessment of neutrophil viability was made after incubation neutrophils (2x105)

with LPS (200 ng/ml), DV or UV-DV at MOI of 1 for 18 h. After incubation, cells were

washed and stained with trypan blue or propidium iodide (PI). The number of

cytometry unit. Neutrophils that did not take up trypan blue or PI were counted as

viable. The percentage viability of neutrophils was obtained by dividing the number

of viable cells by the total number of cells. In addition, cell viability was detected

using an XTT kit (Boehringer Mannheim Biochemicals, Indianapolis, IN). Briefly,

cells (2 x 105 _{cells/well) were incubated with 50 l of the XTT labeling mixture (final}

XTT concentration, 0.3 mg/ml). The microtiter plate was further incubated for 2-4 h

at 37℃ in 5% CO2. The spectrophotometrical absorbance of the sample was

measured at 450 nm using a Vmax ELISA reader (Molecule Devices, Menlo Park,

CA).

Neutrophil Apoptosis Assays

The percentage of apoptotic neutrophils were assessed by the terminal

deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)

assay as well as annexin V stain. Neutrophils (2x105) were incubated with LPS (200

ng/ml), DV or UV-DV at MOI of 1 for 18 h. In addition, some cells were treated with

C6/36 supernatants (C6/36), heat-inactivated DV (H-DV) as negative control. After

for 10 min at room temperature and permeabilized with 70% ethanol at -20℃. The

percentages of apoptotic cells were assessed by TUNEL reaction using ApoAlert

DNA Fragmentation Assay Kit (CLONTECH) according to the manufacture’s

instruction and then analyzed by flow cytometry. In addition, apoptosis was also

assayed by binding annexin-V-FITC to the exposed phosphatidylserine of apoptotic

cells with the Human annexin V Apoptosis Kit (Bender MedSystems) and then

analyzed by flow cytometry.

Detection of Lactate Dehydrogenase

The release of cytoplasmic enzyme lactate dehydrogenase (LDH) of

DV-stimulated neutrophils was measured using a commercial kit (CytoTox Non-

Radioactive Cytotoxicity Assay; Promega). Briefly, Neutrophils (2x105) were

incubated with DV at different MOI as indicated, LPS (200 ng/ml) or without

stimulation at 37℃ for 18 h. The supernatants were collected and assayed for LDH.

Cytotoxicity in experimental samples was measured as % LDH release as

Statistical Analysis

Data are expressed as mean ± SD. The significance of the difference between

the test and the control groups was analyzed using Student’s t-test and the

Results

Kinetics of IL-8 Production in Neutrophils Induced by DV Stimulation

Human neutrophils were incubated with DV for increasing periods of time and

IL -8 production was assessed by ELISA. Figure 1 shows the results from one of

these experiments. Freshly isolated neutrophils produced low levels of IL -8. These

levels were not significantly changed by incubation in the medium alone for up to 24

h. However, a time-dependent increase in the secretion of IL -8 was found in both

DV and LPS stimulated neutrophils. Significant level of IL -8 was detected 6 h after

incubation and reached the peak level between 12 h and 24 h, the longest period of

time tested. A dose-dependent IL-8 production was also found when different MOIs

of DV were used to stimulate neutrophils (data not shown).

Comparison of IL-8, MIP-1α and MIP-1β Release of Neutrophils Induced by

Live and UV-inactivated DV

The concentrations of IL-8, MIP -1α and MIP -1β in the culture supernatants of neutrophils after incubation with DV or LPS for 18 h are shown in Figure 2.

produced only a small amount of IL -8 and MIP -1α while DV or LPS stimulation significantly increased the release of IL-8 by 2 to 3 fold (1000-1500 pg/ml) and

MIP-1α by 4 to 5 fold (2000-2500 pg/ml). UV inactivation of DV abolished the ability of DV to induce IL-8 and MIP-1α secretion in neutrophils. On the other hand, there was a spontaneous release of MIP-1ß in the control neutrophils without any

stimulation or with C3/36 medium (1500-2000 pg/ml). While DV or LPS stimulation

could further increase the release of MIP -1ß to 3000-3500 pg/ml, UV inactivation of

DV did not significantly reduce its activity to induce MIP -1β release of neutrophils (fig. 2).

Neutrophil Degranulation Induced by Both Live and UV-inactivated DV

Since myeloperoxidase (MPO) is one of the enzymes in the primary granules of

neutrophils, we used MPO as a marker to detect the degranulation of the

neutrophils. After 18 h of stimulation, MPO levels in the culture supernatants were

measured. DV and LPS induced significant amount of MPO release (117 ± 9 ng/ml

and 106± 27 ng/ml, respectively) from neutrophils compared to the un-stimulated

viable DV is required to stimulate neutrophil degranulation, neutrophils were

stimulated with UV -DV. Inactivation of DV with UV radiation did not significantly

abate MPO release from neutrophils (101.5± 1.2 ng/ml), in comparison with viable

DV.

Increase the Expression of CD11b and TLR4 on the Surface of DV-stimulated Neutrophils

To further confirm the activation of neutrophils induced by DV stimulation, the

surface expressions of adhesion molecule CD11b and signal-tranducing molecule

TLR4 of DV-stimulated neutrophils were analyzed by fluorescent antibodies and

confocal fluorescent microscopy. DV and LPS significantly increased the expression

of CD11b and TLR4 on the surface of neutrophils (fig. 3). The expression of CD11b

on neutrophils was further analyzed by flow cytometry. About 2% of the control

neutrophils were CD11b positive before stimulation. LPS, DV and UV-DV

stimulation induced CD11b positive neutrophils to 59.9%, 37.8% and 25.2%,

Infection and Morphological Changes of DV-stimulated Neutrophils

After 18 h of incubation with DV, about 30 .4 % of neutrophils were DV E protein

positive compared to only 2.6% in mock-treated neutrophils as detected by anti-DV

E protein antibody and flow cytometry. However, no viral progeny was detected in

the supernatants or cell lysate of neutrophils by plaque assay. In addition, no NS-1

protein can be detected in these cells using anti-NS-1 antibody (data not shown),

indicating DV did not replicate in neutrophils . When neutrophils were cytospined

onto slides and stained with Liu’s solution or fixed for electron microscopy

examination 18 h after incubation, more vacuoles and less granules were found in

DV-stimulated neutrophils than in C6/36-treated cells (fig. 4).

Phagocytosis and Co-localization of DV and FITC-beads in Neutrophils

To assess the phagocytic activity of neutrophils, FITC-beads were incubated

with neutrophils with or without pre-incubation with DV. FITC -beads were detected

in 29.9% of un-stimulated neutrophils as determined by flow cytometry (fig.5). DV

stimulation of neutrophils increased the percentage of neutrophils with FITC-beads

(fig. 5). About 48% and 62% of neutrophils were positive for FITC -beads in the

presence of DV at MOI of 1 and 5, respectively. The fluorescence intensities of

DV-stimulated cells were also significantly increased, indicating an increase in the

number of FITC-beads in these cells . Using PE-conjugated anti -DV E protein

antibody and confocal microscopy, co-localization of DV E antigen and FITC-beads

was found in DV-stimulated neutrophils as indicated by the appearance of yellow

dots (fig. 6). However, there were a few green and red dots in DV-stimulated

neutrophils also; indicating some of the neutrophils contained only DV or

FITC-beads.

Decrease of Neutrophil Viability and Apoptosis in the Presence of Live but not

UV-inactivated DV

The cell viability of neutrophils after DV stimulation was measured by three

different methods, trypan blue stain, PI staining followed by the flow cytometry

analysis and XTT assay. Both trypan blue and PI stain, which mainly detect the

changes of membrane permeability, showed similar results (fig. 7). No significant

neutrophils (greater than 90%) were still survived after 18 h of incubation in vitro

without any stimulation or stimulated with LPS or UV-DV. However, the survival rate

of neutrophils was reduced to 50-60% in the p resence of live DV (fig. 7). Similar

results were also found in XTT assay, which mainly detect mitochondria enzyme

activity. The survival rate of DV-stimulated neutrophils was significantly decreased

after 18 h of incubation as compared with the controls (fig. 7). To further understand

the mechanism of decreasing the viability of neutrophils in the presence of DV, two

different methods were used to measure the occurrence of apoptosis (annexin V

and TUNEL stains). There was no significant change in the apoptotic cells after 3 h

of incubation in all groups. However, a spontaneous apoptosis of un-stimulated

neutrophils (about 50% by annexin V stain, and 35% by TUNEL) was detected after

incubation for 18 h (fig. 8). Incubation of neutrophils with C6/36, UV-DV or H-DV for

18 h did not change the percentage of apoptotic cells significantly. However,

incubation of neutrophils with LPS or live DV significantly decreased apoptotic cells

to 20-30% (fig. 8). In all groups, annexin V stain seemed to be able to detect more

Increase of Neutrophils Necrosis in the Presence of Live DV

Cellular necrosis was detected by the release of cytoplasmic enzyme LDH. After

12 h of incubation with DV, significant amounts of LDH were released by neutrophils

compared to uninfected cells or LPS-stimulated neutrophils. About 20% and 40% of

Discussion

Neutrophils constitute the first line of host defense against infectious agents

and exert a significant influence on the outcome of infection. In this study, we

demonstrated that a variety of chemokines were released from DV- but not

UV-DV-stimulated neutrophils. Chemokines are important mediators of the

immune system with primary chemotactic properties. By recruiting phagocytes as

well as lymphocytes to infection sites, chemokines connect nonspecific with

specific compartments of the immune system and therefore, play a pivotal role in

developing a rapid, focused, and effective immune response [25]. IL-8 released

by DV-stimulated neutrophils can recruit more neutrophils to the local infected

site and induce neutrophil adherence to vascular endothelium and extravasation

to tissues [6, 20], while MIP -1α and MIP-1β induced by DV-stimulated neutrophils can attract other immune cells such as monocytes, T cells, basophils to the

infected lesion [26, 31, 32]. All these immune cells are potential targets for DV

infection, which in turn can release more inflammatory cytokines to amplify the

elevated levels of IL-8, MIP -1α and MIP-1β are found in dengue patients [23, 30]. In addition, MIP -1α can also inhibit hematopoietic progenitor cell growth, which may account for some of the hematological disorders associated with dengue

illness [22]. DV stimulation of neutrophils also induced MPO release, which is a

marker for the degranulation of neutrophil primary granules. This is consistent

with the results from previous reports which showed that another granular

enzyme, elastase was significantly increased in DHF patients with shock [14].

The activation of neutrophils induced by DV was further supported by the

increased expression of C D11b and TLR4 in DV-stimulated neutrophils. The

increased expression of CD11b/CD18 (Mac-1) integrin on the surface of

DV-activated neutrophils may enhance the binding of neutrophils to ICAM-1

positive endothelium [21, 24, 28]. On the other hand, the significance of TLR4

expression induced in DV-stimulated neutrophils is unclear, except to reflect the

status of neutrophil activation. TLR4 plays a very important role in the signal

transduction induced by LPS and serves as a cell-surface co-receptor for CD14,

leading to LPS-mediated NF-κB activation and subsequent cellular events [4]. In a previous study, it has been shown that LPS can i nhibit DV infection of human

monocytes/macrophages by preventing virus entry via a CD14-dependent

mechanism [3]. Whether TLR4 may involve in the binding and signal transduction

induced by DV requires further study.

In this study we also demonstrated that DV was taken up by neutrophils

through phagocytosis and was presumably degraded inside the cell

compartments. This result is consistent with the previous report which found that

neutrophils did not become infected and replicate DV [33]. Similar phagocytosis

and degradation of DV have been previously reported in other human

phagocytes such as kupffer cells and monocytes [7, 19]. However, the

mechanisms of viral clearance by these phagocytes are unclear; probably both

oxygen-dependent and independent pathways are involved [5]. In striking

contrast to previous reports which indicate the stress of DV infection induced

apoptosis of many different cells [1, 7, 13, 18], we report that DV induced human

neutrophils undergo necrosis. This is based on the following observations that

the survival rate was decreased, while less apoptosis was found in DV-stimulated

neutrophils as compared to un-stimulated neutrophils. In addition, the release of

dose-dependent manner after incubation with DV. Normally, neutrophils are

considered to be short-lived, terminally differentiated cells undergo spontaneous

apoptosis (programmed cell death) in vitro if not appropriately stimulated [9, 27].

Apoptosis, which in contrast to necrosis, maintains the plasma membrane

integrity of neutrophils. This may explain why about 90% of un-stimulated

neutrophils were viable as detected by trypan blue or PI, yet a 35-50%

spontaneous apoptosis were detected in these cells at the same time. Cells

undergo apoptosis can avoid inflammatory reactions due to the release of

tissue-injurious granule contents. On the other hand, release of neutrophils’

formidable arsenal of protease and other toxic intracellular contents into tissues

can create significant damage, prolonging the inflammatory response. Taken

together all these results, we think neutrophils may play an important role in the

clearance of DV at first place. However, excessive activation and necrosis of

neutrophils induced by DV may also contribute to the pathogenic development

Acknowledgments

This work was supported by grants NSC90-2320-B006-058 from National

Science Council and grant NHRI-CN-CL-8901P from National Health Research

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Figure legends:

Fig. 1. Kinetics of IL-8 release by neutrophils in response to DV and LPS.

Neutrophils (2x106) were incubated with DV at MOI of 1, LPS (200 ng/ml) or without

stimulation (C) at 37 ℃. The supernatants were collected at indicated times and

assayed for IL -8 by ELISA. The results presented in this figure represent the mean

+ SD of experiments performed in triplicate on neutrophils from three individuals.

Fig. 2. Chemokine release from neutrophils after DV stimulation. The

concentrations of IL-8, MIP -1α and MIP -1β in the culture supernatants of

neutrophils after incubation with LPS, DV or UV inactivated DV (UV-DV) for 18 h

were assayed by ELISA as described in the Materials and Methods. Cells treated

with medium (C) or C6/36 supernatant (C6/36) were also included. Data from three

experiments are shown as mean + SD. *p＜ 0.05, compared with the corresponding

values from un-stimulated neutrophils (C) or mock DV stock (C6/36).

Neutrophils (2 x 104 cells/well) were incubated with LPS, DV or without any

stimulation (C) for 18 h. Cells were stained with FITC conjugated anti-CD11b or

anti-TLR4 antibodies. The expression of CD11b and TLR4 on the surface of

neutrophils was observed by confocal fluorescent microscopy as described in the

Materials and Methods section.

Fig. 4. Morphological changes of neutrophils after incubation with DV. (A)

Neutrophils (2x106) were incubated with DV or C6/36 supernatant (C6/36) as

indicated at 37 ℃ for 18 h. Cells were cytospined onto slides, stained and observed

under light microscopy at 100X magnification. (B) Transmission electron

micrograph of DV-stimulated neutrophils. Neutrophils which showed numerous

vacuoles (V) also showed fewer granules (G) in the cytoplasm.

Fig. 5. DV stimulation increased phagocytosis of neutrophils. Neutrophils (2x 106)

were pre-incubated with medium (C) or DV at MOI of 1 or 5 for 1 h followed by

incubation with FITC-beads for 30 min at 37℃. Free FITC-beads were washed

the Materials and Methods section.

Fig. 6. Co-localization of DV and phagocytized particles in neutrophils. Neutrophils

(2x 106) were pre-incubated with medium (C) or DV at MOI of 5 (DV) for 1 h

followed by incubation with FITC -beads for 30 min at 37℃. Neutrophils were

washed, fixed and permeabilized after incubation. Intracellular DV particles were

detected using biotin labeled anti-E antibodies and PE-conjugated streptoavidin,

and then visualized under a confocal microscope as described in the Materials and

Methods section.

Fig. 7. Evaluation of cell viability after DV stimulation. Neutrophils (2x 106) were

inoculated with LPS (200 ng/ml), DV or UV-DV at MOI of 1 for 18 h. Trypan blue

up-take and PI stain were assayed using light microscopy and flow cytometry,

respectively and XTT assay was assessed using a microplate reader. The data

represent the mean of results from two different experiments of trypan blue and PI

stain, while the results of XTT assay were from three different experiments and are

Fig. 8. Effects of DV on the spontaneous apoptosis of neutrophils as determined by

annexin V and TUNEL stain. Neutrophils (2x106) were incubated with DV (MOI=1),

LPS (200 ng/ml) or without stimulation (C) at 37 ℃ for 18h. In addition, some cells

were treated with C6/36 supernatants (C6/36), heat or UV-inactivated DV (H-DV or

UV-DV) as indicated. Cells were stained with annexin V or TUNEL and analyzed by

flow cytometry as described in the Materials and Methods section. The data

represent the results from three different experiments and are shown as mean ±

SD. *p ＜ 0.05, compared with un-stimulated neutrophils.

Fig. 9. LDH release of neutrophils induced by DV. Neutrophils (2x105) were

incubated with DV at different MOI as indicated, LPS (200 ng/ml) or without

stimulation (C) at 37 ℃ for 18 h. The supernatants were collected and assayed for

LDH by ELISA. The results presented in this figure represent the mean + SD of

experiments performed in triplicate on neutrophils from three individuals. *p ＜

Fig. 1 0 500 1000 1500 2000 2500 3000 0 6 12 18 24 hour IL-8 (pg/ml) C DV LPS

Fig. 2

0

500

1000

1500

2000

2500

3000

3500

4000

C

C6/36

LPS

DV

UV-DV

pg/ml

IL-8

MIP-1α

MIP-1β

*

*

*

*

*

*

*

Fig 3

DV LPS C

DV LPS C

CD11b

Fig 4

Fig. 4

Fig. 6

Fig. 7 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100 Unstimulated L P S DV UV-DV viability (%) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 O.D Trypan blue P I XTT

*

Fig. 8 0 10 20 30 40 50 60 C C6/36 D V LPS H-DV UV-DV apoptosis % Annexin V TUNEL

*

*

*

*

Fig. 9 0 5 10 15 20 25 30 35 40 45 DV(MOI=1) DV(MOI=2) C LPS LDH release (%)

*

**